The methods used by actors to learn an accent or a dialect often overlook the cultural and historical significance of a particular way of speaking. More often than not, the majority of the time spent learning a dialect is only spent memorizing the production of distinctive sounds. And what is learned is a “general” version of a dialect that is often stereotyped and not representative of a speaking community. This comes from a long history of reducing the innate complexity of dialects and the creation of foreignness through language prescriptivism and language prestige systems. These impose ideas such as a “proper” way to speak English on stage or the association of negative characteristics with a particular accent. Many productions will not use dialect coaches due to budget constraints or the choice of a director or producer. When productions run on a limited time schedule, dialect coaching is pushed to the side in favor of other elements of character building. This can lead to an actor not doing the proper research, performing a dialect poorly, or at certain cases the production deciding not to use a dialect that is an integral part of a character’s identity. There is also a frame of mind within society that thinks of accents as either authentic or passable, as opposed to accurate. What leads to poor representation on stage is when productions don’t take into consideration the effect a dialect or lack thereof will have on the production as a whole. This project brings a critical lens to the history of dialect coaching and proposes how as theater makers we can shift our thinking regarding accents and through dialect coaching create better representations on stage.

At initial glance, Americans may envy the apparent androgynous nature of Korea’s fashion and dance world. However, what onlookers fail to understand is how the K-pop world’s creation of such genderless dancing and androgenous appearances may be a consequence of the gender binary enforced on Korean individuals by traditional Korean Confucian Values. In this research, I aim to explore how Americans who indulge in Korean pop culture may have interpretations which differ from Koreans who have personally experienced the gendered expectations in contemporary Korean society. I conducted a literature review that focused on the gender presentation of Bangtan Sonyeondan and how cultural values are still strongly weaved into modern Korean society. I also interviewed American and Korean American individuals to conduct a comparative analysis on how their perspectives on Korean gender roles differ. Preliminary results suggest how there may be a glass ceiling to how far an idol’s gender performance can expand beyond societal gender binary constraints. Through my analysis, I hope to bring an alternative perspective to Americans who enjoy Korean culture that highlights some of the unfortunate faults of the seemingly perfect industry.

Bats are a maligned and misunderstood, yet crucial, part of urban ecosystems. Often attributed as pests, bats are major insect population controllers and biodiversity indicators with complex social systems and language. In Seattle, bats take advantage of the abundance of water and tree canopy, and are often seen in public parks and natural areas. However, the current social climate around bats is based mainly on fear of disease and human injury. Bat populations globally are in decline due to habitat loss, and from a fungus that causes white-nose disease, which can be fatal to bats. Landscape architecture is uniquely poised to address both the environmental needs of native wildlife in cities, as well as the social perception of nature. This project uses landscape design as a lens to interpret scientific research on insectivorous bats and proposes creative methods of landscape intervention to communicate these concepts to the public.The structure of this project is composed of three main elements: a literature review that examines current research being done on bats in cities worldwide; a series of field observation sessions in three Seattle parks to document bat emergence sites; and design proposals for highlighting areas in these parks where bat activity can be safely observed by the public. Data collected includes (but is not limited to) landscape assessment of bat emergence sites, number, and species of bats observed. Results and design proposals are presented visually, including audio recordings and photography of field observations along with digital collages of site design proposals. Also included are interview excerpts, key findings from literature review and an explanation of scientific communication concepts.

Metastasis, or the spread of cancer to a secondary site, is responsible for most cancer-related deaths. The tissue-specific environment that disseminated tumor cells experience influences whether they will proliferate and colonize the tissue, remain dormant, or die. Skeletal muscle (SkM) is one of the rarest sites for metastasis, despite making up nearly half of human body mass. What makes SkM so resistant to metastasis? We set out to test the hypothesis that the mechanical nature of SkM is responsible for the lack of metastases at this site. To do so, we used the mdx mouse model of Duchenne muscular dystrophy to probe if the destruction of SkM structure and function would make it a more hospitable host to disseminated tumor cells. Wild type and mdx mice were intramuscularly injected with EO771 murine mammary tumor cells and monitored for tumor outgrowth using bioluminescent imaging. Preliminary results suggest that tumor growth is increased in dystrophic (e.g. dysfunctional muscle) versus wild-type mice. A complementary and more reductionist approach to test whether mechanics influences tumor colonization of muscle is to employ a culture model that allows tumor cells seeded on top of a SkM layer to experience mechanical stretching akin to the contraction/relaxation movements of muscle. To accomplish this, we constructed a device that applies a cyclic stretch to a 3D organotypic SkM culture model on a flexible silicone plate. We predicted that stretching would reduce tumor cell survival, when compared to no stretching. While these experiments are in progress, we believe that these data may elucidate a relationship between mechanical activity and suppression of tumor outgrowth in SkM. This work will contribute to a more complete understanding of how SkM avoids tumor colonization and could inform future approaches that leverage tissue mechanics to treat or prevent metastasis.

Mutations in myosin may lead to severe muscle disorders that greatly reduce the quality of life. For example, the embryonic skeletal myosin (MYH3) mutation R672C leads to Freeman Sheldon Syndrome (FSS), a rare inherited disorder that causes severe contractures at birth. A comprehensive understanding of the relationship between protein structure and function is urgently needed to treat diseases such as FSS. Computational methods, such as molecular dynamics simulations, can be used to examine the effects of mutations on protein structure and function. However, the Protein Data Bank (PDB) is missing most human skeletal myosin heavy chain structures. We employed homology modelling to construct structures of human MYH3. To inform homology modeling, I generated a multiple sequence alignment of 7 human myosin genes. The extent of sequence identity was used to identify the optimum myosin isoforms to use as templates for model generation. For example, MYH3 and MYH7 were the most distinct at 78.93% similarity, which was expected as they are embryonic skeletal and adult cardiac myosin respectively. Specific sequence consensus at each position in the sequence was used to determine the most and least conserved regions of myosin. The cleft region was the most conserved; the N-terminal Domain was the least conserved. I used MYH7, adult cardiac myosin, as a template structure to derive a homology model of the ATP state of MYH3. A structure of MYH3 R672C was generated via in silico mutation of the wild type structure. Molecular dynamics of the resulting structures will be used to explore how R672C, which is located near myosin’s converter domain, alters myosin structure and function. This computational platform will model all phases of the cross-bridge cycle, potentially reveal new drug binding pockets, guide and be validated by in vitro experiments using human induced pluripotent stem cell derived myocytes (hiPSC-Ms).

The purpose of a newly developed foot prosthesis is to improve frontal plane foot adaptability for over 1 million adults living with lower extremity amputation in the US. On uneven ground, side slope, and turning corners, the anatomical foot can adapt but most prosthetic feet cannot. The innovative prosthesis mimics biomechanical aspects of anatomical joints that use ligaments in tension throughout the range of motion while the joint surface acts as a specialized load-bearing structure similar to that of a cross four-bar linkage. In the current prototype cam linkage, we found deviation between the instantaneous center of rotation (ICR) and the point of contact (POC) as it rotated through the frontal plane. The error in this distance could cause unexpected wear on the prosthesis. The goal of this project was to reduce the error caused by the difference in location of the ICR and POC during rotation and translation of the four-bar linkage. In a crossed four-bar linkage, the ICR is found at the intersection of the crossing links. To find the path of the ICR, we plotted a series of points by rotating the linkage about a fixed link in space. The continuous ICR path was estimated by smoothing the ICR points. The curved shape developed becomes the load-bearing surface of the linkage. To create the upper and the lower load-bearing surface, the upper and lower link must be fixed and the opposite rotated, respectively. Using the method described, we built linkage assemblies that showed an error of 0.96 mm per 20 degrees of rotation compared to 2.17 mm of error in the original prototype. The International Organization for Standardization (ISO) requires that foot prostheses undergo wear and fatigue testing. Reducing potential wear on the prosthesis is advantageous because engineers can design for optimal product durability for the foot.

The dystrophin protein protects cardiac and skeletal muscle from damage during contraction and relaxation. Mutations in dystrophin lead to Duchenne muscular dystrophy (DMD), an incurable X-linked recessive disease affecting 1 in 3500 boys. Previous work has shown that several cardiac symptoms of DMD can be traced to calcium handling defects. To that end, a preliminary drug screen by our lab identified several L-type Calcium Channel blockers (CCBs) that were able to provide a cardioprotective effect. To conclusively determine the effectiveness of these CCBs, a platform that can accurately replicate physiological cardiomyocytes and screen these CCBs at semi-high throughput is needed. A major limitation with current drug screening platforms is that they use cardiomyocytes equivalent to the fetal heart. This is due to the limitations in current differentiation protocols, which fail to induce further maturity. Because symptoms of most inherited cardiomyopathies are exhibited in mature cardiomyocytes, these platforms are unable to predict drug efficacy accurately. Additionally, microelectrode array (MEA) systems - a system for high throughput drug studies - require highly accurate cell plating to provide good quality results, which requires extensive and costly training. Here, we addressed these issues by developing a novel platform that uses ComboMat, a technique used to enhance cardiomyocyte maturity, and designing an assistive device to plate cardiomyocytes in MEA plates. We showed that our platform with ComboMat-treated cardiomyocytes can give a more physiologically relevant response compared to platforms that use untreated cardiomyocytes. A MEA-based drug study is currently being performed to validate the CCBs identified in the preliminary drug screen. We expect to successfully validate a subset of the CCBs analyzed and further test them in animal models. This project will culminate in creating a novel and cost-effective platform that offers superior prediction of drug efficacy for DMD and potentially other cardiomyopathies as well.

In the United States, 1.5 million individuals suffer a fracture due to bone disease each year. In addition, there are many unknown mechanisms behind how muscular disorders and mechanical load adversely affect bone development, such as in the disease distal arthrogryposis. Disease research in human cell models has greater translational potential compared to animal models but have faced challenges when constructing highly-specialized tissues such as bone. We propose a novel, three-dimensional bone tissue model as a platform for musculoskeletal disease modeling that allows for compressive loading. By seeding induced pluripotent stem cell (iPSC) derived osteoblasts and osteoclasts in a 3D, porous, hydroxyapatite-coated poly-L-lactide scaffold, we propose to generate a bone tissue model that replicates human tissue in a laboratory. By applying compression to the novel 3D bone tissue model, we expect to observe phenotypes of bone disorders and bone development under mechanical loading. We propose to induce osteoblast and osteoclasts lineage from mesenchymal progenitor cells and hematopoietic progenitor cells, respectively, and co-culture to identify optimal conditions for cell growth. Preliminary experiments have found success in culturing active osteoblasts from iPSC-derived mesenchymal progenitor cells. By screening for markers of cell proliferation, calcium deposition, bone resorption and secretion, the cultures can be assessed for their robustness. In parallel, a porous scaffold will be fabricated by dissolving poly-L-lactide in chloroform and molding over sodium chloride particles. Coating said scaffold in fibronectin and hydroxyapatite will improve cell adhesion and uptake bone secretion. Seeding osteoclast and osteoblasts cells in a porous scaffold will allow for improved cell diffusion and 3D growth, mimicking the human microenvironment. We expect that combining robust, osteogenic tissue culture on a bioactive scaffold that allows 3D bone growth with mechanical loading will reveal phenotypes of distal arthrogryposis. Thus, this method has significant applications in accelerating laboratory findings to clinical research.

Transcription factors such as TCF-1, as well as inflammatory cytokines such as IL-12, have been shown to play a role in cytotoxic (CD8) T cell differentiation during an immune response. Here, we build a stochastic computational model of the canonical CD8 T cell immune response encompassing experimentally hypothesized transcription factor and inflammation control mechanisms. We hypothesize that the transcriptional factor control of CD8 T cells can be quantified into concrete model parameters that control various aspects of the immune response such as cell effector cell expansion, proliferation, and memory cell formation in various immune challenge conditions, such as inflammation. With a fully developed model, effects of inflammation levels and gene regulation kinetics on CD8 T cell differentiation can be measured in situ, and new hypotheses surrounding cellular differentiation kinetics can be generated. Understanding the factors that control CD8 T cell differentiation is crucial in the development of vaccines and immunotherapies, such as CAR T cell therapy.

Fluorescent biosensors are a vital tool in the goal to decipher the complexity of neural networks. Genetically encoded fluorescent indicators (GEFIs) are protein-based sensors that increase in fluorescence upon ligand binding and allow for passive monitoring of neuronal signals. However, the development of such sensors is limited by the slow throughput of traditional protein engineering which has long engineering cycles of new plasmid variants. Our project aims to tackle this problem by developing a high-throughput sensor engineering platform that can effectively generate and screen unbiased genetic libraries of GEFIs in mammalian cells. Our platform can identify high performing sensor variants on a custom microarray and effectively isolate and recover their genetic material. Our new platform will be used to develop a sensor for the μ-Opioid receptor (MOR), which is a G-protein coupled receptor that is involved in opioid addiction. Our experiments have already developed a MOR sensor that surpasses the standard in the literature and we will continue to optimize it for maximum spatial and temporal precision. The development of a MOR sensor through this iterative process allows researchers to further investigate the molecular mechanisms underlying the pathology of addiction and provides a novel platform for protein engineers to more efficiently develop a wide variety of biosensors.

The purpose of this study is to examine the feasibility of virtual reality meditation (VRM) for symptom management in outpatients with rheumatoid arthritis (RA). The specific aims of this feasibility study include: 1) examining the feasibility of implementing VR meditation; 2) determining the acceptability of using VR-delivered meditation; and 3) exploring a patient’s experience of using VR-delivered meditation for symptom management. RA is a chronic disease that affects more than 1 million people in the U.S. While recent advances in medicine have shown promising results in managing physical symptoms, a large portion of outpatients with RA still suffer from fatigue. Recent studies have found that fatigue may be managed through meditation, but VR meditation has yet to be tested and deployed in this population. This feasibility study implements a mixed-methods design. Eight adults (18 years and older) with clinically-diagnosed rheumatoid arthritis were enrolled from a local rheumatology clinic. Participants used a VR headset incorporated with meditation software over the course of four consecutive weeks. Patient Reported Outcome Measurement Information System (PROMIS) measures of fatigue, pain, depression, anxiety, physical activity, and mood were collected at baseline and weekly intervals for 4 weeks. Two semi-structured interviews were conducted to capture the patient’s experience of RA, fatigue, as well as experience of the virtual environment. I was personally in charge of analyzing the interview transcriptions and adding coding measurement tags for the quantitative analysis. Results are currently pending. Expected results include that participants will find VRM both feasible and acceptable for fatigue management, and that participants will report reduced fatigue levels after using the VR device. Results of this study will inform future clinical trials using VRM, implementation of VRM into clinical use, and give a better understanding of the patient’s experience of utilizing VRM for fatigue management.

Years before the major events that are tied to the New Left in American collective memory, Bay Area college students’ protests against the House Un-American Activities Committee garnered national media attention for their perceived radicalism in the face of the federal government. Student protesters’ altercation with police at San Francisco City Hall in May of 1960 became a turning point at which the Old Left, New Left, and McCarthyism converged, providing valuable insight into the transition of broad leftist activism from union-based to direct action protest. Through secondary sources including histories of early student protest as well as student newspapers, government publications, supporting organizations’ communications, and oral histories from participants, I construct a timeline of the challenges early student activists faced. This critical angle centers how these protests’ complicate the historical understanding of the university as the postwar institutional mediator for left-wing protest and radicalism, thereby revealing the disparities and power relations between students, professors, and administrators in the pursuit of their respective political agendas. This perspective, therefore, challenges the prevailing notion of universities as a natural partner of progressive social movements by emphasizing the institutional obstacles and inadequacies that restrict student political activism and expression.

The international failure regarding humanitarian relief during the 1994 Rwandan genocide has been the topic of many research endeavors. From the lack of response of the United Nations (UN) to the limited media coverage, international actors continually dismissed the genocide in Rwanda as unimportant primarily due to a lack of interest in the country itself. While much of the existing literature focuses on UN documents and humanitarian aid worker testimony, this paper posits that survivor testimony is a source of largely untapped authority regarding the depth and human cost of the failures of the international community to respond to the genocide in Rwanda. Utilizing survivor testimony, this paper explores the extent of the failure of the international community to respond and the broader dehumanization of Rwandan victims during and since the 1994 genocide. The deeply personal perspectives of survivor accounts highlight three distinct failures: 1) the failure to prevent the genocide and subsequent atrocities; 2) the lack of justice brought to the perpetrators; 3) the lack of care and aid given to survivors in the years following the genocide. Survivors are acutely aware of the lack of intervention during the genocide and the enduring lack of aid and attention due to the lack of international interest. Their testimony speaks to the real human cost of the failure to intervene, as well as to ongoing fundamental institutional weaknesses in the international community today. This study thus ultimately suggests that survivor testimony has healing and perhaps even preventative potential.

As a student of both Religion and International Relations, I am intrigued and perplexed by the presence – or absence – of sanctity within international systems. My senior capstone, which I conceived of and researched myself, studies the Tlingit potlatch and its use of the sacred in international relations between the Sitka Tlingit Nation and the Russian Empire (prior to 1867), as well as the United States government (after 1867). Using secondary sources, the anthropological and historical work of Dr. Sergei Kan, and definitions of the sacred as discussed in the works of Mircea Eliade, I ask several questions to evaluate international relations theory. Firstly, what is the sacred and how does it connect us? Secondly, how was the sacred used in the Tlingit potlatch, and how did the Russians and Americans differ in their interactions within this space? Finally, how might a closer look at the potlatch help me understand one form of Indigenous international relations and how can the use of the sacred challenge assumptions made by predominantly white western international relations theorists? The Tlingit potlatch served as the primary space for international relations through the prescribed roles of “host” and “guest.” My research has led me to conclude that the Russian Orthodox Church leaders accepted their role as guests, thereby maintaining their sovereignty and the sovereignty of the Tlingit, and by extension, preserving international relations between the two groups. American Presbyterians, however, sought to conquer and control Tlingit sanctity, effectively dissolving the potlatch, Tlingit sovereignty, and the possibility of maintaining international relations between the communities. With these discoveries in mind, I ask, “What might Tlingit Elders tell us about how to conduct international relations, and what do we do with that?” By asking this question, I attempt to elevate Indigenous voices in the field of international relations and revolutionize the ways in which international diplomacy is conducted by allowing space for empathy, sanctity, and trust.

The representation of genocide in the post-conflict period is paramount in shaping public opinion and, in turn, providing relief, aid, and justice for the victims. Two common forms of representation of violence in genocide are statistics and the memory of survivors. Data and statistics add a viewable representation in graphs, tables, and charts. However, data and statistics can be manipulated and changed to suit one's biases. One of the most prevalent forms of memory in post-genocide periods is testimony. It can be intensely personal and can provide narratives for oppressed people during the conflict. However, testimony can be altered by trauma and memory of the witness and can be influenced by an interviewer. This presentation explores the delicate balance of memory and statistical representation in a post-genocide period, primarily in the Guatemalan Genocide. It contains an analysis of data on victims that was collected by the Guatemalan police during the genocide, widely published testimonials, and personal stories about the genocide. A mix of testimony and data representation can provide additional and nuanced insight into violent conflicts and help to create a peaceful and just society. Considering testimony and data together instead of completely separate entities fits with established and proven genocide prevention efforts.

Bacteria can inherit genes through two modes of transmission: vertical inheritance, in which a cell receives genes from its parent cell during division, or horizontal gene transfer, in which genes are passed laterally between two unrelated cells. Extrachromosomal DNA, called plasmids, can be transmitted from a bacterial cell to a neighboring cell of the same or different species through a form of horizontal gene transfer known as conjugation. These conjugative plasmids can encode for antibiotic resistance genes which allows a host cell to produce proteins that degrade antibiotics. In an environment with these drugs, a host cell with this plasmid would be more likely to survive, compared to a plasmid-free cell, due to the benefits of the antibiotic resistance gene. Additionally, these antibiotic resistance genes can often acquire mutations which increases the level of antibiotic resistance for the host cell. However, these acquired mutations may be more beneficial (i.e., higher resistance for the host cell) in one bacterial species than the same mutation in another species. In other words, the effect of mutation on the plasmid-encoded antibiotic resistance gene may be contingent upon the host species. Given that antibiotic resistance genes are often encoded on conjugative plasmids that are shared among species, my project is investigating how protein evolution may be affected due to plasmid genes existing in multiple bacterial hosts. I performed a series of competitions between three bacterial species containing plasmids with versions of an antibiotic resistance gene to determine which versions of the gene outcompete others in an environment containing antibiotics. In doing so, I can determine how this particular gene may evolve when various bacterial hosts exist in a microbial community. The results of this experiment will improve our understanding of plasmid biology and the evolution of antibiotic resistance genes in diverse communities of bacteria.

Memory has been widely studied for its crucial role in learning and its diverse range of expression. Although the means of acquisition differ, it is generally accepted that memory goes through three encoding stages: sensory, short-term and long-term memory. The retention of memory is important as it enables us to act with the wisdom of past experience. However, although one could not survive without memory, remembering everything is also devastating. In fact, forgetting is an important cognitive feature that allows us to adapt to the constantly changing environments. Despite its importance for cognition, little is known about the molecular nature of forgetting. Here, we investigated the genes behind forgetting by studying an olfactory memory in the nematode C. elegans. Like human, worms can modify their behavior upon acquiring unpleasant experience – their movement towards preferred odor is significantly reduced after prolonged exposure to the odor during starvation. Upon returning to food (e. coli OP50), the odor attraction slowly returns to the worms within 3-4 hours, indicating a diminishing impact of the starvation experience. By contrast, the forgetting process was significantly accelerated by 1-2 hours, when worms were cultivated on pathogenic bacteria, pseudomonas aeruginosa PA14. Our genetic studies showed that a null mutation to the daf-16 gene restored recovery time to 3-4 hours, despite of the exposure to PA14. These data indicate that daf-16 plays a positive role in accelerated memory loss upon pathogen ingestion. Because DAF-16 is involved in innate immunity and stress response, our results provide a potential connection that couples the memory to environmental stressors.

Calcific Aortic Valve Disease (CAVD) is progressive osteogenic changes including calcium deposition and thickening of the aortic valve causing stiffness which impairs normal function ultimately leading to ventricular hypertrophy and death. Runx2, a transcription factor involved in osteogenic changes, has been observed to be upregulated in the diseased valves. RNA sequencing data from the lab had previously revealed several genes regulated in a Runx2 knock out mouse model of CAVD. This study aims to determine if NKX2.5, one of the genes identified in the RNA sequencing data, is an upstream regulator of Runx2. Baboon valve interstitial cells (BVICs), which can be induced to calcify and share characteristics similar to human valve cells, were used in CRISPR knockout experiments to determine molecular and biochemical changes following NKX2.5 deletion. CRISPR-Cas9 is an effective method of gene deletion that uses programmable guide RNA to induce a gene mutation, inactivating its protein product. Initial experiments were aimed at optimizing the conditions for electroporation and gRNA delivery to ensure BVIC survival and efficient gene deletion in the cell line. Gene deletion success was determined by sequencing a ~1kb region around the deletion site. After generating BVIC lacking NKX2.5, cells were cultured in media known to induce calcification. A biochemical assay indicated changes in activation. Cells were assessed for calcification changes using biochemical assay, changes in activation, and Runx2 expression determined via RT-qPCR. We expect to confirm that NKX2.5 negatively regulates Runx2 and in turn calcification.

Mapping the locations where transcription factors interact with DNA is central to uncovering genome regulation mechanisms and gives key insights as to how organisms respond to intracellular or extracellular signals. The current gold standard for characterizing transcription factor binding sites is Chromatin Immunoprecipitation Sequencing (ChIP-Seq). Despite the ubiquity of ChIP-Seq, significant caveats remain. This method requires high-quality antibodies that may be laborious and costly to acquire, and large amounts of material are needed to obtain reliable data. We propose an alternative to ChIP-Seq: exploiting the interbacterial toxin double-stranded DNA deaminase (DddA). DddA catalyzes the deamination of cytosine to uracil, which is replaced by thymine via DNA replication. We harness this activity by designing translational fusions of DddA to the protein of interest. We hypothesize that the target protein carries DddA to its DNA binding region, introducing local C→T mutations that deep-sequencing detects. As a proof of concept, we tested this system with known bacterial transcription factors GacA, GcsR, and FleQ. Our results confirmed that DddA fusions have higher localized C→T mutations where the characterized transcription factors bind. We also tested different conditions and construct designs to maximize the efficiency of this method. Our results show that the use of DddA as a method of analyzing protein–DNA interactions is a broad and powerful tool with advantages over current methods like ChIp-Seq. Through this, we can better understand how organisms control gene expression when infecting a host, competing for resources, or surviving stressors.

Performing computational molecular dynamics (MD) simulations of small-molecule systems has become one of the most prominently used methods in studies of molecular structure and behavior. However, MD is a computationally expensive and time-consuming methodology because of the requirement of computing detailed interactions among atom-atom pairs. There is great interest, therefore, in reducing the time and computational power needed to approximate real-world systems. Most commonly, such efforts have employed machine learning techniques to predict extensive properties of molecular systems. Here, we propose accelerating simulations by predicting conformational changes - a prospect that has not yet been fully explored. Previous work attempted applying a linear dynamical analysis algorithm named Dynamic Mode Decomposition to MD data, which has been shown to be ineffective through a multiresolution analysis. We propose herein the use of Sparse Identification of Nonlinear Dynamical Systems (SINDy), a nonlinear model which has been shown to accurately decipher the governing equations of dynamical systems. We will be testing the effectiveness of SINDy with MD data by performing an iterative error analysis while varying the initial parameters of the dataset, thereby gaining a better understanding of how much data (and in what form) should be inputted to maximize the accuracy of a simulated SINDy model of an MD dataset. If shown to be sufficiently accurate, we then can implement SINDy simultaneously with MD in an active learning loop to save time and computational power while maintaining a high degree of predictive capability for peptide conformations. The current goal is to obtain a deeper understanding of peptide conformational changes that could, in the future, be combined with machine learning techniques to greatly accelerate classical MD simulations.

This project is supported by the UW Computational Neuroscience Center, and the DMREF Program of NSF through the MGI platform under DMR# 1629071, 1848911, and 1922020.

Mitochondria are multi-functional organelles that regulate calcium signaling, an important signaling pathway that controls cellular processes ranging from transcriptional regulation to vesicular secretion. Calcium ions enter the mitochondria through the Mitochondrial Calcium Uniporter (MCU) complex, a highly selective calcium uniporter controlling calcium flux across the inner mitochondrial membrane. Previous data suggests uniporter inhibition alters lipid metabolism and induces a qualitative increase in cytosolic lipid droplets. Stemming from this initial observation, we created two research goals for this project: 1) to quantify and characterize lipids in response to MCU inhibition and 2) aims to explore the role of known or novel lipid synthesis pathways in MCU-dependent lipid accumulation. To understand the role of MCU in cellular physiology, we used CRISPR technology to knockout the MCU gene (MCU KO) in HeLa cell lines. We quantified differential lipid accumulation in single cell images through confocal fluorescence microscopy. In addition, potential mechanisms were investigated by Western Blot analysis and quantitative Polymerase Chain Reaction (qPCR). Here we verify that inhibition of mitochondrial calcium flux is responsible for lipid accumulation, a phenotype rescued by exogenous MCU expression. This accumulation is attributed to the upregulation of a transcription factor – Nuclear Factor of Activated T cells 4 (NFATc4). Gene knockdown of NFATc4 rescues the MCU phenotype, strongly suggesting NFATc4 as a downstream signaling factor of MCU inhibition. In follow-up experiments, we seek to identify MCU-regulated gene expression and understand their roles in regulation of metabolism. Although the function of increased cellular lipid content remains elusive, we theorize it may play a role in mitochondria-to-cytosol stress communication. Futher characterization of lipid-mediated stress pathways may identify novel targets for conveying increased cell stress tolerance.

The European Court of Human Rights (ECtHR) rules on controversial human rights cases in Russia to carry out the country's commitment to the European Convention of Human Rights (ECHR). However, over the past 30 years, Russia has sought to disregard its membership in the Council of Europe by overruling ECtHR rulings. In 2015, Russia passed Law no. 7-FKZ to allow the Constitutional Court of the Russian Federation the ability to overrule the ECtHR’s rulings, when deemed in conflict with the Constitution of the Russian Federation. To decipher the impact of Russian Law no. 7-FKZ, I investigate the implementation of ECtHR rulings before and after 2015. This project examines the consolidating democracy of Russia's attempt to disregard international law by tightening access to courts, overruling and limiting the implementation of ECtHR rulings, and limiting individual freedoms and liberties. To answer, I develop an original case law dataset from 2014 to 2020 that includes the key ECtHR cases involving Russia. I code for the implementation level of ECtHR judgments to determine Russia’s ability to obstruct ECtHR judgments, after the enactment of Law no. 7-FKZ. Preliminary findings suggest that Russia is successful in limiting the reach of international law. These findings suggest de-democratizing nations have the ability to isolate on an international level, highlighting the struggles international courts may face when their legitimacy is challenged.

Mexico underwent a dramatic cultural revolution in 1920, and from this revolution sprung a new type of art. ‘Los Tres Grandes,’ as they came to be known, were three men, Rivera, Orozco, and Siqueiros, who painted legendary murals depicting anti-capitalist and Revolutionary themes. This paper discusses the works of David Alfaro Siqueiros, a revolutionary Mexican mural painter, and his involvement in the Modernist movement in the United States. I first explore Siqueiros’ Los Angeles mural America Tropical and argue that he created it to be a strong political statement in a time of national turmoil. I explore the iconology and iconography of the piece that combined ancient Maya symbolism with modern techniques, creating a wholly new form of publicly available art. I also highlighted the importance of the location of this mural in a Los Angeles district that relies on white tourism for survival, and contrasted it with a mural highlighting the suffering of Indigenous people in the United States and America as a whole. Focusing on his time spent in the United States, I also explore his artist-relationship with the modernist painter Jackson Pollock and Siqueiros’ apparent influences on his style. Finally, I will discuss the influences that Los Angeles art culture had on Siqueiros, including a change in his mural style from individualistic to collectivist.

The concept of the separation of church and state was not a priority for the founders of the Irish Republic. Elements of the Catholic religion were woven into the Constitution, a reality that characterizes the laws in this country to this day. Despite this beginning, Ireland is transitioning away from a legal system and laws based on traditional and conservative religious values and adopting more progressive policies that reflect the current norms and values of society. In order to better understand this transition and how it is reflected in the legal system, I examine the legalization of abortion in Ireland focusing on the case law of the Supreme Court of Ireland and rulings regarding Irish reproductive rights by international courts. In this project, I assess whether the decline of traditional religious values in Irish society allowed the Irish Supreme Court to become more activist or progressive. To answer this question, I develop an original case law dataset from 1973 to 2010 coding the judgments for change in the legality of abortion. Further I utilize World Values Survey data to map changes in religious values during this time period. Preliminary results suggest that international pressure and change in the dominance of traditional and conservative religious values pushed the legalization of abortion. This research informs our general understandings of religious values and the legal system, and the role of courts in bringing change.

In the fields of physics and economics, the conceptualization of time is that it is constant, never changing. However, using a cognitive psychological approach, time is anything but constant. Some days seem incredibly long, while others fly by quickly. Some days we have all the time in the world, while other days we don’t have enough time. This is a result of our perception of time. There is much research in cognitive psychology about manipulation of our Temporal Perception, but there is no research on the application of this phenomenon to how we organize and prioritize our daily tasks. Additionally, there is no current information on whether the perceived Temporal Distance of a task’s due date changes when a person completes a task alone vs in a team. My project seeks to fill both these voids in literature. This project is based on perception, so there is commonly a difference between what participants say and what they do. To address this difference, I intend on conducting a survey about individual participants’ experiences with prioritizing tasks and experiences with teamwork, as well as conducting a task-prioritization activity. The survey will be disseminated over Amazon mTurk’s survey service, while the teamwork task prioritization activity will be conducted throughout courses here at the University of Washington. I am still in the construction phase of this study so I have no preliminary results yet, but one hypothesis is that there will be a positive correlation between team process gain (whether a group of people works better in a team than alone) and perceived temporal distance, with a negative correlation between stress level and these variables. A summary matrix of temporal distance perception and prioritization will be presented that can inform team leaders, as well as individual members about the management of time.

Teamwork, now widely recognized as a vital corporate, academic and social dynamic, requires each team member to share a similar individual understanding of the task at hand. In this research, we analyzed shifts in team members’ individual mental models to gain a deeper understanding of this process. We listened to and coded audio files granted by NASA’s Human Research Program, extracted from a few of NASA’s Human Exploration Research Analog (HERA) mission campaign teams as they were preparing for, executing, and debriefing various simulated missions. We developed a coding scheme to assess the statement’s process, purpose, and contribution to mental model shifts. A statement’s process refers to its topic in terms of planning, acting or evaluating (this idea is backed by literature). A statement’s purpose declares whether what was said was an agreement, disagreement, suggestion, or direction. In accordance with the purpose and process of the statement, we concluded whether each specific statement helped in moving the team towards a shared understanding (positive shift) or away from a shared understanding (negative shift). At the end of each task, based on the gathered data, we deduced whether the team had a shared mental model of the task. This coding scheme, as well as its application, will be instrumental in guiding future research that implements team communication in the understanding of mental model states. In addition, this coding system may reveal patterns that can predict future instances where teams experience a series of negative mental model shifts and instruct future interventions.

All natural features that make up river systems are created through erosion. The energy in the water column that causes this erosion is called turbulence. Turbulence isn’t limited to river systems, but the focus of this paper is on turbulence, specifically within the possession sound. Depending on how water is flowing within a local ecosystem, the terrain and biological components in that ecosystem can change drastically. In this study I examine how turbulent flow in the mouth of the Snohomish River in Everett, Washington changes relative to river discharge. I defined turbulence for this study as the relationship of the direction and magnitude of two vertically adjacent water particles. I also used a variation of the Reynolds number (Re) to more clearly define the difference between Transitional and Turbulent flow. In this study, data were collected with a 3-beam Aquadopp 1MHz Acoustic Doppler Current Profiler (ADCP), which measures the speed of passing particles in the water column at 1-meter increments starting at 1.4-meters above the river bed. I processed the data in RStudio and Excel. From preliminary research I know that during periods of high flow, the difference of adjacent flows is more dramatic at depth than it is anywhere else in the water column. I also have observed constant random direction of water flow towards the surface. It is expected that this will also be observed while doing tests with the Re, meaning that there will be continuous high Re at the surface due to outside influences, with low Re at depth during periods of normal flow rate. It is also expected that there will be prolonged high Re throughout the water column during and after periods of high flow rates.

Salinity is a fundamental component of all estuarine environments, affecting water chemistry and density-driven flow dynamics. In the Snohomish River Estuary in Washington State, saltwater from Possession Sound and freshwater from the Snohomish River stratify, forming a partially-mixed salt-wedge estuary. Haloclines, zones of rapid salinity-change, vary in depth depending on season, temperature, and freshwater influx. Above and below the halocline, the water column displays relative homogeneity in salinity. An established relationship between tide height and salinity may allow researchers to use accessible tide data as an indicator of salinity. If such a relationship were to change, any difference from the baseline relationship may be used as a measure of change in the ecosystem to track climate change and other factors. I examined salinity data collected throughout the water column, at surface, halocline, and deep zones, to detect the influence of tides on salinity. I anticipated increased tide height, paired with corresponding saltwater encroach, to correspond to an increase in the salinity of brackish water at the halocline. I expected surface and deep zones would be relatively unaffected, owing to their separation from this immediate area of change. I predicted any relationship between tide height and salinity to strengthen with increased distance from the Snohomish River, as saltwater would be less diluted by freshwater, implying a more noticeable influence on it. My analysis of readings taken at field sites in Possession Sound from 2017-2020, restricted according to site and season, did not present any consistent correlation between degree of tide-salinity correlation and distance from the Snohomish river. I detected varying correlation between salinity at restricted depths and tide height. Further research will entail further elimination of confounding variables.

 As global ocean temperatures rise, there has been a worldwide increase in frequency, intensity, and geographic range of harmful algal blooms (HABs). HABs can have detrimental effects on both natural and anthropogenic factors. Diatoms of the genus Pseudo-nitzschia are known to cause HABs in Puget Sound, Washington. This toxigenic diatom is a particularly concerning species, as they produce domoic acid (DA), which can be transferred up the food web through biomagnification, resulting in illness and death of birds and mammals in the surrounding area. The goal of this project is to identify trends in the relationship between the population density of Pseudo-nitzschia and water quality indicators such as surface temperature, dissolved oxygen (DO) levels, chlorophyll, and pH. This study focuses on data collected by the Ocean Research College Academy (ORCA) during State of Possession Sound (SOPS) cruises from the five sites in Puget Sound from the years 2016 to 2020. Students from ORCA conduct vertical plankton tows using a 335-micrometer net and horizontal plankton tows using a 20-micrometer net. Plankton enumeration is conducted in the lab, and plankton density is calculated. Surface temperature, DO, chlorophyll, and pH data are collected during EXO2 Sonde deployments during SOPS cruises. Based on preliminary research, a greater population density of Pseudo-nitzschia is expected during years with higher surface temperatures and lower DO levels. Researching the conditions that allow Pseudo-nitzschia to thrive may allow for increased accuracy in predicting the locations and time in which harmful blooms are most likely to develop. Understanding the impact and expected severity of Pseudo-nitzschia blooms can aid in signifying whether concern should be raised regarding water quality within the Puget Sound ecosystem.

When fast-moving water flows into a basin with weaker tides, a highly stratified salt-wedge estuary occurs. Calculating the travel time of water in an estuary can be useful when predicting pollutant spills, erosion, and depositional effects at the river mouth. This study examines the Snohomish River, which is part of a salt-wedge estuary that encompasses the Port of Everett Marina and deposits into Possession Sound in the Whidbey Basin of the Salish Sea. When the tide floods, water goes north. River travel time calculated by previous Ocean Research College Academy (ORCA) students was nearly 10 hours of delay between a United States Geological Survey monitor located 12 miles upriver and water quality data at the river mouth. This previous research shows there will be less travel time and less water speed during high tides. A more accurate travel time can be found by cross-correlating river discharge with water speed. Through retrieving water speed and direction at the river mouth, one can develop current vectors to compare with tide stage. Using the distance to the water's surface from the Acoustic Doppler Current Profiler (ADCP) near the riverbed, one can determine the tidal effect on river speed and travel time through extreme water levels correlating to low and high tides. This study investigates July 2020 ADCP North/South vectors at the river mouth and correlates them to river discharge upriver. Using RStudio statistical analysis, this correlation is then compared to ADCP water height to model for tides. It is predicted that the faster the river discharge and the larger the Southern vectors, then the more drastic decrease in ADCP’s height in the river. Further research would include adding more ADCP data and precipitation to consider seasonal patterns. Since runoff feeds the Snohomish River, examining precipitation would create a more accurate model.

When massive stars die, they explode in violent spectacles known as supernovae, specifically core-collapse supernovae. These cataclysmic events produce and distribute a large fraction of the heavy elements in the universe, but the properties of the massive stars that produce them have historically been difficult to measure. I have made new measurements constraining the masses of stars that have produced core-collapse supernovae, also known as supernova progenitors. I have done this by measuring the ages of stars at the location of supernova remnants: the nebulae of excited and enriched gas left behind by supernovae that have occurred over the past 20,000 years. Assuming the progenitor was associated with these stars, I am able to estimate the age of the star that exploded. Using theoretical models, I am able to infer the mass from this age. I used images taken by the Hubble Space Telescope to investigate the stars responsible for producing hundreds of these remnants in the nearby galaxy NGC 6946. In addition to the remnants of supernovae, this galaxy has hosted ten observed core-collapse supernovae within the past hundred years, leading to it being referred to as the “Fireworks Galaxy”. I was able to constrain the progenitor mass distribution for 175 remnants, eight of the historically observed supernovae, as well as the progenitor of the first direct black hole formation candidate in NGC 6946. I found the distribution of progenitor masses was consistent with mass distributions measured for massive stars in other galaxies, including our own Milky Way. These new measurements allow NGC6946 to be included for the first time in statistical studies of the masses of stars that produce supernovae.

Progress in addressing the simultaneous demands for increasing speed and miniaturization in electrical and computer engineering is to its greatest extent bounded by material stresses under thermal shock. Higher speeds require higher power dissipation, and smaller unit volumes make adequate power dissipation more difficult to achieve. Although there already exists a large body of research concerning endpoint thermal failure in semiconductors, there is little research available on the topic of pre-failure behavior of circuit designs containing semiconductors. Our goal is to subject a circuit containing a semiconductor-based diode to the failure mechanism of thermal shock and test the conductivity of the circuit under drastically changing ambient thermal conditions. We will then use this data to experimentally determine any observable behaviors that qualify as pre-failure symptoms. The resulting observations will be used to determine the efficacy of simulation softwares like LTspice in predicting thermal behavior of a diode circuit under extreme and rapid temperature fluctuations. Our theory is circuit simulation softwares do not account for extreme ambient thermal changes. After completing statistical analysis we will compare the experimental results to simulated results of a duplicate circuit subjected to equivalent temperature parameters and determine if we can reject our theory.

 Human biospecimens have undeniably paved the way for medical progress, but historically also surfaces problematic ethical considerations around consent, compensation, and privacy. Marginalized communities have been historically exploited as test subjects and sample sources; unknowingly or unwillingly playing a lead character role in scientific research. This results in a legacy of distrust in science and medicine as well as health disparities. Broadly, I hope to examine cultural impacts and health legacies that result from exploitive research practices, specifically examining biospecimen collection from the Rongelap people during Project 4. in the Marshall Islands during the 1950's and other examples, such as Henrietta Lacks, and the Tuskegee Syphilis Study. By comparing these case studies, I endeavor to interrogate historical discourse around biospecimen collection in research to inform the relationship between scientific exploitation and population health disparities in marginalized communities today. In doing so, I hope to broaden the space for amplifying Marshallese voices and incorporating the population health impact of Project 4.1 into current dialogue around issues in scientific research.

Throughout Washington, there is a lack of awareness about the Hanford nuclear site and its impacts on Native American tribes. Since 2015, teaching of Since Time Immemorial (STI) - a tribally developed curriculum covering Hanford and other issues - has been required throughout Washington schools.Yet, there is a lack of information on how this curriculum has been implemented and to what extent Hanford is being taught about. Thus, this research project aims to gather data on current teaching practices on the Hanford site as well as general tribal education throughout Washington schools. We also attempt to understand what measures can be taken to help teachers implement native centered curriculum, especially about Hanford. Initial research was conducted through semi structured interviews with stakeholders about current education practices and efforts to implement education about Hanford. This preliminary research was used to develop a survey for middle and high school teachers across Washington. The survey contained 15 multiple choice and short answer questions addressing the STI curriculum and education on Hanford. The survey also included an educational research guide for teachers who were interested in learning more about STI or Hanford. The survey was administered to 234 districts by emailing school administrators with the request that the survey be forwarded to teachers in their school; contact information for administrators was gathered through the WA OSPI website. Based on current data, it’s anticipated that there is very limited coverage of Hanford in schools due to time constraints, lack of interest, and omission from standard curriculums, suggesting a need to ensure that existing tribally developed curriculum is taught in classrooms. This is an urgent matter because lack of public awareness about the history and impacts of the Hanford Site on Native Americans allows for the harms to continue to be perpetrated. 

Hydrogen sulfide (H₂S) is a toxic gas in the environment, but it is also an important cellular signaling molecule. Our goal is to understand the genes and pathways that mediate the physiological effects of H₂S. Previous work from the Miller lab has shown RHY-1 to be a component of a pathway that mitigates H₂S toxicity. RHY-1 is an integral membrane protein with predicted acyltransferase activity that localizes to the endoplasmic reticulum. We are attempting to identify proteins that work with RHY-1 to promote survival in H₂S. In this project, we have optimized biotinylation by antibody recognition (BAR), a proximity-labeling approach, to identify proteins that may physically interact with RHY-1. In these experiments, we introduced an antibody conjugated to horseradish peroxidase (HRP) into C. elegans that expresses the epitope-tagged RHY 1::FLAG::GFP protein, so that upon addition of biotin peroxide, biotin radicals were formed only in proximity to RHY-1. These biotin radicals react with other proteins that are localized near RHY-1. We visualized biotinylation using a fluorescent label and showed that the biotinylated proteins colocalize with RHY-1, indicating a successful BAR reaction. To our knowledge, this is the first use of BAR in C. elegans. Going forward, we will use mass spectrometry to identify the proteins that are biotinylated, and then test the functional role of these proteins in mitigating H₂S toxicity. Identifying the genes and pathways downstream of RHY-1 that promote survival in H₂S will reveal new factors that can modulate H₂S signaling in cells and may lead to treatments for H₂S poisoning.

Herpes Simplex Virus 1 (HSV1) is a prevalent lifelong virus transmitted through oral-to-oral contact resulting in painful oral sores. HSV1 may also give rise to encephalitis, or inflammation of the brain, and has even been linked to sporadic Alzheimer’s Disease (AD). Typically, HSV1 enters through the oral cavity, replicates, invades nearby clusters of sensory neurons that innervate mucosal membranes called the trigeminal ganglia, and can ultimately lead to infection of the brain. HSV1 can establish latency in these trigeminal ganglia and other neuronal nuclei, and can reactivate in the brain in response to external stresses at any point in time. Previous studies have even shown presence of HSV1 in the brain. However, this process of reactivation is still not understood. Our goal is to comprehend the mechanisms of HSV1 reactivation in the brain by generating two recombinant HSV1 through the CRISPR-Cas9 gene editing system. One strain will contain an insertion into the HSV1 genome of luciferase and mEmerald, a green fluorescent protein, for detection of cells with reactivating HSV1 through in vivo imaging of bioluminescence. The second strain will have an insertion of Cre recombinase and mEmerald. When this strain is injected into a mutant mice line with a Cre-dependent reporter gene, the Cre recombinase can lift genetic repression of a red fluorescence protein. This prompts all cells with an HSV1 infection, including those with past reactivation, to be detected. Through these novel recombinant HSV1 strains, we can monitor viral reactivation in brain cells in response to different external stresses in vivo, examine the time course of the infection, and distinguish areas of the brain with high viral load, especially cell types vulnerable to HSV1 infection. These experiments will be the stepping stone for improved investigation of viral latency and reactivation and understanding the connection between HSV1 and AD.

DNA damage occurs regularly, and although the cell has numerous repair mechanisms to counteract it, some DNA lesions may persist. Many of these lesions can be bypassed by DNA and RNA polymerase, with replicational mutagenesis leading to permanent mutations in the genomic sequence, and transcriptional mutagenesis leading to mutant transcripts that may direct the production of mutant proteins. We hypothesized that transcriptional mutagenesis is a mechanism for initiating adaptive mutagenesis, which occurs in nondividing cells in direct response to a selective pressure, allowing cells to overcome the selection and resume cell division. Our lab has previously demonstrated that DNA damage leading to adaptive mutagenesis in yeast is biased to the template strand relative to transcription, supporting this hypothesis. Unexpectedly, DNA damage leading to replicational mutations in our system is similarly skewed to this strand, although the sequences surrounding these two classes of mutations seems to differ. We hypothesize that sequence context is important for determining whether certain mutation sites are more likely to contribute to replicational or adaptive mutagenesis. To address this hypothesis, our aim was to create Trp5 mutant yeast strains using CRISPR/Cas9 in which the mutation occurred in a sequence context-specific manner, and the reversion of which could be scored for both replicational and adaptive mutagenesis. The CRISPR/Cas9 system required generation of a plasmid containing the target sgRNA (via molecular cloning techniques) as well as a linear repair template with the mutation of interest (via a two-step PCR protocol). Creating both of these components proved challenging, and optimization of the protocols will be required. Once we have all the components for CRISPR/Cas9-mediated mutagenesis, future research involves creating the mutant strains and assessing whether the sequence context affects the mutation frequency with respect to either replicational or adaptive mutagenesis.

Environmental stressors trigger physiological adaptations in organisms that allow them to dynamically remodel tissues. As many of these processes in the gastrointestinal tract of Drosophila melanogaster are studied in the context of stem cell proliferation, we instead chose the novel approach of investigating visceral muscle adaptations in response to stressors to the gut. The three stress conditions we employed were starvation, damage, and aging. For starvation, we treated adult flies with only water following nutrient-enriched recovery. For epithelial damage, we fed them with the chemical damaging agent, bleomycin. Finally, for aging, the flies were subjected to different prolonged time periods on normal media. The flies were then dissected and processed for confocal imaging and phenotypic analysis, allowing us to categorically define and quantify the phenotypes in order to measure the degree to which the visceral muscle has changed. We discovered that various responses of the visceral muscle are induced by the types of stressors, and will further look into whether it is adaptive remodeling, muscle repair, or other undescribed mechanisms. We will investigate the molecular mechanisms that contribute to the remodeling of the visceral muscle using RNAi approach. This research would provide valuable resources as a model system for studying complex tissue responding to environmental challenges and understanding of its mechanisms utilizing the vast genetic tools of Drosophila.

IMPDH1 catalyzes the rate limiting step of de novo guanine synthesis. cGMP is a critical signaling molecule involved in phototransduction in rod and cone photoreceptors. In humans, nine mutations in IMPDH1 lead to Retinitis Pigmentosa and Leber's Congenital Amaurosis; however, the cause of retinal degeneration is unknown. In zebrafish, IMPDH1a is the major variant in the retina, exclusively expressed in rod and cone photoreceptors. To understand the function of IMPDH1, we utilized an IMPDH1a knock-out (KO) zebrafish line. Loss of IMPDH1a does not lead to retinal degeneration, and cGMP levels remain unchanged. However, retinas lacking IMPDH1a show a 61.6% reduction in guanine. Since photoreceptors do not undergo cell division, mitochondrial DNA (mtDNA) synthesis would be a major use of guanine during mitochondrial biogenesis and turnover. Under normal conditions, the amount of mtDNA between the KO and WT zebrafish was the same. To eliminate the potential that KO fish could supplement guanine from their diet, fish were starved for 24 hours. Starved IMPDH1a KO showed a trend of increased mtDNA compared to WT fish. Interestingly, starved fish had about four times the amount of mtDNA for both WT and KO compared to fed fish. Starvation may increase mitochondrial DNA copy number to increase efficiency of ATP production by increased utilization of oxidative phosphorylation. Reducing guanine levels by knocking out IMPDH1a in zebrafish retina does not affect mtDNA or nDNA levels, and the photoreceptor cells appear healthy. Although more research is needed, silencing the gene encoding for IMPDH1 could be a possible therapy for those suffering from the effects of its mutation.

Cancerous cells have a modified metabolism that supports their demands for increased proliferation. One of the essential molecules in cancer cell metabolism and proliferation is the amino acid aspartate. Aspartate is not only incorporated into proteins, but is also a substrate for nucleotides and other amino acids, including asparagine. Aspartate availability can constrain tumor growth rate, and the consumption of aspartate to generate downstream products can alter aspartate levels. One gene that draws from the aspartate pool is asparagine synthetase (ASNS). ASNS converts aspartate into asparagine, which is used in the production of proteins, but does not increase cell proliferation. Thus we hypothesized that ASNS expression and activity can affect aspartate levels. With this, we aimed to determine if ASNS expression could alter aspartate availability and change sensitivity to aspartate suppressing therapies. Since cancer cells express ASNS to varying degrees, my project sought to determine if ASNS expression could be used to identify those cancers that are most amenable to aspartate suppression therapies. To test this I generated cell lines that express ASNS in different ways and treated them with multiple electron transport chain inhibitors. Preliminary results suggest that cells that express ASNS to a higher degree are more susceptible to mitochondrial inhibitors. More broadly, this research sought to better understand the conditions that determine aspartate levels, and how to exploit those conditions to inhibit tumor growth in association with asparagine synthetase.

Aortic aneurysms form when the aorta, the large artery that carries blood from the heart, weakens and expands. Smooth muscle cells (SMC) in the aortic wall regulate aortic structure and contractility and help preserve normal aortic diameter. Transforming growth factor beta (TGF-β) signaling is integral to SMC function and abnormal TGF-β signaling and is implicated in aneurysm formation. However, whether excessive or deficient SMC TGF-β signaling promotes aneurysms is controversial. To determine if increased SMC TGF-β signaling is sufficient to cause aneurysms, we expressed a Constitutively Active Type I TGF-β Receptor (TβR1-CA) transgene in SMC of male and female mice (both hemizygous). Because the transgene is integrated into the X chromosome, and half of female X chromosomes undergo inactivation, expression of TβR1-CA in males and females may differ. This difference could affect the interpretation of studies that investigate the role of SMC TGF-β signaling in aneurysmal disease. We hypothesize that hemizygous females will have lower levels of TβR1-CA expression than males due to random X-inactivation. To test our hypothesis, we will quantify the mRNA levels of TβR1-CA relative to that of Gapdh (a reference gene for normalization) in aortic SMC of male and female hemizygous transgenic mice using real time (RT)-qPCR. To confirm that we can detect X chromosome inactivation using RT-qPCR, we will also measure mRNA levels of a gene that is known to undergo X-chromosome inactivation (Pgk1) and a gene that is known to escape X-chromosome inactivation (Kdm5c). If our hypothesis is correct, we will find that the amount of aortic TβR1-CA mRNA is ~50% lower in aortas of female versus male transgenic mice. The results of this project will be essential in helping us to interpret results of parallel experiments that investigate whether TβR1-CA expression (and activated SMC TGF-β signaling) causes aneurysms in male and female mice.

Non-human primate (NHP) research has become an essential step in the translation of medical technologies from animal models to clinical trials. This is especially so in neural research, as there is a large discrepancy between rodent and human brains in both anatomy and size. For some techniques such as optogenetics, which requires viral transduction of neurons, traditional diffusion-based viral injection approaches are effective in rodent brains but are impractical for large NHP ones. Convection-enhanced delivery (CED), a large-scale injection approach, currently lacks a practical quantitative bench-side injection modeling method to guide neurosurgical preparation. We aim to develop a gel model of the NHP brain and replicate surgical injections of it in order to reduce the risks of directly injecting into a NHP without sufficient preparation. We are testing the validity of our model by monitoring the spread of the injection through the gel and comparing the data with those from MRI scans of the injections in NHP. Since CED can behave differently depending on the location of injection in the brain, we are testing bench-side injections at different depths to validate the versatility of our model. We are seeing that the injections in the gel model mirror that of the injections in NHP brains as expected. Our next steps are to test the effectiveness of smaller injection cannula sizes with our bench-side model to assess if injection results remain consistent. This would indicate that tissue damage could be minimized in surgeries while still achieving desired injection parameters.

Optogenetic stimulation is a technique that modulates the activity of genetically modified neurons with light of a particular wavelength. Optogenetic modulation has high temporal resolution and cell-type specificity that enables precise stimulation of cortical neurons and allows us to conduct artifact-free recording during stimulation. Using a large-scale optogenetic interface, we stimulated and recorded across the primary somatosensory (S1) and motor (M1) cortices of non-human primates (NHP). We conducted our investigation on NHPs because their cortical organization is particularly similar to that of humans. The goal of this study is to determine the effect of various spatial and temporal patterns of optogenetic stimulation on the neural response and network dynamics across the two cortical regions. Delivering stimulus pulses via two lasers placed on top of the cortical surface, we found that stimulation of one cortical region evoked neural responses across both S1 and M1, which we then classified into primary and secondary responses based on their delays. While our previous work has established that optogenetic stimulation strengthened functional connectivity between S1 and M1, we wanted to further investigate the distribution of primary and secondary neural responses after repeated stimulation. We examined two measures of neural responses, the temporal delay between the trough of evoked response and onset of light stimulation, and the distribution of power across cortical networks up to 50ms after the stimulation. Our preliminary results indicate that optogenetic stimulation changed the delay of the primary and secondary response. We also observed that different temporal patterns of paired laser pulses evoked distinct neural activity. Identifying different neural responses after complex spatiotemporal patterns of stimulation would help us predict network changes post cortical modulation and contribute significantly to the development of stimulation-based clinical therapies and rehabilitation strategies for neural disorders.

Non-human primate (NHP) research is a pivotal step in the progression of neuroscientific and neural engineering research from animal models to human trials. In most NHP neuroscience experiments, neurosurgery is required to implant devices such as head posts, recording arrays, and optical windows. Current practices for these surgeries use methods for surgical preparation that carry a degree of unavoidable uncertainty. This comes from an inability to visualize and test the physical compatibility of complex components and anatomy prior to neurosurgery. This project details methods for creating 3D printed models of a subject’s brain and skull, as well as an agarose gel model of the brain. These models can be obtained from magnetic resonance imaging (MRI) using brain extraction software for the brain model, and custom code for the skull. The preparation protocol takes advantage of state-of-the-art 3D printing technology to combine models of the brain and skull with neuroprosthesis. With the addition of a craniotomy using the custom code, the skull and brain models can visualize brain tissue inside the skull, enabling better preparation for surgeries. Using the methods outlined in the protocol, the accuracy of the 3D printed brain, skull, and craniotomy placement were successfully validated through a comparison to the original MRI scan. The gel brain was additionally used to visualize delivery of a mock viral vector through the craniotomy of a skull model. By preoperatively fitting a headpost to the physical model of the skull, we successfully shortened the implantation surgery time by 40% and greatly reduced the risk of operative complications. These methods are designed for surgeries involving neurological stimulation and recording as well as injection in NHPs, but the versatility of the system allows for future expansion of the protocol, extraction techniques, and models to a wider scope of surgeries.

Stroke, when the blood supply to the brain is reduced or disrupted, is a leading cause of disability among adults. Brain plasticity, also known as neural plasticity, can aid in the way that the brain recovers from a traumatic injury such as a stroke by creating newer, stronger synapses (connections) between neurons and thus increasing cell functionality. This literature review explores how brain plasticity informs new bioengineering solutions to stroke rehabilitation and asks, “What type of novel stroke treatments have been developed using the concept of brain plasticity?” Preliminary findings indicate that a breakthrough in this field is using optogenetics to trigger and control the neural connections that the brain can make through promoting motor function after ischemic stroke. Other innovations in this field include repetitive transcranial magnetic stimulation, transcranial direct current stimulation, and epidural cortical stimulation, which have all been shown to make permanent changes in neural synaptic transmission. These methods partially restore brain function while being less invasive and more effective in comparison to older interventions. This literature review indicates that new bioengineering treatments informed by brain plasticity are promising and could promote better rehabilitation outcomes for those suffering from stroke and potentially other traumatic brain injuries.

Chronic non-bacterial osteomyelitis (CNO) is a rare disease where the immune system attacks healthy bone, leading to inflammation and destruction. Currently, the use of inflammation markers - which consists of a blood test for erythrocyte sedimentation rate (ESR) and c reactive protein (CRP), a clinical visit, and magnetic resonance imaging (MRI) are used to monitor the state of CNO. However, the reports from these assessments are inconsistent as longitudinal data allowing for a detailed analysis is lacking.Thus, we aim to investigate the association among these disease monitoring modalities using Seattle Children's Hospital CNO research database. The database which contains patients under 21 years old, spanning from January 2014 - January 2021 is one of the largest composed. We hypothesize that an increased presence of lesions, as observed through MRI, correlates with a greater value of inflammation markers and greater physician global assessment (PGA) score. Through blood samples and MRI scans, ESR/CRP values and number of active lesions within 30 days of the visit were recorded. General statistical methods were used to summarize the data and determine correlation. We expect a strong positive correlation between active lesion count and ESR/CRP values. ESR/CRP values measure the rate of inflammation. As inflammation is a common physiological symptom stemming from lesion presence, a positive correlation between these variables must occur. Additionally, we expect a strong positive correlation between active lesion count and PGA scores. PGA scores are determined during clinical visits and are drawn from assessing a patient’s current condition. The prominence/severity of lesions are factored into the PGA score. As CNO lacks a reliable technique to monitor the disease, we expect that the results from this study will shed light on a reliable and robust assessment tool to monitor disease activity.
 

The blood-brain barrier (BBB) is a highly specialized interface of brain microvascular endothelial cells (BECs). The main functions of BECs are to protect the brain against exposure to harmful substances in the blood, and to transport and secrete nutrients and other molecules that support normal brain functions. BECs can also alter their functions in response to signals from the brain or blood compartments. GLUT-1 is the predominant transporter at the BBB that regulates glucose entry into the brain, and does so by a mechanism of facilitated diffusion, which permits glucose transport in the blood-to brain or brain-to-blood direction. GLUT-1 dysfunction occurs in and may contribute to Alzheimer’s disease.The main hypothesis is that GLUT-1 malfunction in the BBB occurs as it deteriorates with age, which causes errors in other regulation methods of the BBB leading to the development of Alzheimer's. However, it is difficult to study mechanisms of GLUT-1 dysfunction at the BBB specifically because of the involvement of multiple cell types that regulate glucose uptake into the brain in vivo. In my research, I have utilized a model of iPSC-derived brain endothelial cells (iBECs) in order to study aspects of GLUT-1 regulation in an in vitro model of the BBB. Currently, my main findings have shown that the GLUT1 transporter is functional in our system, and that GLUT1 protein expression is increased and glycolytic enzymes are decreased when iBECs switch from a proliferative state to a quiescent state. Glucose transport in the blood-to-brain direction is decreased in the quiescent state.  Additionally, high glucose exposure causes downregulation of glucose transport.  My future studies aim to determine whether proliferating vs. quiescent iBECs respond differently to high or low concentrations of glucose. In summary, we have shown that iBECs are a translatable and effective model that allows us to further investigate the regulation of GLUT-1 at the BBB to further understand the physiological mechanisms involved in Alzheimer's Disease. 

Duchenne Muscular Dystrophy (DMD) is a severe muscle wasting disease caused by the deficiency of dystrophin protein affecting ~1 in 3500 boys. Exercise has been investigated as a potential therapy but has shown conflicting effects on dystrophic muscle. Animal models that have been studied but do not fully mimic the disease and cardiac phenotype. Here, we provide the first known evaluation of exercise in a new DMD mdx rat model that mimics skeletal muscle and cardiac pathology. Having an animal model that better reflects the DMD cardiac phenotype is crucial in establishing physical activity guidelines that minimize potential damage to the heart. In Aim I of this pilot study, DMD mdx rats (n=3) and sibling wildtypes (WT; n=3) were assigned to wheel or treadmill exercise training groups (high intensity or low intensity) or unexercised (sedentary) for 6-weeks. Throughout this 6-week period, I was responsible for executing the rat treadmill exercise training protocol at low-to-moderate intensity (5x/week), and monitored the rat wheel running activity. Physiological measurements were conducted pre- and post- training (wheel running, treadmill testing, grip testing, echocardiography, and hindlimb force testing). Compared to wheel running or unexercised rats, treadmill training produced the greatest gain in treadmill exercise endurance testing and in fatigue-resistance in in-vivo hindlimb force testing for DMD rats. However, the treadmill program was associated with severe cardiac effects in DMD mdx, indicated by significant fibrosis and inflammation and echocardiography (Myocardial Performance Index, % fractional shortening and ejection fraction), a finding which will be investigated in subsequent cohorts.

Mycobacterium tuberculosis (Mtb) is a bacterium that kills nearly 2 million people annually and continues to be a large threat to the health of individuals around the world. T-cells play a key role in the immune response to Mtb infection, but there is still much we do not understand about their role in mediating protection. CD4 and CD8 are glycoproteins found on the surface of T cells. CD4 and CD8 T-cells recognize glycolipids associated with the bacterium, such as glucose monomycolate (GMM), that are presented by the cell-surface glycoprotein CD1b on a human antigen presenting cell. I am focused on identifying the T-cell receptor (TCR) of a T-cell line from the Rhesus Macaque species. Using newly validated CD1b tetramers, I sorted for T-cells that can bind to the GMM lipid antigen using flow cytometry. The tetramer is made up of four CD1b molecules which present the antigen and bind to T-cells that have a matching receptor. I used various molecular techniques such as RNA extraction, 5’ RACE PCR (a procedure for amplification of nucleic acid sequences using a messenger RNA template), and in fusion cloning to determine the TCR gene sequence of the alpha and beta chain regions. By identifying the sequence of a T-cell receptor in the Rhesus Macaque species we will be expanding our knowledge of how T cells recognize Mtb lipids in non-human primates. Since we have previously published that CD1d-restricted TCRs are very similar between NHP (non-human primates) and humans, we expect the same is true here. We can use tetramers and the NHP model to understand how lipid-specific T cells are important for controlling Mtb.

Enteric alpha defensins, such as human defensin 5 (HD5), are antimicrobial peptides secreted by Paneth cells in the lumen of the small intestine as part of the innate immune response. Defensin activity effectively inhibits many bacterial and viral pathogens during infection. However, not all pathogenic human viruses are neutralized in the presence of defensins. For example, among the seven human adenovirus (HAdV) species, infections of certain serotypes are enhanced by HD5, while other serotypes are inhibited. Our goal is to identify the molecular determinants that confer HAdV neutralization or enhancement by HD5. We have previously identified regions of the three major capsid proteins that form the outside of the virus, hexon, penton base, and fiber, as key determinants. We identified these determinants through rational design and the creation of chimeric viruses in which we swapped portions of the three major capsid proteins between two serotypes with opposite HD5-dependent phenotypes. From these data, we have created a model of the enhancement and neutralization mechanisms. To further test these models, I am creating a panel of chimeric HAdVs by swapping capsid components from additional HAdV species and serotypes. I will study the concentration-dependent effect of HD5 on each chimera’s infectivity compared to the wild type viruses, which will uncover new mechanistic detail and allow us to create a more generalized model.

  My research focuses on ‘tauopathy’—the pathological effects of misfolding of the tau protein—using the fruit fly, Drosophila melanogaster, as a model system. In particular, I am interested in how the expression of tau affects locomotive function. Tau is a protein found in both humans and flies that is associated with stabilizing neuronal microtubules. However, under certain physiological conditions, human tau proteins form neurotoxic aggregates in the brain. This neuronal damage leads to dementia, a hallmark of Alzheimer’s Disease (AD). Aging comes with a multitude of age-related functional deficits, including decline in locomotor function. Some individuals with AD pathology are never diagnosed, however, because the cognitive impairment they experience is not sufficient for a clinical diagnosis of dementia, the most common symptom of AD. Consequently, it is imperative we understand AD as more than dementia. I study the climbing abilities of transgenic tau and control flies through negative geotaxis assays. Negative geotaxis refers to the tendency of flies to move vertically upward when startled. For my project, I am measuring the climbing performance of the flies to see the effects of tau on locomotor function as flies age. I hypothesize that the neurotoxic tau aggregates that form will interfere with neural activity involved in the flies’ motor function, resulting in decreased climbing performance. This research holds an abundance of biomedical implications and a capacity to better the lives of many. By using motor function to flag at-risk individuals early in their lives, they have the opportunity to participate in preclinical AD clinical trials that may prevent them from developing AD pathology later on. It is important that we, as a scientific community, strive to better understand the effects of aging and tauopathies, as it is through this understanding we can provide elderly individuals with care that transforms their quality of life.

Aging is the most prevalent risk factor behind many common diseases such as cancer, cardiovascular disease, and various neurodegenerative diseases. The changes that take place with age are complex and affect most of the human body; age-related changes in gait, in particular, have been found to be associated with the onset of disease. While it is generally understood that there is an effect of aging on walking speed in humans, the mechanisms underlying age-related locomotor impairment have not yet been fully characterized. The focus of my research is using Drosophila melanogaster as a model to investigate such mechanisms. My hypothesis is that D. melanogaster exhibit similar age-related changes in gait as those seen in humans, which includes a decrease in walking velocity and duration, and a significant change in limb coordination over their lifespan. I followed cohorts of D. melanogaster over their lifespans and recorded videos of their walking in an arena. I then used these videos to study factors such as walking velocity and duration by analyzing the trajectories of each fly. In addition, I investigated more in-depth limb coordination of individual flies by analyzing the movement of each individual leg. Previous studies using D. melanogaster have found a significant decrease in climbing behavior with age, but have not looked at gait on a finer scale. Therefore, upon completion of this study, I expect to see a decrease in limb coordination and population walking velocity as the flies age. With the findings from this study, I hope to establish a foundation for how gait changes with age in D. melanogaster and to further use gait analysis to help predict the onset of disease and to distinguish between diseased and healthy flies.

Alzheimer’s Disease (AD) is a neurological disease that causes memory loss and neurodegeneration, and is one of the leading causes of death within the United States. Alzheimer’s is linked to the presence of neurofibrillary tangles within the brain, generated by hyperphosphorylation and aggregation of a protein called tau. However, the specific impact that tau has on biochemical pathways in neurons is largely unknown. My project attempts to fill this gap. I compared samples of wild type fruit flies, Drosophila melanogaster, with a strain which expresses the human tau gene in neurons. Despite the difference in human and fly brains, determining the affected biochemical pathway in flies could suggest similar effects in humans. I am using metabolomics, which quantifies the levels of approximately one hundred metabolites, to try to identify the biochemical pathways that are affected by expression of tau in the neurons. Head and body samples of these two fly strains were frozen at 12 days of age and assayed for a targeted set of metabolites. I analyzed these metabolome data using statistical methods in Python to search for potential differences between wild type and tau flies. Any metabolites found to be different will then be analyzed to see if they tend to represent similar biochemical pathways, allowing us to speculate about the effect of tau on those pathways, both in flies and humans. Future work aims to analyze fly brains and individual neurons, thus mapping the affected metabolic pathways more precisely. Importantly, metabolic pathways are often identical between organisms, allowing us to speculate on the effect tau has on similar pathways within humans, thus this work will inform our understanding of the mechanism of AD in humans.

Parkinson’s Disease (PD) is a neurodegenerative disease that affects movement and often leads to tremor, rigidity, and balance issues. Speech impairment, or dysarthria, is also common and may influence many aspects of speech production, such as respiration, articulation, phonation, and prosody. There are currently multiple methods for measuring speech impairment severity over time. Often, severity of dysarthria is captured by neurologists during administration of a global measure of PD severity, the Unified Parkinson’s Disease Rating Scale (UPDRS). This scale uses one question to assess speech impairment severity on a perceptual level, but does not formally evaluate specific characteristics, such as intelligibility. Alternatively, speech-language pathologists may use measures of speech intelligibility to assess dysarthria severity. This method systematically quantifies the extent that speech was understood by a listener. The connection between these two approaches is currently unknown. Therefore, the purpose of this project is to examine the relationship between UPDRS scores and speech intelligibility metrics, two commonly-used indicators of dysarthria severity in PD. Connected speech samples for 27 individuals with idiopathic PD were collected. UPDRS ratings were completed independently by a speech expert who was blinded to intelligibility scores. Speech intelligibility ratings were based on 100-word speech samples and averaged across three listeners. Statistical analyses will include Pearson correlation to determine the strength of this relationship. Expected findings include a significant negative correlation, suggesting that increased severity on the UPDRS (higher score) corresponds to increased severity on the speech intelligibility calculation (lower score). Determining the relationship between these two indicators of speech severity will unite clinical understanding of speech impairment across the fields of neurology and speech-language pathology. This will also indicate whether a single speech severity question equates to more time-intensive intelligibility ratings.

This paper aims to explore the gender wage gap in the University of Washington. We use data collected from College of Arts and Sciences to analyze how faculty members' salary might be affected by different factors. The number of women who entered into the labor market grew exponentially after the Second World War and women tended to spend longer hours for work. But this fact did not eliminate the existence of gender wage gap. Researching the gender wage gap in academia is unique because academics employed usually obtain relatively homogeneous education and job trainings such as earning Ph.D. degree. The factors we examine which may have an impact on the salary difference include gender, department, and the years of attainment of Ph.D. degree. This observational document study also intends to show how the number ratio of female faculty members and male faculty members differ in different department and discuss whether implicit discriminations exist. Women may spend longer time to complete Ph.D. degree due to that female's presentations and works somehow seem to face more biased judgments comparing with male by mentor or adviser and thus have less potential job experience. Women professors may experience more years in the process of tenure promotion which can lead to direct salary increase than male peers. 

While technology advances and continues to increase its influence on the world, we need to scrutinize the process in which these technologies and digital platforms are created. Throughout history, there have been repeated offenses of colonization in design that erase or undermine the work of underrepresented communities. With this research, our goal is to destabilize design practices in Human-Computer Interaction (HCI) reinforced through pedagogies that seek to uphold unquestioned and dangerous societal norms. Our research centers around analyzing how colonization is enabled in HCI pedagogy, both inherently from academia and uniquely to HCI, and what the impacts of such colonization are on students in HCI learning spaces. For our methodology, we employed methods of literature review, reflexive writing, and storytelling to design a toolkit facing HCI students. We reviewed literature concerning critical race theory, queer theory, feminist theory, postcolonial and anticolonial design approaches, and design justice in HCI that evoked reflection on our positionality as student designers. Our discussion of past literature and personal stories informed the creation of a design toolkit with the goal of encouraging students to identify and critique current HCI practices in pedagogical materials. The toolkit explored tangible methods and frameworks to deconstruct colonized systems in HCI pedagogy and decentralize whiteness in design, as well as articulated the burden and responsibility of resistance in marginalized communities. Throughout the toolkit, we facilitated daily probe activities and interviews with student communities, in which we asked students to document their thoughts and learning process in a multimedia format. With this study, we hope to enact change within HCI student communities by highlighting colonial concerns in modern HCI pedagogy and providing actionable steps for inclusion, advocacy, and resistance.

Cultural spaces are essential for creating a sense of belonging in our neighborhoods and cities. In rapidly growing metropolitan areas, gentrification threatens to displace spaces deemed significant by Black, Brown, and Indigenous communities. Initial research on Seattle’s cultural spaces produced an inventory skewed toward white-centric, dominant cultural spaces in the fine arts. The initial inventory failed to represent the full range of community culture spaces, especially those cherished by local Black, Brown, and Indigenous communities. What is the best process for the City of Seattle to equitably engage with communities to identify cultural spaces? In partnership with the City of Seattle Office for Civil Rights, answered this question using the analysis of ethnographic data collected through qualitative interviews and surveys, and mental mapping exercises, conducted with 100 Black, Brown, and Indigenous community members. Our research team observed trends of several types of identified cultural spaces, including small businesses, outdoor and spiritual spaces, and spaces that are either stable or facing displacement. We represented our analysis, synthesis, and coded data spatially in an interactive storytelling map and report called “Holding Space: An Interactive Ethnographic Map of Cultural Spaces in Seattle’s Black, Brown, and Indigenous Communities”. Both have been made accessible online as anti-displacement advocacy tools for community-based organizations. The results of this study will help inform the City of Seattle's cultural spaces indicator, more equitable allocation of city government funds toward Black, Brown, and Indigenous-led community culture spaces, and policy recommendations to prevent further cultural displacement in Seattle.

 In the United States, there are approximately 2.5 million people with spinal cord injuries (SCIs). Depending on the location and severity of the injury, SCIs can result in long-term motor and sensory impairment. A very promising technology in neurorehabilitation for people with SCIs is transcutaneous spinal cord stimulation (tSCS). tSCS is a novel, non-invasive technique that stimulates the spinal cord through the surface of the skin. Recent clinical studies have already shown that tSCS is effective in helping to rehabilitate people with SCIs. However, while the rehabilitation method is sound, the physiological effects of tSCS on muscle recruitment are not well understood. Therefore, we are investigating the modulation of the spinal networks after the intervention with tSCS. Six patients with cervical SCIs underwent physical training paired with tSCS. Before and after training, SCS was used to induce motor evoked potentials which were measured with electromyogram. Evoked responses were extracted and analyzed by comparing peak to peak amplitude. After training and tSCS, both motor function and motor evoked potential amplitude increased, providing evidence that tSCS improves rehabilitation outcomes by modifying spinal networks. This research could lead to innovations in neural engineering and rehabilitation medicine and could greatly improve the quality of life for many people with SCIs.

Leigh syndrome (LS) is a progressive neurological disorder which often manifests within the first year of life and is characterized by the gradual loss of mental and movement abilities accompanied by epilepsy. LS has been associated with loss-of-function (LOF) mutations in genes that encode for proteins present in complex 1 of the electron transport chain. LOF mutations in one such gene, NADH dehydrogenase (ubiquinone) iron sulfur protein 4 (Ndufs4), are strongly associated with LS. Mice carrying a deletion of this gene exhibit symptoms similar to those found in humans, creating a relevant mouse model of LS. In this study, we investigated the effects of an Ndufs4 knockout on the neuronal excitability of both inhibitory and excitatory neurons located in different regions of the brain in LS mouse models. Two LS mouse models were generated by knocking out Ndufs4 in inhibitory or excitatory neurons utilizing LoxP/Cre technology. Mice carrying floxed alleles of Ndufs4 were crossed with VglutCre or GadCre driver mice. The progeny with excitatory or inhibitory neuron-specific Ndufs4 knockout and their control littermates obtained were perfused with phosphate buffered saline (PBS), then fixed with 4% paraformaldehyde (PFA). Brains from these mice were sliced and stained with c-Fos immunocytochemistry, then imaged to quantify neuronal activity. We hypothesize that neuronal excitability in both inhibitory and excitatory neurons will decrease after the Ndufs4 knockout, as mitochondrial defects would reduce the activity of both subsets of neurons. Findings from this study will potentially help understand the mechanisms for development of seizures in LS.

Reactive astrogliosis is a condition where astrocytes, a type of brain cell, undergo morphological – shape - changes upon exposure to brain injury. Morphological changes of astrocytes are a key indicator of activation and can be beneficial in stopping initial brain injury effects, but chronic activation can drive glial scarring, which is detrimental for full recovery of normal brain function. Glial scarring has been linked to several diseases, including traumatic brain injury, Alzheimer’s Disease, and dementia. The purpose of this work is to quantitatively analyze the relationship between frequency and extent of reactive astrogliosis with relation to distance from the primary site of brain injury. My approach is to build a Python-based image analysis pipeline to quantify astrocyte cell features. The Nance Lab’s prior work using Python packages was effective in developing a pipeline to identify and quantify microglial - a different type of brain cell - shape properties. We are now building a pipeline to study astrocytes in brain slices from the injured preterm ferret brain, which were stained with an antibody for glial fibrillary acidic protein (GFAP). Images of cells at 20x magnification are provided by Dr. Tommy Wood. Since response to injury can be brain region and animal sex dependent, I analyze astrocyte cell features in each region of the brain from both sexes of ferrets. I used SciKit-Image along with other packages to segment, label, and quantify features of our cells, including perimeter, area, and circularity, among others. Expected results include a significant reduction in area and an increase in perimeter (larger surface-to-volume ratio) of cells that are closer to the injury. This image analysis pipeline will give us quantitative information about the cells morphology which are associated with biological markers that can be targets for future therapeutic treatment. Clear biological markers help researchers develop better treatment.

This paper seeks to analyze 13th century poet Mevlana Celaleddin-i Muhammed Rumi’s role in modern Turkey. Composed of twelve interviews conducted in Istanbul, Turkey during 2019, the report focuses on two events as starting points for an ethnographic research project. The first event, the Gezi Park protests, occurred in the summer of 2013 when Istanbul saw demonstrations demanding civic freedoms sparked by the attempted destruction of Istanbul’s Taksim Gezi Park. Secondly, a flurry of small protests across the Middle East in the summer of 2016 followed Turkey’s joint UNESCO claim on Rumi’s seminal work, stirring an international discussion about which country - if any - had a right to the poet, Rumi (1207-1273).

Touching on Turkish popular culture, Sufism, the Mevlevi Order, and Rumi’s legacy, the paper analyses 12 interviews with a cross section of Turkish citizens and residents with varying levels of interest in Rumi. These interviews’ findings which reflect popular reception of Rumi’s legacy are juxtaposed with the government’s implementation of his image. Addressing how Rumi’s legacy is represented in Turkey, in the participants’ lives, and through his translated works, the paper draws on the interview recordings in combination with scholarly and documentary sources.

In summary, the project seeks to answer the following question: In today’s Turkey, what role does Rumi’s legacy play in Istanbul’s society, and how is that cultural and societal sense of ownership specific to Turkey? Hypothesizing that a strong cultural presence associated with Rumi exists in modern Turkey, this paper uses field research and on-site evidence to explore issues of cultural ownership and legacy. Providing insight into the reinterpretation and uses of historical figures, these findings are applicable to studies of cultural translation and revitalized legacies in the globalized world.

Talanoa (Tongan compound word—tala meaning to talk and noa meaning balance: form of conversing in a relational learning space) has been used within Pasifika cultures to engage with each other in respectful and balanced ways. This research not only acknowledges the importance of talanoa (and its analogous forms throughout the Pacific Islands) within the context of schools, but also reveals the possibilities of dismantling the power dynamics that occur within Western educational institutions and settings through conversations and classroom discussions. The following research question: “Does talanoa within a relational learning space impact Pacific Islander (PI) and non-PI learners?” We have observed and conducted conversations with PI and non-PI students in the ANTH 306 - The Power of Representation: Pacific Islander Voices course which centers the talanoa framework. Additionally, we will be using Indigenous decolonizing methodologies by Tuhiwai-Smith (1999), Wilson (2008) and more to critically analyze discourse used throughout our observations. Our anticipated research results will highlight the significance of talanoa to educators and learners who have been introduced to and/or adapted to this framework. Talanoa being a space where relationships and connections are of value and emphasized—creating transformational learning environments for, but not exclusively to, marginalized communities. As Pacific Islander students, we bring awareness to our ways of sustaining and supporting our knowledge-making dialogue. Talanoa is one of many Indigenous tools for understanding how we, Pacific Islander students, are able to communicate more meaningfully and beyond traditional discussions.

Research Family (R.F.) is a student led group focused on Pacific Islander (P.I.) culture, diasporic identity, policy issues, and educational upbringing- to name a few. In this learning space, we emphasize relationship building and discourse within a safe space. We develop weekly curriculums; responsible for gathering resources, media, and often finding guest speakers. The purpose of our research is to reframe the narrative behind “research” from the Western notion of individualistic learning to a shift towards collaborative collective learning. Research should not be extractive, knowledge should constantly be moving respecting where, and whom, it comes from; research should value reciprocity, respect, and relationships. We ask the question, “does R.F. impact P.I. learning and representation within traditional education systems?” To reflect on R.F. impacts, we turn to our own community to learn about the experiences of past, and current, R.F. members. Interviews are conducted to answer this question. We find that R.F. helps students utilize their resources in our educational system creating space for our community and help build our capacities to further beyond the school grounds. Being in this position promotes strong leadership, communication, and collaboration that will greatly benefit members long past their time at the University of Washington.

Our group, the King Lab at the University of Washington, computationally designs self-assembling protein nanoparticles for therapeutic applications. These nanoparticles make great candidates for therapeutics because of their ability to both encapsulate molecules as “cargo” and display antigens on their surface. One of the problems we face in vaccine and therapeutic design is targeted nanoparticle delivery, or making sure nanoparticles are delivered to a specific location, in vivo. Understanding where certain nanoparticles localize in vivo will be useful for determining what influences a nanoparticle’s interactions inside an organism. One approach our group is taking to examine nanoparticle biodistribution is miniprotein library display on synthetic nucleocapsids. These miniproteins are 20-50 amino acids long and were designed to produce stable, folded structures with surface patches that could facilitate binding to target receptors. Synthetic nucleocapsids, or nanoparticles designed to encapsulate their own RNA genomes, display these miniproteins in the library. The library was injected into healthy and tumor-ridden mice. RNA sequences were then obtained from blood, brain, heart, spleen, kidney, liver, lung, tumor, and dose samples. Using unsupervised and supervised learning algorithms such as Principal Component Analysis, Hierarchical Clustering, and Random Forests, I am building mathematical models that can analyze the biochemical properties of these library variants and determine why certain nanoparticles vary in biodistribution. I will also be analyzing another synthetic nucleocapsid library to answer similar questions about nanoparticle biodistribution. By constructing these models, I hope to provide tools that aid in experiments regarding targeted drug design.

Mitosis produces two genetically identical daughter cells, each inheriting their own nucleus and a full set of replicated chromosomes from the parent cell. Inaccurate chromosome segregation can result in severe consequences like cancer and developmental defects. Microtubules are dynamic cytoskeletal components that provide the forces necessary to segregate chromosomes into their respective daughter cells during mitosis. The kinetochore is an assembly of proteins and protein complexes located on the centromere that binds to microtubule ends to attach chromosomes to the force-generating microtubules. The accurate segregation of chromosomes relies on the ability of the kinetochore to strongly bind chromosomes to microtubule ends. Ndc80 complex is an outer kinetochore component that binds microtubule ends and is required for proper chromosome segregation. Emerging cellular data suggests that multiple Ndc80 complexes interact with one microtubule end to facilitate chromosome separation. In vivo data suggests multiple Ndc80 complexes are arranged around microtubules. To closely model the native kinetochore-microtubule interface, we have begun to assemble a structured particle of multiple Ndc80 complexes in vitro using designed proteins that form oligomers. The particles have different geometries and stoichiometries. A method to couple Ndc80 complex to the designed protein was developed. We then tested the coupling efficiency under different temperatures and concentrations to optimize the reaction and ensure complete particle assembly. We found that the reaction goes nearly to completion with a 3:1 ratio of Ndc80 complex to designed protein at room temperature with a reaction time of thirty minutes. We will now measure the stoichiometries of the particles, which are designed to have four, five, or six Ndc80 complexes. Successful formation of these organized particles will allow us to measure the effect of geometry and stoichiometry on the ability of the Ndc80 complex to make strong attachments to microtubules.

During the Victorian period, a growing middle class and widespread industrial advancements led to the development of serialized fiction and the modern novel. Well-educated writers who had previously referenced classical antiquity in poetry now introduced a new generation of readers to Ancient Greek and Roman authors via the realistic novel. Charlotte Yonge’s best-known novel, The Heir of Redclyffe (1853), has often been interpreted as a promotion of her own religious beliefs in the context of the Oxford Movement and Tractarianism. In addition to Christian themes, Yonge also makes several classical allusions throughout the novel, including a double reference to Vergil’s Aeneid, where she likens the characters Philip Morville and James Thorndale to Aeneas and Achates respectively. The comparison of Philip to Aeneas, one of the most highly esteemed and recognized classical heroes in the Victorian era, centers Philip as the primary protagonist and titular heir of the novel. I also propose an additional parallel between Philip’s cousin Guy Morville and Aeneid’s Pallas to both reinforce Philip as the focus of the novel and to demonstrate Guy’s role as an archetypal hero’s companion. For this project, I compare Philip and Aeneas’ journeys to Redclyffe and Latium, respectively. I also analyze the differences in companionship between James/Achates and Guy/Pallas. The dynamics between each trio lead to a reading of The Heir of Redclyffe as an epic journey and more broadly demonstrate how classical influences shaped story and character in a traditionally Christian morality tale.

Good mental health and well-being are essential to fulfillment in all aspects of life, including the workplace. Although there are many factors that influence mental health and well-being, a new, universal stressor has emerged in the past year: the COVID-19 pandemic. While many are socially distancing and working remotely, those employed in the restaurant industry continue to work in a high-risk environment. Restaurant workers were already mentally and financially vulnerable pre-pandemic, earning low wages and experiencing high rates of substance abuse-- and they now face the additional stressor of potential exposure to COVID-19. Ongoing research suggests that restaurant employees are facing unprecedented pandemic-related challenges to their mental health and well-being. This study explores the experience of restaurant employees in Western Washington who interact with the public regarding their communication about mental health and well-being. The main research questions are: a) Are restaurant employees having conversations about mental health and well-being with their coworkers and with their managers? If not, why not?; b) How do restaurant employees describe communicating about mental health and well-being with their coworkers and with their managers?; c) How do the descriptions about communication with coworkers compare to that with managers? To answer these questions, an exploratory cross-sectional survey was conducted to gather self-report data from a nonrandom sample of restaurant employees in Western Washington. At the time of writing this abstract, all data has been collected and the data analysis phase is beginning. By the time of the symposium, I will be able to report key findings on measures including the prevalence of restaurant workers’ conversations about mental health and well-being, with whom they occur, why they may not occur, the specific topics discussed, when and how these conversations take place, and motives prompting these conversations.

Chinese adoptees have played a significant role in the makeup of transnational adoption in the United States. Due to the intense measures of China's One Child Policy from the 1980's to early 2000's, a large influx of Chinese adoptees were brought to the US and became part of many multiracial families. From this, a unique population of Americans today engage in difficult conversations to clarify and defend their own identity in a multiracial family. These conversations can lead to negative psychological and behavioral outcomes that qualitative investigation can aid in addressing. This study aims to investigate how Chinese American adoptees respond and process these comments and conversations that question their legitimacy as a member of their family.  Methods used for research include qualitative interviews and thematic analysis. Analysis will focus on common patterns in adoptees' responses and types of interactions described during the interviews, grounded in the Communication Theory of Resilience. Communication Theory of Reslieience is the interpretive method that highlights the communicative processes by which people reconstruct a new normal after facing adversity. Results will inform us how Chinese American adoptees choose to respond to these comments, and/or how they would like to respond and construct these conversations in a form of resilience to disruptive narratives around adoption. By understanding how adoptees respond to and handle these conversations, we hope to provide insight for other Chinese American adoptees and their social networks in how to navigate these situations and best support adoptees in communicating their story. 

Audio description (AD), an additional narration track that conveys essential visual information in a media work, is imperative for improving video accessibility for people who are blind or visually impaired. While large streaming services such as Netflix, Disney+, and AppleTV have begun offering AD on new titles, current processes are manually done. Movies can take more than 60 hours to describe, with a cost of $10-$70 per minute. To investigate the need for AD, we conducted extensive user research with people in the blind and visually impaired community. We learned when they use AD, how they use it, where they use it, and most importantly, what they value in a high quality audio description experience. Existing literature regarding AD does not address these questions of user preferences, no projects have specifically targeted the area of user-generated content or smaller budget video content, and there is minimal existing work on automating the AD process. We received over 100 survey responses and conducted 40 interviews with stakeholders, including leading industry accessibility experts. Of these interviewees, 18 identified as blind or low vision. Our findings show that the most prominent challenge is the lack of available AD. Some interview participants preferred brief descriptions, wanting to fill in the gaps with auditory information, while others favored longer, more expressive audio descriptions. Based on our interview insights, we developed an audio description software to automatically describe videos from a user-provided link. For our prototype, we identify key frames, use existing APIs from Microsoft and Google to describe and read the text from each frame, and use text-to-speech to generate a second audio track. Through this project, we have extended knowledge of audio description preferences and developed a service to provide automatic audio descriptions based on novel user insights.

Hate speech classifiers, which are machine learning models that detect hate speech, are important tools for content moderation online and help keep online communities safe. However, these models show evidence of bias against certain groups of people. For example, current research shows that state-of-the-art hate speech classifiers show significant bias towards African American Vernacular English (AAVE). In response, new research has produced novel datasets with more complex label spaces compared to previous hate speech classification datasets. By having additional labels (i.e. more than a single label determining whether the text is hateful or not), these datasets aim to capture more social context embedded in natural language. In this project, we investigate this aforementioned claim. In particular, we investigate which features of language are the most salient in the decisions they make based on the datasets that they are trained on. We use multiple methods to determine feature saliency to discover biases in hate speech classifiers. Through these methods, we introduce a set of words that hate speech classifiers determine as highly salient which represent these biases. We also investigate whether these sets of words differ across datasets with varying label space complexity to understand whether these biases persist regardless of dataset. Finally, we will discuss the implications of the results and address future directions for the project.

Data-driven predictions of the future often suffer from error propagation and overconfidence. In many scenarios where forecasting is practical, the data follows a quasi-periodic pattern, which means that data from one point in time relays useful information about the data one period later. Through a Koopman theoretic approach, we make use of this feature to make long-term probabilistic forecasts that do not suffer from error propagation. Overwhelmingly, data-driven forecasts attempt to predict the exact value of a quantity into the future; however, such point-forecasts fail to describe the uncertainty in that quantity. Even when models are designed to make probabilistic forecasts, they are often overconfident and rely on the model to supply all sources of uncertainty. By forecasting the parameters of a probability distribution describing a quantity, rather than the quantity itself, we are also able to overcome the problem of overconfidence. Furthermore, our model relays novel and useful information about temporal patterns in the uncertainty of a dataset. We apply our model to electric load data from the 2017 Global Energy Forecasting Competition and show significant improvements compared to competing forecasts. I implemented the approach and ran experiments to explore the ways in which Koopman theory can be leveraged to robustly mitigate error and overconfidence in electric load forecasting. Our contributions to probabilistic forecasting of energy demand have the potential to lessen global warming, and the approach can be used in forecasting problems more broadly.

Amidst the turn of the twentieth century, few women were accounted for witnessing the advancement of archaeology, later known as the "Golden Age'' of Egyptology. In fact, much of the source material by women in this period have been misplaced or yet to be published. Mrs. Emma B. Andrews wrote nineteen volumes of diaries while observing the unearthing of over twenty tombs in the Valley of the Kings. The Emma B. Andrews Diary Project seeks to introduce Mrs. Andrews's works—and other unpublished historical documents composed by women during the time—to modern platforms, such as an online reader and social media. The transcribed texts are encoded into XML, using Text Encoding Initiative Guidelines, and the structural and contextual elements are denoted. The TEI documents are currently being used to create a comprehensive database for scholars and students alike to read the compositions of the lesser-known women of Egyptology. Accompanying the transcribed texts are biographies to equip the readers with additional context. However, we interns stepped further into this project by attempting to answer a fundamental question—how did Mrs. Andrews's accounts of her day-to-day interactions reflect the manner she viewed her life, and therefore herself? Guided by this question, we used research methods involving an analysis of Mrs. Andrews's language and identification via XML-TEI of the various contextual elements referenced—the people, places, hotels, and boats she encountered. These components were also cross-evaluated with further sources, including a sentimental analysis tool, to provide supplemental information to our study. After converging Mrs. Andrews's life history with recognized research in memory retrieval, we have noted observations on how her writing reveals her distinct perspective of the world and how her past may have influenced that point of view.

 Understanding complex and dynamic inter-specific relationships is key for informing and developing conservation policies. Accurately identifying the diet of various predators across Washington can provide insight into these relationships in terms of resource needs and predator-prey dynamics. DNA metabarcoding studies present a comprehensive way to surmise the complete diet profile of the American black bear (Ursus americanus), an opportunistically omnivorous predator that provides several important ecosystem services. As one of the only large omnivores in Washington, they also require large home ranges, making them an umbrella species that can be used as a guideline for overall conservation efforts. Our preliminary metabarcoding study with scat comparing the American black bear’s diet in northeastern and central Washington using a mitochondria-specific marker suggested their diet could be greatly influenced by human activity and that diet preference could delineate along geographic and ecological means. As the samples containing atypical food sources were within proximity to anthropogenic structures, these results suggest the pervasiveness of human-based food source availability to wildlife. These results were corroborated in our comprehensive study using both mitochondria and chloroplast specific markers. Analysis of the herbivorous portion identified the native and introduced plant species and their frequency of occurrence. This allowed us to understand the American black bear’s reliance on specific native species, as well as the extent of how local bear populations have incorporated non-native plant species into their diet, thus identifying their ecosystem intrusion. Additionally, the study’s sample collection spans a multiyear period, providing information on the seasonality of the American black bear’s diet, giving insight into seasonal prey switching and prey partitioning. This total diet profile allows for the examination of the influence of the wildlife-urban interface on food availability and resource selection in the Washington American black bear population, increasing our understanding of Washington predator dynamics.

 Long-toed salamanders (Ambystoma macrodactylum) are common amphibians throughout the Pacific Northwest, making them an ideal species to use for restoration assessment. A successful wetland restoration project would be expected to have a healthy long-toed salamander population, but it was not previously known whether long-toed salamanders were present in the Union Bay Natural Area (UBNA), a Seattle park with many ongoing restoration efforts. The Amphibian Corridor restoration project in UBNA, installed in 2015, aimed to provide amphibian migration habitat. To assess its efficacy, I examine long-toed salamander movements and woody debris microhabitat preferences within and around the corridor. The study also acts as an inventory of amphibians present in the corridor and throughout UBNA. The corridor and surrounding areas are being monitored throughout the 2021 breeding season (January through May) for adults, larvae, and egg masses. To find larvae and egg masses, dip net aquatic surveys are conducted once per week, alternating between day or night surveys. To find adults, visual encounter terrestrial surveys are conducted once per week, alternating between day or night surveys. Individual salamanders are photographed and identified by their unique spot patterns so that individual movement can be tracked using a mark-recapture method. The location and microhabitat of all amphibians are recorded . My surveys have shown that long-toed salamanders use the corridor. To date, 18 individuals have been recorded in the corridor with up to 10 recaptures per individual, and 11 individuals have been recorded in other parts of UBNA with up to 8 recaptures per individual. Three egg masses have also been found in UBNA. UBNA management and future restoration could be informed by this study’s insights about long-toed salamander microhabitat preference and movement strategies. Given this information, similar restoration projects to the Amphibian Corridor in other urban parks may be effective for amphibian conservation.

Seed dispersal is a crucial phase of plant lifecycles. Effective dispersal is important to the ecosystem as a whole because it affects composition of the community, ecological succession, and response to climate change. Given the importance of seed dispersal, understanding the factors that contribute to the evolution of varied dispersal modes and promote convergence on specific dispersal strategies is particularly important to understanding plant ecology. We think habitat might be one of these important factors. Here, to explore the relationship between habitat and dispersal mode, we are studying the onion grasses (Melica), a small genus of perennial grasses primarily distributed in temperate regions, and their relatives. These grasses are found in a wide variety of habitats and possess a remarkable diversity of seed dispersal strategies. These traits make them a useful case study for better understanding the factors that influence the evolution of dispersal strategy. We are testing the hypothesis that evolution in traits associated with seed dispersal is correlated with changes in habitat. In particular, we hypothesize that the evolution of wind dispersed seeds follows transitions into open habitats. Seed dispersal structures (diaspores) were collected from 28 grass species (14 Melica and 14 outgroup). To assess wind dispersal potential, we quantify falling velocity by filming seed descent at 1000 fps. Lower falling velocities are associated with higher wind dispersal potential. Diaspores were photographed and the images were used to measure surface roughness, which is associated with adhesive dispersal potential. Habitat data were obtained for each species via a literature survey. These data, along with several other traits associated with seed dispersal processes, were mapped onto the evolutionary tree of the onion grasses. Initial results indicate that convergence upon wind dispersal may be in part driven by convergence upon disturbed habitat types.

After an 80-year absence, gray wolves have been returning to Washington state over the last decade. Mapping their population growth and reproductive activity is key to understanding their recovery and to assisting wildlife conservation management. Recognizing an established pack requires identification of the presence of breeding females. Accurate noninvasive identification of pregnant wolves through scat could greatly assist such efforts. A reliable index of pregnancy in most mammals is progesterone levels (ng/g) in feces; progesterone rises post-ovulation, but will only be elevated above a “pregnancy-threshold” level among pregnant females. However, this metric is less definitive in canids. Progesterone levels remain elevated in all post-ovulatory females, regardless of whether the females become pregnant or not. Since gut microbiome diversity has also been shown to differ between pregnant and non-pregnant large mammals, in this study I examined whether the combination of progesterone levels and gut microbiome diversity can refine this pregnancy metric in free-ranging wolves. Fecal samples from female wolves (n=62) with known progesterone levels were provided by the Center for Conservation Biology from a 2015-2017 study in Northeast Washington. Samples with progesterone levels above 2000 ng/g were defined to be from potentially pregnant females. I generated gut microbiome profiles by amplifying and sequencing the V4 16S rRNA gene region in each sample. I analyzed the sequences using Qiime 2 and R with the Silva reference database for microbial taxonomy classification. Gut microbiome profiles were compared to progesterone levels. Pregnant wolves are expected to have a different phyla diversity in bacterial communities from non-pregnant wolves. Results may guide future studies to focus on identifying a particular bacteria species or a certain proportion of diversity combined with progesterone levels to further refine pregnancy diagnosis. Accurate identification of pregnancies in wolf packs can improve population growth estimates, leading to informed Washington wildlife conservation policies.

Approximately 10-20% of adolescents globally struggle with mental health conditions. Due to multiple physical, social and emotional changes, adolescents are particularly vulnerable to mental health illnesses. Untreated or inadequately treated mental health conditions can be exacerbated by stigma, discrimination, and isolation. The inability to effectively address mental health problems can manifest both short-term and long-term consequences into adulthood, impairing both quality of life and having negative impacts on health outcomes and mortality. Mental Health for Every Adolescent (MHEA) is a programme we, as students at the University of Washington, created as an early intervention to destigmatize and educate students about mental health. Through this project, we sought to understand the impact and need for community based mental health intervention for young adults. We partnered with faculty to create curricula that addressed stress, anxiety, depression and other mental health related topics. We then recruited volunteers through social media advertising and virtually trained them to facilitate workshops. Using feedback we obtained from local stakeholders at our workshop sites, we modified our curricula to ensure they were culturally appropriate and sensitive. Over the course of 3 years, we completed 74 workshops across 3 countries - India, the United Arab Emirates and the US - and we reached more than 3100 middle school and high school students. We found that the needs of students varied across communities and therefore, the workshops had to be tailored appropriately. In April 2020, the MHEA team leveraged local relationships and published a COVID-specific guide aimed at adolescents which was launched at 6 school districts in the United States. The MHEA project describes a successful implementation of a community-based mental health intervention for children and young adults. Programmatic materials developed can be disseminated and incorporated into middle and high-school curriculum. Future work will evaluate the impact on participants in the program.

The goal of the Kaeberlein lab is to understand the underlying molecular mechanisms of aging and how they can be perturbed in order to beneficially alter the aging process. To do so, we use the unicellular model S. cerevisiae or brewers yeast. In order to expedite the process of studying aging in large numbers of cells, the Kaeberlein lab has been using microfluidic devices that immobilize yeast cells throughout their lives so that visual data can be collected without manual interference of the cells. The question being investigated here is whether a neural network can be trained to automate the scoring tasks required for processing the image data generated by these microfluidic devices. Using python and the Yolov3 neural network architecture, I have created an AI-based screening tool to quickly analyze data generated with these microfluidic devices, such that the pipeline as a whole produces lifespan data for about 120 cells at a time, using stacks of image files as raw data. This work is significant as it provides insights into the issues of training a neural network on similar objects, and more importantly, insights into how these issues can be mitigated. In addition to increasing the speed with which data can be scored, this system will also increase the capacity for scoring experiments with large numbers of experimental groups. The system itself can also be used in order to expedite biology research.

Microglia, the resident immune cells of the brain, become activated and mediate neuroinflammatory responses in response to traumatic brain injury (TBI). This neuroinflammation can be detrimental to the health of the brain; thus, inhibition of this natural response can benefit TBI patients. Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are a technological advancement that allow manipulation of specific cellular signaling pathways. The hM4D DREADD gene was inserted in a viral construct downstream of the CD68 promoter, which is markedly upregulated in activated microglia following TBI. This construct is activated with clozapine n-oxide (CNO) to downregulate secondary messengers in activated microglia and thus attenuates the inflammatory response by reducing microglial activation and proliferation after injury. After viral transfection to deliver the construct to cells, rats underwent a controlled cortical impact (CCI) - an experimental model of TBI - and were treated with a CNO injection at varied intervals after injury. After the rat brains were dissected and sectioned, we used immunohistochemistry techniques to label microglia with an anti-Iba1 antibody, proliferating cells with an anti-BrdU antibody, and cell nuclei with DAPI. This allowed for the visualization of microglia using fluorescence microscopy and microglia were quantified using stereological principles. The aim of this research project is to use pharmacological DREADD receptor-mediated inhibition of microglia at different intervals post-injury to quantify the proliferation of microglia following CCI. We hypothesize that increased duration between CCI and CNO injection leads to more pronounced microglial activation represented by increased numbers and morphological changes. We also hypothesize that microglial activation can be observed as a gradient with the greatest proliferation closest to the CCI epicenter.

The Wnt pathway is a critical regulator of osteoblast function. Activating and inactivating mutations in the Wnt pathway lead to high and low bone mass phenotypes in humans and animals, respectively. To evaluate the role of Wnt signaling in facial skeletal development, a transgenic strain of mice (daβcatOT) was generated in which the Wnt intracellular mediator, β-catenin, was constituitively expressed in osteocytes and mature osteoblasts. The craniomaxillofacial (CMF) tissues of daβcatOT mice and their littermate controls were collected at two time points: one where mice had reached sexual maturity e.g., 2 months old and the other where mice had reached adulthood e.g., 4 months old. Thirty-eight anatomically distinct landmarks were identified on three-dimensional volumetric renderings of control and daβcatOT (each N=8) head skeletons. Landmarks typically corresponded to intersections of skeletal elements and were predominantly located at CMF growth sites. Through the open-source visualization software Drishti, we performed 28 separate measurements. Morphometric analyses of the dataset were conducted using Microsoft Excel. Inter-rater reliability was qualitatively assessed to ensure reproducibility. Two tailed T-tests were performed, which revealed statistically significant (p<0.01) differences in the lengths of the maxillary, premaxillary, and mandibular bones (p<0.01) which comprise the jaw skeleton. Equivalent analyses revealed limited asymmetries. The resulting facial phenotype of daβcatOT mice was a largely symmetrical but severely flattened midface, accompanied by a widened, retruded mandible. Comparative analyses revealed that the daβcatOT facial anomalies phenocopy an exceedingly rare human malformation known as craniodiaphyseal dysplasia. Ongoing analyses of daβcatOT tissues are focused on the molecular/cellular basis for the facial malformations caused by constitutive activation of Wnt/β-catenin signaling and in doing so, gain more insights into how the CMF skeleton normally develops.
 

Currently, in the United States and worldwide, the population is aging, living longer, and the increase in people living with dementia (PLWD) will continue to rise. Many PLWD receive care from their primary care clinicians, who face unique challenges caring for this population given decline in cognitive function, overall health and unpredictable behavior requiring more attention. Gaps in care could be addressed through the development of a cadre of dementia nurse specialists, as much of dementia care is within the scope of nursing practice. However, interest in the aging population is one of the least desirable disciplines to work for new nurses as well as currently practicing nurses. The purpose of this study was to evaluate nursing students’ and practicing nurses’ perceived knowledge and attitudes about providing care for people who are living with dementia and their family caregivers. Based on existing literature, we identified the factors contributing to the attitudes and conducted semi-structured focus group interviews with nursing students and current practicing nurses. Quantitative and qualitative data was analyzed to identify themes and factors that would encourage nursing students and practicing nurses to receive specialty training in dementia. This research will aid in development of a multimodal dementia education training program for nurses in ambulatory care.

Alzheimer’s Disease (AD) is a neurodegenerative disease that is the most common cause of dementia. One hallmark of AD pathology is hyperphosphorylation of Tau protein. Tau is a neuronal-specific protein that stabilizes microtubules. Hyperphosphorylation of Tau leads to loss of its normal function and promotes aggregation into neurotoxic fibrillary tangles. While Tau aggregation is well-documented, the exact role of Tau loss-of-function in AD pathogenesis remains uncharacterized. The goal of our project is to determine the mechanism by which Tau loss-of-function contributes to AD pathogenesis. We hypothesize that Tau loss-of-function contributes to AD pathogenesis by activating the cellular stress response in neurons, characterized by DNA damage, stress granule formation, and the upregulation of heat shock proteins, chaperones that stabilize and refold proteins damaged by cellular stress. To test this hypothesis, we cultured neural progenitor cells (NPCs) and neurons derived from human induced pluripotent stem cells (hiPSCs). We generated three cell lines: one in which the gene encoding Tau was knocked out (Tau KO), another in which Tau expression was knocked down (shTau), and a control line. To determine whether genes implicated in the cellular stress response are upregulated in Tau KO neurons, we used RNAseq and RT-PCR. Then, we used immunocytochemistry to detect protein markers of cellular stress in NPCs and neurons from all three lines. Preliminary results indicate that several genes encoding proteins involved in the cellular stress response are upregulated in Tau KO neurons, including the small heat shock protein HSPB8 and BAG3, a gene that is upregulated in the aging rodent brain and regulates the autophagic response. By immunostaining, we show that dsRNA aggregation, which may indicate stress granule formation, is more prevalent in Tau KO cell lines than control. By elucidating the role of Tau loss-of-function in AD pathogenesis, this research could inform therapeutic targets for AD.

People with autism spectrum disorder experience varying challenges in sharing social-emotional reciprocity and the establishment and maintenance of social relationships (American Psychiatric Association, 2013). Having social relationships is important for a variety of reasons, including but not limited to, building and solidifying lifelong skills in conflict resolution, improving self-perception, and promoting healthy emotional and cognitive development (Bukowski & Sippola 2005). There is growing research that has provided characteristics of autistic individuals’ peer relationships and friendships, however, these studies often must approach these complex relationships through a focused lens. In order to better understand the characteristics  of social relationships in this community, we must comprehensively examine this literature base, and to our knowledge, no such literature review exists. Thus, the current project explored 31 studies of autistic individuals’ social relationships with friends, peers, or acquaintances, finding connections in the existing literature including friendship reciprocity, social network centrality, and how severity of diagnosis affects friendships. Results from these studies show that the majority of autistic participants maintain friendships, but there are significant differences in friendship quality, the friends’ characteristics, duration and frequency of interaction, and satisfaction and enjoyment of friendships. Furthermore, several factors can influence how these friendships are formed and sustained, including parental involvement, the participant’s diagnosis and other characteristics, environment, and technology. This review will allow for further understanding of ASD and the social aspects of the disorder, which could have implications for academic supports, clinical interventions, and further research on social relationships among individuals with ASD. 

Over the last 50+ years, American Crow populations have increased in urban areas in the United States, taking advantage of abundant food sources and protection received under the Migratory Bird Treaty Act. However, heightened urbanization is correlated with increased anthropogenic noise, which can negatively impact bird communication. Some avian species compensate for this by increasing their call frequency to improve sound transmission. Previous work has also shown that avian social behavior can vary with urbanization, with individuals displaying increased boldness in more urban environments. In this study, we assessed the vocalizations, number of approaches per crow, latency to respond, and closest approach of groups of American Crows in response to a predatory owl decoy and playback of a distressed crow call, across an urban gradient in Western Washington. To quantify urbanization, we scored aerial photographs of each site, assessed levels of pavement, vegetation, building cover, and water, and used principal component analysis to create an urbanization score. Analysis of the first 12 sample sites revealed no statistically significant relationship between behavior or the frequency of vocalizations against urbanization. Surprisingly, ambient noise did not vary across our urbanization gradient, so our lack of significant findings may be the result of decreased anthropogenic noise due to the Covid-19 shutdown. However, we did detect a significant negative relationship between average pause duration, the spans of silence between separate calls, and urbanization, so crows may have adapted to produce more predator-related calls in the once noisier urban areas. We have increased our sample size to 31 sites and are currently continuing our analysis. Understanding how human impacts on the environment influence how wildlife responds to threats is important given the continuous expansion of urban areas. Our results may aid with ecologically-focused urban planning and urban wildlife efforts.

Despite research supporting the efficacy of certain mental health practices, many mental health care providers in community mental health clinics are not utilizing evidence-based practices (EBPs) consistently when treating patients with mental health needs. Clinical supervisors play a critical role in EBP implementation given that they have regular oversight with clinicians and can gauge how often clinicians use EBPs and how effective they are at delivering them. The purpose of our project is to examine the feasibility and self-reported usefulness of case-based consultation for clinical supervisors at child-serving community mental health clinics to support the implementation of EBPs. In our pilot study, nine clinical supervisors and two supervisor consultants were recruited from community mental health clinics to participate in supervisor consultation calls. The purpose of these calls was to improve clinical supervision skills and perceived competencies for clinical supervisors. These calls consisted of: (1) a brief 15-minute didactic lead by the supervisor consultant that covered various content areas relevant to their roles as supervisors; and (2) 45 minutes of case-based consultation among the consultant and the clinical supervisors where they discussed real-life supervision scenarios, barriers and challenges that they faced, and proposed solutions or strategies. Pre and post surveys with clinical supervisor participants showed the consultation calls resulted in greater self-reported supervision competency. To build on the quantitative analyses, my mentors and I have been conducting a qualitative thematic analysis of eleven of the case-based consultation calls, which were recorded and transcribed. We will analyze the qualitative data to identify reoccurring themes discussed on the calls coded as barriers or solutions to supervision and clinical challenges to EBP implementation. This thematic analysis will help us gain insight into one potential support strategy to improve EBP implementation and sustainment to increase the quality of behavioral health care for youth.

Chronic pancreatitis (CP) is a significant source of morbidity, mortality, and reduced quality of life. A common symptom of CP is severe abdominal pain, and it is associated with adverse effects on physical, psychological, and social functioning. Despite these concerning impacts, pain self-management programs have not yet been developed or evaluated for individuals with CP. Instead, pancreatitis pain is typically managed with opioids which can have limited efficacy and negative side effects. In other chronic pain conditions, cognitive-behavioral therapy (CBT) programs have improved patient outcomes (e.g., pain-related disability, depression) without pharmacological treatment. Therefore, this study aimed to evaluate the feasibility and acceptability of an internet-delivered CBT program for individuals with CP pain, as well as its impact on pain outcomes (pain interference, pain intensity, and quality of life). The study consisted of 30 adult participants with CP. Participants were randomized to either internet-delivered CBT (Pancreatitis Pain Course) or a wait-list control. The Pancreatitis Pain Course delivered 5 lessons over a span of 8 weeks that gave participants information to understand their pain, a range of CBT skills to manage their symptoms, and homework assignments to practice the skills. Pain outcomes were assessed at pre-treatment, post-treatment and 3-month follow-up, and qualitative interviews were conducted at post-treatment. Eighty percent of participants rated the program as highly acceptable and 64.3% completed all 5 lessons. The qualitative interviews showed that participants found the program relevant and helpful, and patients in the treatment group experienced reduced pain intensity and pain interference. Because this is the first trial of a CBT pain self-management intervention for adults with CP pain, future trials are needed to further assess its ability to reduce pain and disability.

Humans hear through the conversion of pressure waves vibrating in the inner ear into chemical signals released by activated hair cells. Loss of function of hair cells due to damage can result in permanent hearing loss. Zebrafish are one of the model organisms used to study hair cells, since zebrafish have hair cells similar to humans. Unlike humans, zebrafish can regenerate their hair cells after damage. Regeneration occurs when support cells differentiate into hair cells. Determining how zebrafish support cells differentiate into hair cells is important to understand if human support cells can be induced to differentiate in a similar way. This requires comparing the structural differences between support cells and hair cells. One important aspect of neuronal signaling is the release of calcium ions from the cell’s endoplasmic reticulum (ER). This experiment looked to answer whether the ER in zebrafish hair and support cells was quantifiably different. Since structure mediates function and support cells do not signal to neurons, their ER structure should not be equivalent to the hair cells’. To test this, I manually segmented and reconstructed serial block-face scanning electron microscope (SBF-SEM) images of both cell types’ ER into 3D. Using SBF-SEM as a reconstruction method allowed for more detailed visualization of both the cell volume and the ER, in contrast to other methods such as confocal microscopy. Support cells had an ER volume of 50.51 µm3, while hair cells had an ER volume of 29.09 µm3. Support cells had a higher ER to cell volume and ER surface area to volume ratio than hair cells. It can be concluded that ER structures of support and hair cells are quantifiably different. Quantifying ER differences between support and hair cells is an important step toward discovering solutions to deafness caused by damage to human hair cells.

The skin is a highly innervated sensory organ, providing our bodies with the vital ability to understand and respond to external stimuli, such as pain, temperature and touch. However, many injuries to the skin result in the severance of somatosensory axons, causing temporary or permanent loss of feeling. To reestablish innervation, skin and neuronal cells launch wound healing responses. Although these responses are known to occur after an injury, the exact biological pathways and cellular components involved remain poorly defined. This project aims to characterize the transcriptional responses to skin injury using zebrafish (Danio rerio) as a model organism. Unlike human skin where complete regeneration is not observed, zebrafish have almost perfect regenerative abilities. To address our question, we plucked fish scales to induce a rapid regenerative response in somatosensory neurons and skin. I previously conducted RNA-seq analysis on populations of neurons and skin cells over the course of an induced injury response and curated a list of differentially expressed genes. Using gene ontology annotations to inform on these transcriptional changes, I have begun to identify the enrichment of well defined pathways, processes, and cellular components that are up or down regulated during the injury response. Thus far, I have identified a number of upregulated biological processes associated with changes in the extracellular matrix of skin-resident cells. The identification of enriched biological processes will help guide future experimental designs that study the effects of manipulations in these processes. With a deeper understanding of genes and mechanisms involved in zebrafish skin repair, I hope to unlock regenerative secrets that could apply to the treatment of human tactile maladies. 

We can use human pluripotent stem cells to derive cardiomyocytes (hPSC-CMs) in vitro, with the goal of transplanting them into the hearts of individuals who have suffered from heart attacks and restore contractile function. After transplantation into animal models, however, hPSC-CMs produce arrhythmias (irregular heartbeats), likely caused by the immature state of hPSC-CMs. This immature state is associated with low expression of cardiac genes regulating heart muscle contraction and electrical properties. We aim to mature hPSC-CMs in vitro by controlling the expression of these genes, so we can engineer them to behave more like adult cardiomyocytes. To do this, I am looking at DNA methylation, a modification occurring at cytosine nucleotides that is associated with transcriptional repression or gene silencing. My project goal is to determine if DNA methylation plays a role in regulating gene expression patterns of cardiac genes in hPSC-CMs. To investigate this, I have treated hPSC-CM genomic DNA with bisulfite reagent, which converts unmethylated cytosine nucleotides to thymine nucleotides. This treatment will allow me to differentiate between unmethylated versus methylated DNA, and determine whether cardiac maturation genes are methylated at their promoters (where gene expression is typically regulated) by running PCR. Additionally, I have cultured hPSC-CMs with the DNA methylation inhibiting drug 5-azacytidine. By blocking DNA methylation, I will be able to determine if methylation has a direct effect on the expression of cardiac genes by measuring gene expression via quantitative real-time PCR. I hypothesize that DNA methylation regulates cardiac gene expression, and inhibiting methylation will cause expression to increase. Thus, if DNA methylation represses cardiac gene expression, we can mature hPSC-CMs by inhibiting methylation. Ultimately, we hope to prevent arrhythmias that occur after hPSC-CM engraftment and develop cell therapies using mature hPSC-CMs to restore heart function after a heart attack.

DNA glycation is the DNA damage induced by reactive carbonyls (such as methylglyoxal and glyoxal) in plants and mammals. Glycation damage is quantitatively as important as oxidative damage. It is one of the major in vivo DNA damage sources associated with increased mutation frequency, DNA strand breaks, and cytotoxicity. In humans, glycation damage may contribute to Parkinson’s disease, cancer, and oxidative stress-induced diseases. Glycation damage in plant organelle (Chloroplasts and Mitochondria) DNA remains poorly understood. We recently showed that the demise of plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) during maize seedling development is associated with an increase in DNA damage resulting from oxidative stress caused by reactive oxygen species. As oxidative and glycation stress are closely linked in plants, we hypothesize that glycation might be one of the causes of ptDNA and mtDNA damage during development. Glycation damage can be prevented by the activity of the protein deglycase DJ-1, also known as Parkinson's Disease Protein 7 (PARK7). Our objective is to quantify glycation lesions in the organelles of maize plant tissues in the presence and absence of DJ-1. Our approach involves the quantification of glycation and deglycation in ptDNA and mtDNA using PCR analysis and the enzyme-linked immunosorbent assay (ELISA). This research should better understand glycation damage in plant organelles and might lead to insights concerning human pathologies and neurodegenerative disease caused by glycation.

In the US over 100 million people live with diabetes or pre-diabetes. The economic burden of this is approximately $327 billion every year. This study seeks to establish an alternative mode of insulin production using a polyethylene glycol (PEG) transformation of Pleurotus ostreatus. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins, rapid growth, and fully sequenced genome. In this study, I transformed P. ostreatus using PEG with a plasmid containing the human insulin gene, a green fluorescent protein (GFP) reporter gene, and a selectable resistance gene. Transformed cells were selected using hygromycin, extracted, and regenerated on growth media. Confocal microscopy confirmed the presence of the GFP and presumably the human insulin gene. An ELISA for insulin and proinsulin will be used in the upcoming months to test for genetic expression, and to determine the efficacy of protein folding in the transgenic fungal cells. This has the potential to not only expand the market for diabetic treatment options, but it initiates a valuable conversation about the importance of diversifying production methods and costs in the treatment of diabetes.

The locus coeruleus (LC) is a small nucleus of noradrenergic neurons in the pons, which, despite its size, has broad projections throughout the central nervous system (CNS). Functionally, the LC is believed to be involved in various critical functions, including the physiological response to stress, as well as mediating arousal. Previous investigations have demonstrated that optogenetic activation of the LC at a tonic frequency promotes wakefulness in rodents. While this observation causally implicates LC function in wakefulness, it is still not known how the LC is endogenously controlled to mediate arousal. One potential candidate in this control is the peptide nociceptin and its cognate receptor, the nociceptin opioid peptide receptor (NOPR), both of which are highly expressed around the LC. To investigate, we first conducted two pharmacological experiments using the NOPR agonist Ro64-6918 to assess its effects on locomotion and on the activity of LC noradrenergic neurons. To determine where the endogenous nociceptin signal to the LC originates, we performed an intracranial injection of a Cre-dependent retrograde virus (AAV2-DIO-eYFP) into the LC of a mouse expressing Cre recombinase in nociceptin-expressing neurons. We observed that Ro64-6198 (10 mg/kg) strongly reduced open field locomotor activity compared to vehicle treatment. Using in vivo 2-photon calcium imaging (GCaMP6s), we found that Ro64-6198 (5 mg/kg) profoundly reduced LC noradrenergic neuron activity. Wakefulness appeared reduced in both in vivo experiments. Finally, we identified nociceptin-expressing cells projecting to the LC in the peri-LC as well as a long-range projection from the bed nucleus of the stria terminalis (BNST). Together, these studies suggest that nociceptin acting on LC noradrenergic neurons reduces arousal, and that the endogenous sources of nociceptin may originate in the peri-LC and BNST. Future studies will investigate nociceptin-expressing neuron activity during sleep/wake transitions and whether this activity is sufficient to alter wakefulness.

The goal of this project is to develop a dynamically evolving connectionist model that more closely resembles the brain through its information-processing. Over the years, AI has shifted from the first generation of feedforward systems to the use of recurrent or convolutional Neural Networks. The third and newest generation of AI models, the brain-based models, and the Spiking Neural Network (SNN), attempts to bridge the gap between Neuroscience and ML using biologically realistic models like Θ-model, LIF, Izhikevich, HR, HH. These models, however, are still a black box leaving very little control or understanding on the learning process within the system without the access to the inner structure of the network. In addition, these systems are highly inefficient, slow, and very complex due to the limitations imposed by the hardware and explicit simulation of partial differential equations. Real world problems require “flexible learning and dynamically adaptive connectionist systems” that are capable to adapt and accommodate new input in real time. Current solutions have focused on varying the weights within a system rather than focusing on how connections within the system are formed. Based on our understanding from organismal brain structures, our approach, called biomimetic information codec, .bic, is a morphologically-adaptive coding hierarchical network that form in accordance with energy minimization - driven by dissipation of "heat" generated by the training data - constructing cortices and connectome for processing of information. My first objective herein is to quantitatively compare detailed structures between biological (fly brain) and .bic. networks using a random matrix approach.

This paper investigates the impacts that asymmetric information has on private pediatric health insurance coverage. In order to do so, I use the Rothschild and Stiglitz model of equilibria in perfectly competitive insurance markets and apply it to pediatric health insurance. It examines the shifts that occur in the model due to exogenous changes from asymmetric information between insurers and parents. These information problems occur in three forms; moral hazard, when people consume more care because they have health insurance; the principal-agent problem, when a task is delegated to an agent and the principal cannot directly observe the agent's actions; and adverse selection, when the consumer of health insurance has more information than the provider. These information asymmetries affect the behavior of both consumers and suppliers of pediatric health insurance, and so results in changes of supply and demand of insurance contracts. Ideally, in an equilibrium contract, consumers would recieve maximum utility, firms would make non-negative profits, and no other possible contracts would have the potential to make the firms greater non-negative profits. However, due to the changes in supply and demand from asymmetric information, this efficient outcome can become harder to obtain. By examining the changes that happen in the market with the modified Rothschild and Stiglitz model, I examine the the existence of equilibrium points, as well as the anticipated effects on health insurance coverage and health insurance prices. Because there are so many possible events that can occur, such as changes in parental income and risk, and the uncertainty of the magnitude of those events, no concrete conslusions can be made. It can however, be said that pediatric health insurance coverage may change in the presence of asymmetric information. 

Nanofibers have broad capabilities in biomedicine, e.g. drug delivery and tissue engineering, because of their diverse tunable properties. For these purposes, nanofibers often must be patterned to be integrated in devices or to optimize their function. Current nanofiber patterning methods lack user-control over design or alter fiber integrity. To optimize and expand the applications of nanofibers, there is a need for a versatile nanofiber patterning strategy that maintains material integrity and function and can be generalized for patterns of different complexity and dimensions. This project aims to address this need with an in situ patterning strategy that allows for complex, three-dimensional patterning at the milli/microscale with different fiber materials. The approach consists of a two-layer composite electrospinning collector with an insulative layer and conductive recessed patterns. An inexpensive collector fabrication method was designed for rapid prototyping. Collector design features predicted to affect fiber deposition were evaluated by quantifying fiber selectivity. Optimal formulations of nanofiber materials were experimentally evaluated based on reproducibility, fiber yield, and selectivity to delineate key polymer solution properties affecting patterning. This project offers a guiding foundation to adapt this patterning strategy to various applications of nanofibers by tuning fiber formulations and specific collector design features.

Implanted biomedical devices are becoming increasingly common for the treatment of tissue defects and organ failures. However, there is an ongoing issue of biocompatibility, where the host body constantly attempts to degrade the foreign object while it can’t most of the time, especially in the case of synthetic polymers. Consequently, virtually all implants will undergo an immune response named foreign body reaction and eventually get encapsulated in collagen, which can be detrimental to the device’s designated function, especially for drug delivery systems. Extensive research attempting to improve biomaterial integration has been conducted in the past decades. Recent studies suggested that the in vivo vascularization within and around a porous polymeric biomaterial is partially driven by the local phenotypic expression of macrophages, where the M1 macrophage was specifically shown to play an angiogenic role. It was also observed that the in vitro pre-vascularization of biologically-derived constructs accelerates and enhances tissue vascularization in vivo. Enlightened by these findings, I proposed to modulate both macrophage phenotypes at the material-tissue interface and scaffold pre-vascularization to restore tissue homeostasis. Two tools previously developed by my mentor’s research groups are vital for my study: the engineered M1-inducible macrophages (i-M1macs) and sphere-templated porous poly(HEMA) biomaterial scaffolds. Three main aims were constructed, where the first two aims are to optimize in vitro the scaffold pre-vascularization from endothelial cells and activation of i-M1macs in scaffolds respectively. The third aim is to determine the effect of macrophage modulation and scaffold pre-vascularization on the foreign body response to biomaterials in vivo. Findings from my research are expected to improve our understanding of the correlation of macrophage plasticity, material porosity, scaffold pre-vasculature, and tissue vascularization, which can be crucial for the development of a novel cell therapy that improves biomaterial integration and ultimately, the quality of life of people with biomedical implants.

Loss of periodontal ligament tissue (PDL) and attachment is a serious complication of periodontal diseases - the most prevalent dental health problems. PDL-degeneration leads to alveolar bone degeneration, infection, gingivitis, and eventual tooth loss. There is currently no product that can cure PDL-degeneration as regeneration requires the combinatorial process of regenerating cementum, signaling the existing relevant cells to proliferate and form PDL, and its integration into a functional system. Current restorative treatments utilize cell-based tissue regeneration, synthetic scaffolds, tissue grafts with limited, temporary success. A market product, e.g., claims to restore periodontium using harvested fetal swine periodontal tissue with highly variable clinical outcomes. Although these traditional procedures are well-established and show some success, their efficacy is limited due to the lack of structural and functional integration of a deposited layer with the underlying tooth, specifically integration into the remineralized cementomimetic layer. GEMSEC labs have developed a proprietary technology dubbed “peptide-guided remineralization” which facilitates new mineral formation using protein-derived peptides and have successfully restored dental hard tissues via several case studies including enamel, cementum, dentin under in-vitro and in-vivo conditions. Translating this technology into a daily-use product, we propose a PDL-regenerating chimeric construct which includes a biomineralizing peptide, ADP5, derived from the key enamel protein, amelogenin, with cell signaling moieties. Herein, we aim to use established bioinformatics, machine-learning tools, and high-throughput experimentation to identify peptides from proteins involved in PDL development cell-signaling towards controlled biomineralization, bioadhesion, and cell-signaling functionalities necessary for PDL regeneration. Addressing current treatment protocol limitations, the interdisciplinary approaches developed in this project are designed for the regeneration and formation of fully functional PDL. 

Group B Streptococcus (GBS) are beta-hemolytic, gram positive bacteria that colonize the vaginal tract of 1 in 4 pregnant women, and can cause preterm birth and neonatal infections. A key factor involved in GBS pathogenesis is an enzyme known as hyaluronidase (HylB). HylB is secreted from GBS during infection, and allows GBS to evade the host immune response by interfering with pathogen associated molecular patterns (PAMPs) recognition through toll-like receptors (TLRs) 2 and 4. The goal of this project is to use a biochemical colorimetric absorbance assay to quantify HylB activity in GBS isolates by comparing 50 maternal commensal isolates to 200 neonatal invasive isolates. Following quantification, the isolates will be stratified based on the epidemiological and clinical data to determine how HylB contributes to disease outcome. Although HylB’s role in GBS pathogenesis has been partially described by in vitro experiments and animal models, this virulence factor is not assessed clinically when patients are tested for GBS during pregnancy. Thus, this project allows us to identify new correlations between HylB activity and real-world clinical outcomes, which may be important for predicting disease progression. Currently, about 24% of invasive isolates exhibit HylB activity, though analysis for these isolates are still in progress, and we are continuing this study to include commensal isolates. We hypothesize that the commensal isolates will have reduced HylB activity compared to invasive isolates, as the ability for GBS to evade the host immune response may allow for greater dissemination. Knowledge from this study may contribute to the design of novel therapeutics, such as those targeting HylB, that may help prevent poor outcomes during human infection. 

The technique of Indirect Calorimetry (IC) allows for the non-invasive and continuous measurement of metabolically relevant functions of an animal. The Michael Schwartz lab uses IC techniques in the investigation of how brain regions, neuronal populations, and the larger neuronal circuitry that they connect with, defend either blood glucose or body weight. Taking IC measurements of treatment mice allows for quantification of glucose consumption and energy homeostasis. My project was to develop a programming pipeline for the analysis of circadian metrics from rodent populations placed on a 14-hour light and 10-hour dark cycle and placed on control or high-fat high-sucrose diets. The circadian cycling of oxygen consumption and respiratory energy ratio was quantified, daily maxima and minima were measured, and latency from dark cycle onset to peak was measured. Using C#, I generated a parsing software that could combine data from experiments, recalibrate time to the day-night cycle used, and pull out these parameters for future research.

Prior to the outbreak of SARS CoV-2, enveloped RNA viruses had been successfully detected in municipal wastewater. Since SARS CoV-2 emergence, researchers have further validated this technique. By March 2020, research suggested viral concentration via wastewater sampling was predictive, by 3-5 days of a subsequent rise in COVID-19 infections. Thus, measuring municipal wastewater quickly became a useful epidemiological tool for COVID-19 management. Despite relative validation, these data provided population level trends, leaving officials little time, or location-specificity, to make use of this warning signal. To address this problem, we partnered with the City of Tacoma Environmental Services Division and Tacoma Pierce County Department of Health (TPCDH) on behalf of RAIN, a Tacoma based biotechnology non-profit, and designed a neighborhood-level rapid response plan. By analyzing health disparities data, wastewater infrastructure and active case rates we sought to illuminate sites of active transmission. To conduct this study, we sampled weekly from two wastewater treatment plants in Tacoma, WA, and five geographically disparate neighborhood sites to monitor SARS CoV-2 levels. We collected population-specific biological samples in each neighborhood for a population-specific SARS-CoV-2 analysis. Using quantitative real-time polymerase chain reaction (qRT-PCR), we successfully isolated SARS-CoV-2 at variable levels in all sample populations between April 2020 and March 2021. Taken together, our data reveal infection trends within a municipal sub-population in absence of individual testing. Additionally, by this method we collect information on the outstanding question of asymptomatic infections. Overall, these data provide health officials geographically specific information to best mobilize limited healthcare resources.

The ability to selectively and efficiently insert DNA into the genome has been a long-standing goal to potentially fix disease associated variations and add tags to proteins for biochemical assays. The advent of CRISPR-Cas9 technology has allowed for the ability to specifically target any region of the genome with a single guide RNA (sgRNA) and lead to DNA cleavage. This double stranded break by Cas9 can be repaired either through nonhomologous end joining (NHEJ), which creates insertions or deletions, or homologous recombination (HR), which requires DNA that matches the region where the break occurred. The goal of this project is to create a tool that favors HR repair over NHEJ repair after cleaving with CRISPR-Cas9 to allow for targeted gene insertion. One way to do this is to generate large amounts of multicopy single-stranded DNA (msDNA), an RNA-DNA generated by a reverse transcriptase, which transcribes RNA back into DNA. We want to determine if combining CRISPR-Cas9 technology with a reverse transcriptase to generate msDNA will produce enough homologous DNA strands to drive HR repair over NHEJ repair in vivo. To test this method, we have replaced the tyrosine hydroxylase (TH) gene, the rate limiting enzyme that produces dopamine, with green fluorescent protein (GFP). We have generated a virus containing a reverse transcriptase with a sgRNA targeted to TH and msDNA containing homology arms flanking GFP. Four weeks after co-injection of this virus with a virus containing Cas9 into the ventral tegmental area of adult mice, we analyzed brain slices to determine the loss of TH and expression of GFP. Future studies will aim to quantify gene insertion and target other genes to insert point mutations. The findings of this study may further research in the repairment of disease associated variations in genes. 

A survey in the existing literary sources says that there are three main diet-related patterns in Blue Zone communities that contribute to their longevity. After I cross-referenced the micronutrients in Blue Zone diets with the average American diet, I found that the Blue Zone diets are all enriched for some common vitamins and nutrients, specifically: retinol, thiamin, pyridoxine, ascorbic acid, vitamin E, vitamin K, magnesium, and potassium. All of these vitamins and minerals are known to affect cellular metabolism through a wide array of factors, potentially linking them to bettering health and increasing lifespan. I initialially will confirm that these compounds indeed play a vital role in extending the functionality of the biomechanical systems in C elegans. I will then further pursue whether combinations of the nutrients increase effectiveness, and which specific areas in the body they individually impact for promoting longevity. My first step in investigating the biomechanical effects of these micronutrients will be to incorporate each one individually into the C elegans’ diet. My long-term goal, once I test the wild type model, is to focus on how these micronutrients combat age related diseases. To do this I will use my results for optimal micronutrients to test the effectiveness of the specific micronutrients in the Alzheimer’s degenerative pathway. Specifically, I will use the GMC-101 transgenic worm, (genetically modified strain to exhibit the degenerating effects of Alzheimer's disease), which expresses human amyloid- beta - a protein implicated in the progression of human Alzheimer’s Disease - in the body wall muscle and becomes increasingly paralyzed with age. I will use a similar experimental set up, looking for a delayed onset of paralysis. I hope this research will be able to shed light on how micronutrients interact and affect degenerating biochemical pathways in aging C elegans; specifically, in a strain that models Alzheimer’s disease.

Caenorhabditis elegans is frequently used as a model organism for testing the effects of various compounds on longevity. A current limitation of running these experiments is the tremendous amount of work needed to collect large sample sizes of data when testing for several compounds in different genetic populations. Fortunately the Kaeberlein lab has developed the WormBot, an image capture robot used to take high resolution images of hundreds of experimental plates each containing ~30 worms. Researchers still have to rely on humans manually annotating tens of thousands of frames to extract valuable metrics for their analysis. I have developed the implementation of a neural network to automatically analyze these images so that the number of experiments and compounds that can be tested is increased exponentially. I utilize a network architecture known as Yolov3 to allow the computer to identify and track individual worms from the images. The results obtained from our new computational method extract data from the images that is equal to or even better than the human method while also requiring a fraction of the time. Using this novel platform, we are analyzing a broad spectrum of natural and synthetic compounds for their effects on longevity and health span in C. elegans.

Synapses between neurons are crucial for all of human behavior as they underlie the ability to form thoughts, make precise movements, learn skills, and form new memories. Spinules are finger like projections from one neuron that are embedded within the information-sending end (i.e., presynaptic bouton) of another neuron. While recent data suggests that spinules are abundant within synapses across the brain, we have a limited understanding of their synaptic specificity or function. It is speculated that synaptic spinules may act as anchoring mechanisms or as a novel form of neuronal communication. Here, we sought to determine how presynaptic boutons that contain spinules differ from those that do not, as a first step towards uncovering whether spinules target specific subsets of synapses in the brain. We performed extensive quantitative 3D reconstructions and analyses of 138 excitatory (i.e., releasing glutamate) presynaptic boutons within the CA1 Hippocampus (center for memory formation) of an adult rat, including 87 spinule-bearing boutons (SBBs), and 51 presynaptic boutons without spinules (Non-SBBs). We found that on average, SBB volumes were 2.6-times larger than Non-SBB volumes (p<.00001), and that the size of the synapses made by SBBs were 1.6-times larger than those made by Non-SBBs (p<.00001). Since the anatomical size of a bouton and its synapse are highly correlated with its physiological strength, these data suggest that spinules may increase the physiological strength of excitatory synaptic connections in the CA1 hippocampus, and may therefore play an important role in memory formation.

5-hydroxymethylcytosine (5hmC) is a DNA modification studied in mammalian cells and tissues that has been implicated in embryonic stem cell differentiation, neuronal development, and formation of tumors. However, much is still unknown about the regulatory function of 5hmC and its genomic localization in diverse cell types. Recently, a novel, enzyme-based method was developed for 5hmC identification, APOBEC-Coupled Epigenetic Sequencing (ACE-seq), that is highly accurate, requires low input, and does not degrade DNA as commonly used bisulfite treatment methods do. ACE-seq is especially relevant to cellular environments that are epigenetically dynamic, including brain tissue and embryonic cell cultures. My project uses ACE-seq to generate high fidelity 5hmC characterizations in the Elf1 cell line across culture conditions that mimic early human embryonic development in which dramatic changes in DNA methylation occur. ACE-seq will be used on three human embryonic stem cell (hESC) conditions that mimic the transition from before embryonic implantation, naïve hESCs (two conditions), to near the time of embryonic implantation, primed hESCs. We hope to gain insight into 5hmC localization and regulatory function during this physiologically significant developmental event occurring during early embryogenesis. A more robust understanding of 5hmC regional abundance during this transition will help us elucidate the regulatory circuitry underlying early development. Knowledge gained in this project is especially relevant to the field of personalized medicine, as thorough understanding of pluripotency transitions will be significant to future applications of stem cell based therapies and precision healthcare. 

 According to the Centers for Disease Control and Prevention, birth defects affect 1 in every 33 babies born in the United States, and severity within each condition varies widely. However, the genetic basis of isolated birth defects (iBD), that is having a single major birth defect, remains largely unknown. The research question for this study is, “What are the underlining genetic causes of different iBDs?” To answer this question, I visually screened patient data obtained from the University of Washington Center for Mendelian Genomics. I looked at the DNA sequences in families to confirm the presence of a genetic variant in the affected child and identify patterns of inheritance for children affected by glaucoma or omphalocele. Glaucoma is a condition affecting vision and omphalocele is a defect of the abdominal wall that causes organs to lay outside of the stomach. I looked at these birth defects because they are common causes of infant mortality in the first year of life. Early findings show that in our cohort, 7 out of 19 individuals with congenital glaucoma have overlapping genotypes with other disorders affecting vision. In the omphalocele cohort, there were no overlaps in their genotype with other similar disorders. These results suggest that within glaucoma, there are other possible candidate genes of interest for targeted sequencing, and that these individuals may be affected by other medical conditions relating to vision. Given these findings, I will continue to analyze data for other defects to expand our understanding of the phenotypes of iBDs. The next major defect I will study is tricuspid atresia which is a defect in the heart. This research will contribute to knowledge of the genetic basis for iBDs by narrowing down the candidate genes which can be used to identify targeted sequences for DNA sequencing.

MicroRNAs (miRNAs) are short 21-23 nucleotide (nt) nucleic acid species that have a seed-sequence complimentary to target RNAs which can then be negatively regulated via the RNA silencing pathway. miR-122 is the most highly expressed miRNA in the liver, accounting for 70% of the miRNA reads and is involved in hepatocyte differentiation and cholesterol/fatty-acid synthesis. Previous studies have indicated that total abrogation of miR-122 or competition of miR-122 by short hairpin RNAs (shRNAs) in mice caused an increased expression of the Dlk-Dio3 locus, which is highly expressed in development but downregulated in adults and ultimately leads to hepatocellular carcinoma (HCC). Furthermore, it is known that activation of the Dlk-Dio3 locus alone has been shown to lead to the development of HCC but the specifics of miR-122’s regulatory role have not been elucidated. To determine the effects of miR-122 on the Dlk-Dio3 locus, I am using human HCC cells that contain a 3-bp deletion down-stream from the miR-122 transcript. We predict that the 3-bp deletion prevents proper miR-122 transcription. By transfecting the cells with a miR-122 mimic transcript to introduce its expression, I can compare the levels of RNA expression in liver cells containing miR-122 to those without by using qPCR and small-RNA sequencing. Preliminary data indicates that cells transfected with the miR-122 mimic have decreased levels of expression of RNAs located in the Dlk-Dio3 locus such as Rtl1 and positive control targets such as Aldoa. To further confirm this relationship, I am planning on using CRISPR-mediated homology-directed repair to replace the 3-bp deletion in my cell line and endogenously re-express miR-122 in order to study its regulatory role at the Dlk-Dio3 locus.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is caused by the death of dopamine-secreting neurons in the midbrain. The onset of symptoms, including progressively worsening tremors, movement difficulty, and dementia are thought to be caused by protein aggregates called Lewy bodies, mitochondrial defects, and neuroinflammation. Mutations in the GBA gene, which codes for the enzyme glucocerebrosidase which breaks down the lipid glucosylceramide, accounts for 5-10% of all PD cases. Our lab has a GBA mutant fly model that features symptoms similar to human PD – neurodegeneration, shortened lifespan, motor deficits, and increased protein aggregation. We hypothesize that GBA mutants fail to break down glucosylceramide, a lipid that is common in membranes of many pathogens. This glucosylceramide accumulation is responsible for triggering the innate immune response system, in turn causing inflammation leading to neural death. I explore innate immune system activation in the Toll, lmd, and Jak/Stat pathways and determine if a GBA mutation causes glucosylceramide accumulation to trigger the activation causing neuron loss, and the associated phenotypes. To do this, I will use reporters to test expression of these transgenic lines in a chemical assay, observe gene activation and protein expression following manipulation of glucosylceramide production, and use RNA interference to understand the effect of specific genetic perturbations in mutants. It is extremely valuable to understand the pathway of neuroinflammation to neurodegeneration, the mechanisms behind this, and the cascading influences they may have. My novel research on the impact of innate immune response pathways streamlines our comprehension of the mechanistic influences of PD and many other neurodegenerative diseases, leading to treatment and prevention development.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 and can result in preterm birth or severe disease or death in the mother. The research objective was to quantify the SARS-CoV-2 viral load in placental tissue and evaluate whether the placenta mounts an antiviral innate immune response. Furthermore, the study seeks to understand whether the timing of a COVID-19 infection during pregnancy correlates with viral load at the time of delivery and placental pathology. I, along with the two research technicians I am working closely with, hypothesize that a SARS-CoV-2 infection modulates the placental innate immune pathway in pregnant women and result in high viral loads in the context of placental pathology. The virus may amplify or dampen the innate immune response, significantly impacting viral clearance and potentially inducing substantial injury to maternal and fetal tissue. In the study, I helped extract RNA from two tissue types in the placenta, chorionic villous tissue and chorioamniotic membranes, from pregnant women with and without COVID-19 at different trimesters. I have begun to extract RNA, synthesize complementary DNA (cDNA), and perform quantitative polymerase chain reaction (qPCR) to quantify the viral load of SARS-CoV-2 per mg of tissue. In SARS-CoV-2 positive samples, I have also begun to quantify the relative gene expression of ifnb, mxa, ifit1, and il6, which will allow my team and I to evaluate the innate immune response. Our preliminary results indicate a low, but significant frequency of SARS-CoV-2 in placental tissues with rare high viral loads associated with a significant IL-6 response. I will help analyze our data through visual graphs and statistical analysis once the data is uploaded to the lab’s database. This project will not only improve our understanding of pregnancy pathologies, but also make significant strides in what is known about SARS-CoV-2’s impact on pregnancy health.

The lifetime prevalence of PTSD is approximately 6.8% among adults in the United States, with an estimated 36.6% experiencing serious impairment. While increased reactivity to trauma stimuli, or hyperarousal, is heavily researched and well-understood, differences in the appraisal of neutral stimuli are minimally studied. Hostile Assessment Bias (HAB) is a measure of the extent to which a person views others’ actions as hostile or threatening towards them. People with higher levels of Hostile Assessment Bias may be at greater risk of decreased functionality and increased emotional distress due to their disproportionately negative reaction to neutral stimuli. Utilizing one of our existing studies which investigate the use of Prazosin, an alpha-1 adrenergic receptor antagonist, in treating subjective symptoms and functional distress in veterans diagnosed with post traumatic stress disorder (PTSD), we investigated the relationship between PTSD severity and increased Hostile Assessment Bias while a participant is not receiving treatment for PTSD, examine the role trauma type and substance abuse play in hostile cognition, and evaluate functional impairment in veterans with both PTSD and increased HAB.We also evaluated if medication (prazosin) improves HAB and if the improvement is associated with biomarkers of noradrenergic signaling. With a positive relationship established and prazosin effectively normalizing hostile assessment patterns, it could provide a new way to target functionally impairing symptoms. We expect this research to have applications in understanding and preventing police brutality, given police officers’ repeated exposure to trauma. The next steps would include participating in the design of a clinical trial based on first responders, including the police.

Sleep problems in adolescents are commonly associated with bedtime media use due to subsequent psychological and cognitive arousal. The purpose of Sleepazoid is to better understand the impact of mind-body interventions on mitigating the effects of media use on sleep in adolescents. Participants virtually attended assessment visits and played pre-selected mobile video games while we conducted arousal level measurements during varied time intervals. We conducted assessment visits at baseline and follow-up to establish arousal levels through Heart Rate Variability (HRV) and Electrodermal Activity (EDA) at rest, and during the gameplay and recovery phases. The Actiheart device measures HRV through heart rhythms and variability in time between each individual heartbeat while the Empatica E4 Wristband measures EDA through the degree to which skin conducts electricity. We monitored the participants during the various phases and tracked their activity for media-induced arousal task compliance. After the remote assessment visit, participants continued to track media use, sleep, and their arousal responses on designated study nights. The pandemic necessitated major protocol changes such as mailing the measurement devices, remotely downloading games, and conducting Microsoft Teams sessions in comparison to the original in-person design. This created barriers such as unstable internet connection and improper camera positioning which hindered our ability to obtain reliable data during assessment tracking. Despite the challenges of implementing virtual assessments, it is possible to transition a psychophysiological experimental protocol to remote administration by revising participant instructions, introducing instructional videos for visual reference, and ensuring a more robust protocol with closer collaboration with the participants. Ultimately, state arousal levels are predicted to return more quickly to baseline in the mind-body intervention group (e.g: bedtime yoga, breathing exercises) during follow-up assessments, during and after ceasing evening media use, and at bed time in comparison to the control group.

Midfacial hypoplasia (MFH) is a disorder characterized by underdevelopment of the upper jaw, nose, and cheek bones which can impede feeding and breathing. The causes of MFH are not yet fully understood, but a novel pig model suggests that the posterior nasal septal cartilage is ossified, or converted to bone, prematurely in pigs with MFH. Thus, we predict that there is an increase in the amount of ossified septum in pigs with MFH. To test the hypothesis that pigs with MFH have increased septal ossification, we measured the area of the entire septum and the fraction occupied by bone on CT scans taken from 20 pigs with MFH and 10 normal pigs ages 3-10 months using ImageJ. All measurements were standardized for size by dividing by skull length. We compared MFH and normal pigs with t-tests using excel. As predicted, the fraction of ossified septum was greater in MFH pigs (0.39 ± 0.08) than normal pigs (0.25 ± 0.06, p<0.0001). The nasal septal cartilage is thought to be the primary driver of facial growth. A decrease in septal cartilage due to increased ossification may hamper normal growth and lead to MFH. Premature ossification of the nasal septal cartilage may also be a cause of MFH in humans and this finding could be used to develop better treatments for this disorder.

Endocrine therapy is used for women with estrogen receptor-positive breast cancer (cancer cells that grow due to the hormone estrogen). The standard duration of endocrine therapy is five to ten years to prevent recurrence and improve survival. However, studies show that endocrine therapy has distressing side effects. Nearly half of patients do not adhere and more than two-thirds discontinue endocrine therapy before recommended. The purpose of this study is to evaluate if there is an association between intent-to-adhere to endocrine therapy and anticipated sleep improvement. To conduct our research, focus group interviews of 14 breast cancer survivors were recorded, transcribed, and coded for themes around sleep quality and medication adherence. We hypothesize that the potential for improved quality of sleep will increase breast cancer survivors’ intent-to-adhere to endocrine therapy. Preliminary results show that survivors taking endocrine therapy reported sleep issues such as hot flashes and difficulty falling asleep. Some participants recognized that they experienced poor sleep before they were diagnosed with breast cancer or started endocrine therapy, making it less clear how endocrine therapy affects their quality of sleep. Other survivors did not attribute their poor sleep to endocrine therapy use but reported other distressing symptoms, such as joint pain, that began when starting this medication. We anticipate that despite each survivor's experience being unique, their intent-to-adhere to endocrine therapy would improve with the potential for better quality of sleep, as well as fewer distressing symptoms. Our study will provide information on the association between intent-to-adhere to medication and sleep quality, and can serve as a platform for addressing the side effects of endocrine therapy. Long-term, this line of research will provide information to measure actual levels of medication adherence following a sleep intervention, and can potentially be utilized to improve the quality of life among breast cancer survivors.

Adolescence is a critical developmental period for many with regard to initiation of marijuana use. With increasing nationwide trends toward legalization of marijuana and known negative consequences associated with an earlier age of marijuana use onset, it is imperative to identify adolescent risk factors associated with motives for using marijuana. Previous research among adults has found that motives for marijuana use vary based on personality dimensions, with higher levels of neuroticism being significantly associated with greater coping motives. Despite these findings, little research has been done on this topic with late adolescents. The overarching aim of this study was to examine associations between personality risk factors and motives for marijuana use among late adolescents. The current study included 170 late adolescents (15-18 years old, M_age = 16.86, 50% female) recruited from Washington State with stratified sampling to enroll participants who ranged from never having used marijuana to those who report heavy, regular marijuana use. Participants completed online assessments that included the Mini-IPIP Big Five Factors of Personality Scale and the Comprehensive Marijuana Motives Questionnaire to assess personality and marijuana use motives, respectively. We examined associations between personality dimensions (extraversion, agreeableness, conscientiousness, neuroticism, imagination) and marijuana use motives (enjoyment, conformity, coping, celebration, perceptions, anxiety, risk, sleep) using correlational and regression analyses. We hypothesized several positive associations between personality and motives including between 1) neuroticism and using to cope with depression/anxiety, 2) imagination and using to alter perceptions, and 3) agreeableness and using to conform to peers. Further, we predicted that late adolescents high in neuroticism that use marijuana to cope would report more marijuana-related consequences. The results inform whether screening for personality dimensions in adolescents could help predict future motives for marijuana use and thus be beneficial in preventing negative marijuana consequences and providing early interventions for marijuana misuse.

Poor sleep is a common complaint during pregnancy that is associated with worse maternal and fetal outcomes including increased depression symptoms before and after pregnancy. Mindfulness-based interventions are effective in improving sleep, but little is known about the relationship between mindfulness and sleep during pregnancy. This cross-sectional study explores the associations among mindfulness, objective and subjective sleep parameters, and mood (depression, anxiety, and positive affect) during pregnancy. Participants are between 12 weeks and 28 weeks gestation, enrolled in my mentor’s pilot randomized controlled trial, Online Prenatal Trial in Mindfulness Sleep Management (OPTIMISM). Data include self-report questionnaires, actigraphy, and daily sleep diaries collected at baseline before receiving a sleep intervention. We hypothesize that mindfulness and sleep quality are positively correlated, whereas mindfulness and mood symptoms are negatively correlated. This study's findings would support mindfulness as a potential non-pharmacologic intervention to improve sleep during pregnancy.

The process of development in mammals is always accompanied with high amounts of brain plasticity. Similarly, the maturation of sex organs during puberty in mammals serves as the foundation for important cognitive development. Researching centers of high plasticity could lead to key discoveries for the molecular basis of behavior. The medial preoptic area (MPOA) is an example of a very active site in pubertal gene expression. Estrogen-receptor 1, or Esr1, is a gene that is essential for hormone binding. We aim to gain insights on the neural basis of behavior by analyzing Esr1’s level of control in the MPOA, and on sexual behavior. This was completed by identifying single-cell types, determining the full transcriptome of the MPOA, and then observing any changes in gene expression after selectively knocking out the Esr1 gene. We utilized scRNA sequencing to identify single-cell types. The mating behaviors of mice was also studied to pinpoint any phenotypic differences. We have found that the deletion of Esr1 leads to severe hinderances in maturation, as well as sexual behavior, due to reduced function of Cis-regulatory elements. This provides important clarity regarding the key effectors of brain development in the pubertal stages. We aim to apply this approach to uncover the transcriptional dynamics of other areas in the brain, eventually forming a full, detailed representation of the brain. This would be a massive step forward in understanding the neural foundation governing sexual behavior.

Osteoporosis is an orthopedic disease in which old bone begins to dissolve, but is not replaced by new bone. This reduces overall bone density and increases a patient’s risk for fractures. Our lab studies genes that contribute risk to osteoporosis. One human gene associated with osteoporosis is WNT16, which is also expressed in zebrafish. Previously, we mutated the wnt16 gene at four different sites to produce a range of skeletal phenotypes. These studies show that wnt16 mutant fish have skeletal defects. The aim of my project was to find how wnt16 influenced spine segmentation in mutant zebrafish. Through GNOMAD, a Genome Aggregation Database, I collected data on the types of mutations that naturally occur in human Wnt genes. Across all human Wnts, there was no strong bias for stop-gained mutations in the terminal coding exon; however, in WNT16, stop-gained mutations were underrepresented in the terminal coding exon, which was of interest. Using CRISPR-based gene editing, we used a guide RNA to mutate the terminal coding exon in zebrafish wnt16 and found a range of severe skeletal abnormalities along the spine. Segmentation of the wnt16 mutant zebrafish was disrupted compared to the controls. I determined how wnt16 influenced segmentation of the spine by quantifying centrum fusions and other morphological abnormalities in these mutant zebrafish. Our results implicate the wnt16 gene as essential for spine morphology and contributes to understanding human phenotypes of osteoporosis.

Some environments hinder microbes from gaining the nutrients they need for a chemical reaction. Species in this circumstance collaborate to produce energy by clearing toxic substrates or providing nutrients. This is mutualism, where two species benefit from each other’s fitness. Previously, Desulfovibrio vulgaris and Methanococcus maripaludis were forced to rely on mutualism for survival over time. D. vulgaris catabolizes lactate, producing hydrogen as a byproduct. Hydrogen is inhibiting at high concentrations. M. maripuladis consumes this hydrogen and uses it for energy, producing methane as a byproduct. To learn how these microbes adapted to mutualism over time, 22 communities were propagated for 5000 generations. Each species was also propagated alone in environments that were as similar as possible to the mutualistic evolution environment. The whole genomes of the entire population of D. vulgaris or M. maripaludis were sequenced for the first 1000 generations using Illumina sequencing. This was done from 8 mutualism-evolved and 8 solitary-evolved cultures. Our research goal is to identify alleles that were substituted because they had a beneficial effect on mutualism fitness instead of other generic features of the environment. We can rule out generic adaptations in the population data by comparing mutations in community versus solitary-evolved clones. Clones were collected from each population that had been stored at –80 °C by streaking them on plates. Plates were also used to test if the clones could grow on sulfate. The DNA of these clones will be sequenced to confirm the presence and determine linkage of alleles that are beneficial to mutualism. It is anticipated that the mutation frequency of catabolic proteins will increase within D. vulgaris solitary clones in comparison with community D. vulgaris clones. This experiment has greater implications for mutualistic interactions between microbes, specifically in terms of the methane greenhouse gas that microbes produce.

Loss of neurons in the retina underlie many blinding diseases, such as macular degeneration, glaucoma, and diabetic retinopathy. These retinal neurons are not replaceable. The Reh Lab has developed a strategy to achieve functional regeneration of neurons in the adult mammalian retina, but not enough regeneration to restore vision loss. One of the potential limitations to restoring vision is the inflammation that occurs during retinal injury or disease. All mammalian retinas contain microglia, which are the primary immune cells of the nervous system that respond to pathogens, injury, and disease. My goal is to determine how microglia respond to damage-induced regeneration in the retina. After damaging mice retinas with an excitotoxin (NMDA), we induced neuronal regeneration with a proneural transcription factor called Ascl1. Using immunohistochemistry and confocal fluorescence microscopy, I stained various markers that represent different inflammation states of microglia. I quantified microglia and analyzed their response to neural regeneration. Data from confocal fluorescence microscopy revealed that microglia surround newly regenerated neurons and display a variety of subtypes during regeneration. Follow up single-cell sequencing experiments confirmed that these immune cells show a molecular heterogeneity of states. This study provides a better understanding about how microglia function during retinal regeneration. Results from this study can help reveal targets for manipulation to improve regeneration of the retina. Further work can be done to analyze how these microglia behaviors impact regeneration of lost retinal neurons.

Tauopathies are a set of neurodegenerative diseases identifiable by the aggregation of insoluble intracellular deposits of the microtubule associated protein tau. Among others, this class of diseases includes frontotemporal dementia (FTD), Pick’s disease, and supranuclear palsy. Though the precise pathology of tauopathies is poorly understood, most forms are associated with hyperphosphorylated tau and impaired clearance of tau through the ubiquitin-proteasome and autophagosome pathways. We attempt to explore the degradative properties of one specific tau variant, a serine to threonine missense mutation (S356T), which is strongly associated with an early onset behavioral variant frontotemporal dementia (bvFTD). Posttranslational modification at the S356 residue in tau controls its proteasomal degradation. Therefore, we hypothesize that the S356T variant exhibits impaired tau clearance compared to wildtype tau. To test this hypothesis, we use the pharmacological inhibitor Cycloheximide that prevents protein synthesis in both human embryonic epithelial cells (HEK293) and neurons transfected with wildtype and S356T tau protein. Cycloheximide has been shown to inhibit translation by binding to the E-site of the 60S ribosomal subunit and preventing protein elongation. This drug allows us to measure protein degradation without being confounded with new protein synthesis. We expect the amount of tau, relative to total protein, to be elevated in the S356T transfected cells after application of cycloheximide. If our hypothesis is correct, we plan to explore possible mechanisms behind the aberrant degradative properties of the S356T tau variant. By exploring the mechanisms through which tau mutants contribute to neuropathology, we hope to provide molecular insight into tau proteostasis that if successful could offer a promising therapeutic target.

Leptin and ghrelin are two hormones essential to maintaining and regulating energy levels and food intake. These hormones have opposite effects on feeding behavior where leptin suppresses feeding and ghrelin potentiates it. While the roles of these two hormones have been widely researched, their relative effects on distinct neural populations are still largely undetermined. Previous electrophysiological and in vivo imaging experiments have shown that the activity of individual populations of glutamatergic hypothalamic projection neurons are differentially affected by feeding hormones. This data leads to the question of whether or not the projection populations have a bias for leptin and ghrelin receptors that could account for the difference in sensitivity. We hypothesize that Lepr and Ghsr will be expressed at different levels within the different projection populations. To study this, we injected two retrogradely trafficked viruses into the target locations, lateral habenula (Lhb) and ventral tegmental area (VTA), in Mus musculus and performed fluorescent in situ hybridization experiments in the lateral hypothalamus (LHA), dorsomedial hypothalamic nucleus (DMH), ventromedial hypothalamus (VMH), paraventricular nucleus (PVH), and arcuate nucleus (ARC) for viral based fluorophores as well as leptin and ghrelin receptors. The viral expression was imaged using fluorescence microscopy and quantified for within individual hypothalamic neurons. Analysis is currently underway to reveal any differences in fluorescence between the two projection populations and therefore, determine any disparities in hormone receptor expression. The objective of this research is to understand whether differences in expression of Lepr and Ghsr exist within LHb- and VTA-projecting glutamatergic hypothalamic neurons. This study could be indicative of how hormones regulate feeding behavior and their particular effects on the hypothalamus projection neurons. Further research concerning the downstream impacts of these opposing hormonal pathways could shed light on the neural networks that govern food and energy balance.

Leigh Syndrome (LS) is the most common pediatric mitochondrial disease, and it is associated with loss-of function mutations in genes that encode for proteins in Complex I of the electron transport chain. Mutations in a gene called NADH dehydrogenase (ubiquinone) iron sulfur protein 4 (NDUFS4) is linked with LS which results in many neurological symptoms and neurodegenerative biomarkers in afflicted patients. Prior studies have discovered that many neurodegenerative diseases are characterized by disturbances in circadian function, which can impact disease symptoms and worsen quality of life. However, it remains unknown if disruptions of circadian function are a characteristic phenotype of all mitochondrial diseases and Leigh Syndrome in particular. In this study, we investigated the integrity of the circadian rhythm in conditional knockout (KO)-models of LS. KO-models carried the mutation in either excitatory (glutamatergic) or inhibitory (GABAergic) neurons. We generated mice with Ndufs4 KO restricted to glutamatergic or GABAergic neurons using LoxP Cre technology. To examine circadian rhythm patterns, we placed each mouse in an individual cage with a running wheel and infrared (IR) sensor. Mice were maintained on a 12:12 hour light-dark schedule where the light period began at 7:00 AM. Mouse wheel activity and home cage locomotor activity were recorded and subsequently analyzed offline using ClockLab Analysis. Our initial results showed that Ndufs4 KO in excitatory neurons leads to severe disruption of circadian rhythms in which locomotive activity was not synchronized with a light-dark cycle, whereas Ndufs4 KO in inhibitory neurons had no detectable effect on circadian rhythm. These results reveal that disruptions in circadian function are present in Ndufs4-related LS, particularly due to excitatory neurons. A better understanding of circadian rhythm disruptions in LS can lead to further research on a molecular level to discover underlying characteristics of LS and become an identifier for the progression of LS.

Anxiety, a heightened state of arousal without the presence of an immediate threat, can be incapacitating once it reaches a disease state. Located in the extended amygdala, the Bed Nucleus of the Stria Terminalis (BNST) has been implicated in sustained fear states and other anxiety-related conditions. BNST neurons have been shown to be diverse, co-releasing neuropeptides and other neurotransmitters, but the quantification and localization of these neuromodulators is not clear. In order to identify the co-expression of neuropeptides I employed in-situ hybridization in the mouse BNST, where RNA sequences specific to relevant peptides are recognized with a fluorescent probe. Then, I created pipelines that identify and group amplified peptide signals to cells, quantifying intensity and presence, and collect them by co-expression and subregions within the BNST. I hypothesize that co-expression of these peptides is regionally biased. Finally, identifying differences in cell-type distribution has implications for anxiety-behavior and may provide insight towards treatment of anxiety conditions.

Everyday environmental exposure can lead to the cell’s DNA to undergo double-strand breaks (DSB). If left unrepaired, these DSB are toxic to the cell’s survivability, since it can lead to the breakdown of the genetic code that is essential for the production of proteins vital to cellular life. As such, organisms have developed mechanisms to account for these common DSB situations. These mending processes are evolved to strongly combat these breaks, as failures or mistakes in the process can result in error-stricken genetic information, resulting in faulty proteins. Not only that, DSB and its repair are observed to be important to the contribution of genetic diversity, as it allows for chromosomal crossovers. This proves that the DNA repairing mechanisms to be a complex yet significantly important process for organismal life. In Escherichia coli, DSB repair processes are carried out by a trimeric protein complex, RecBCD. The mechanism behind RecBCD is not completely understood. Previous studies in the Smith lab have proposed a working model of the protein. Here, we combine both computational prediction and genetic assay to further understand the specifics of its structure-function relationship. Past experiments have identified a structural change that affects the protein’s function. Then, using a protein-protein docking algorithm, we have identified the possible conformation and interacting amino acid docking pair accounting for it. We then follow up with site-directed mutation of these pairs to test the validity of our prediction to elucidate the exact model. These experiments will allow us to further understand the conformational effects on the complex’s function. Proper understanding of RecBCD will enable us to generate new drugs to target bacterial infections whilst also reinforcing our understanding of DSB repair.

Engineered solid binding peptides can be used as molecular tools for a variety of bio/nanotechnology applications, especially in interfacing biology with solid-state devices at bio/nano soft interfaces. The control of surface organization, and therefore peptide-solid interactions, is critical and involves surface phenomena such as binding, surface diffusion, and self-organization on atomically flat solids. Each of these phenomena requires the knowledge of peptide’s folding patterns which are, however, difficult to study both experimentally and computationally. Molecular dynamics, MD, has been used to computationally model peptide/solid interactions, but without information regarding the energy landscape of peptide conformations the challenge of predictive design remains. While several methods exist for finding the energy landscapes of single peptide systems, currently no approach handles multi-peptide/surface systems. Here we use Time-Varying Autoregression with Low Rank Tensors, TVART, to efficiently explore the energy landscapes of such systems, aiming to find accurate linear approximations for predictive design of peptides at bio/nano interfaces. Using TVART, with each slice representing a discrete time window, allows for temporal smoothness and high predictive accuracy. It is anticipated that some descriptions of conformation will be better suited to describe peptide conformation energy landscapes than others; based on this premise, we examined interatomic distances/adjacencies and peptide backbone torsion angles as descriptions of peptide conformation. Through such analyses, it is becoming possible to describe how peptide conformations in multi-peptide/surface systems evolve through the energy landscape and settle into energy minima (stable conformations). These conformations can then be corroborated with experimental validation of peptide self-organization on the surface using scanning probe microscopy techniques with sub-A resolutions. The combination of computational modeling and high-resolution experiments is expected to aid predictive design platforms for future applications in biosensors, bioelectronics, and logic devices.

SARS-CoV-2 spreads across the globe, infecting more than 128 million people and claiming over 2.7 million lives with an absence of definitive treatment up to date. Therefore, there is an immediate need to develop treatments fighting against the COVID-19 global pandemic. The goal of my project is to generate and assess an innovative treatment for the SARS-COV-2 virus infection. Our treatment formulates computationally designed proteins, and we want to evaluate its therapeutic effects using human induced pluripotent stem cell (h-iPSC) derived cell lines and organoids. The designed protein is a combinatorial cage (mosaic cage) containing spike binders previously shown to significantly inhibit SARS-CoV-2 viral infection and F-domains that were shown to activate the Tie2 pathway. The Tie2 pathway is a key regulator of vascular stability, where active Tie2 can strengthen cell-cell junctions and enhances endothelial cell survival, thus enhancing blood vessel stability. We hypothesize that the designed protein would neutralize the spike protein to block viral entry and activate the Tie2 pathway to alleviate sepsis in COVID-19 infected patients. We will test spike-binding activity and determine the activation level of the Tie2 pathway of this mosaic cage in iPSC-derived spike-overexpressing endothelial cells. We expect to measure a strong spike-binding affinity of designed proteins and strong downstream pathway signals pAKT, pERK, pFAK in designed protein-treated iPSC-derived endothelial cells. We also plan to test the mosaic cage’s activities using Kidney Organoids. If our hypothesis is correct, we will apply the findings clinically for their potential intranasal administration as a COVID-19 therapeutic.

Interactive add-ons to computers offer the potential to enhance user experience and productivity, but these peripherals are often expensive and uncustomizable for normal users, designers, and differently abled individuals. AcousTickBoard uses cardboard, a ubiquitous household material due to widespread online shopping deliveries, to address accessibility in human-computer interaction (HCI) through tangible widgets that are both cheap and easily reconfigurable. First, we created the design of a pressable button made of cardboard that emits a “tick” sound when released. Then, we explored different ways of modifying the cardboard buttons in order to produce unique enough acoustic signatures to be independently recognized such that multiple buttons could be used at the same time. The modifications made to the buttons generally involved adding or subtracting material in order to manipulate the “tick”’s frequency and amplitude. Next, we developed a machine learning (ML) pipeline to recognize these different buttons through the computer’s microphone, with several modifications on the buttons producing detectably different audio signatures with an average of 90% accuracy per button. Finally, we conducted a user study in which participants replicated a subset of our button designs and provided both quantitative feedback in terms of how well their button worked as well as qualitative feedback in the form of survey questions. By understanding which modifications to the cardboard buttons yield the most accurate detection results, we can create and use the fabricated buttons as peripheral computer widgets. These low-cost widgets are easy to create and customize for educational purposes, temporary computer setups, and people with different abilities who need regular adjustments to their digital setups.

Homomorphic encryption is a type of encryption which allows computations to be performed on encrypted data, without the need for the data to be decrypted first. Recent works have shown that homomorphic encryption can be used to train encrypted machine learning models on untrusted hardware. Federated learning enables distributed training of machine learning models on remote devices with their own private datasets. Existing federated learning techniques focus on protecting the privacy of the remote data, but not on protecting the content of the model being trained. Homomorphic encryption can be used with federated learning to protect the model. Homomorphic encryption is very computationally expensive, however, it has been shown that GPU (Graphics Processing Unit) acceleration can be used to decrease its required computation time. Modern GPUs, including those in mobile devices, can be used for general purpose computing. GPUs are well suited to data-parallel tasks in which one operation is applied to many items. Many of the computations involved in homomorphic encryption are well suited to a data-parallel approach. This research investigates the use and implementation of GPU accelerated homomorphic encryption on mobile devices and examines the potential for its use in federated learning tasks. There are many types of homomorphic encryption, some of which are better than others for certain types of computation. For instance, HEAAN (Homomorphic Encryption for Arithmetic of Approximate Numbers), also known as CKKS (the initials of the authors of HEAAN), is well suited to computations involving vectorized fixed-point values. We implement and benchmark HEAAN for mobile GPUs. We anticipate that the practically achievable model depth will be very limited even with GPU acceleration, but that it will be significantly better than CPU implementations. We also anticipate that memory requirements will be a significant limiting factor for practical model depth.

Deep embedded clustering (DEC) utilizes deep neural networks (DNN) in order to learn feature representations using an autoencoder which is optimized for clustering. This is done by integrating a clustering loss using Kullback-Leiber divergence (KL divergence). Autoencoder models have been successfully applied to many types of data in order to enable unsupervised representation learning. Recurrent neural networks and long-short term memory (LSTM) networks have been utilized in learning representations of audio data. In many cases, convolutional autoencoder algorithms (CAE) have been used in processing audio data, in order to extract their feature representations. However, for the purpose of clustering similar learned embedded features from audio data, there has not yet been an integration of DEC in the LSTM autoencoders. This research project focuses on implementing DEC for audio signals. For this purpose, we have integrated a clustering loss using KL divergence into a LSTM autoencoder. Mel Spectrograms of the audio data are then extracted. This time-series data is fed into the network. We have evaluated our model performance using enormous data sets of audio signals collected from deep and shallow water hydrophones. With the decrease of hardware costs, stationary hydrophones are increasingly deployed in the marine environment to record animal vocalizations amidst ocean noise over an extended period of time. Bioacoustic data collected in this way is an important and practical source to study vocally active marine species and can make an important contribution to ecosystem monitoring. However, a main challenge of this data is the lack of annotation which many supervised neural network models rely on to learn to distinguish between noise and marine animal vocalizations. In contrast to the previous works done in this field, our approach is designed for unsupervised representation learning, allowing us to use a large volume of unlabeled data.

With the rise of social media and video sharing platforms, many people are turning to sources like Youtube as their main source of information. However, these platforms have become easy targets for misinformation campaigns. Credibility is hard to ascertain on a video sharing platform like Youtube come from the wide base of content creators. On a typical Google search for a topic, the leading sources are usually mainstream-media companies. However, on Youtube, it is significantly easier for an individual person, or channel, to overtake mainstream media and become popular. There is also no standard way for creators to display credibility factors or cite their sources on video-sharing platforms. This leads to creators using a workaround to cite sources or simply foregoing citations all together. The implications of this for viewers is that they are unable to quickly identify the credibility of the source without cross-referencing other places on the web, or lateral reading. Currently, the only standardized information displayed about a channel is its display name, profile picture, subscriber count, and occasionally a verification indicator. The verification indicator, in particular, can be misleading as it only indicates whether a channel is who they claim to be. This project is researching and developing citations for Youtube videos. Video citations will allow creators to display credibility within individual videos and allow viewers to conduct lateral reading with ease. Citations specific to this format could also take on advantages of social media platforms, specifically user ratings and collaborative creation of citations. There are many possibilities in how citations can be introduced that we will explore through this project, each with pros and cons. For instance, sharing and creating credible videos could contribute to the pre-existing communities currently on Youtube through crowd-sourcing citations.

Misinformation and disinformation online — and on social media in particular — have become a topic of widespread concern. Recently, Facebook and Social Science One released a large, unique, privacy-preserving dataset to researchers that contains data on user interactions with URLs shared on Facebook, including how users interact with posts and demographic data from those users. We analyzed this data through the lens of U.S. mis/disinformation and cross-referenced three lists of domains: Third-Party Fact Checked URLs marked within the dataset, 2016 Election Fake News sites from Grinberg et al. (2019), and Broad Misinformation domains from Zeng et al. (2020). We calculated average interaction by user demographic group for these URL subsets, finding distributions for views, clicks, and shares among other metrics (taking into account the differential privacy noise added to the data). Furthermore, we compared interaction metrics between the three mis/disinformation subsets and baseline U.S. URLs, finding that posts containing mis/disinformation URLs draw significant user engagement. We also find that older and more politically conservative U.S. users are more likely to be exposed to (and ultimately re-share) potential mis/disinformation, but that those users who are exposed are roughly equally likely to click. We discuss the implications of our findings for platform interventions and further study towards reducing the spread of mis/disinformation on social media.

The diet of the common raven is generally known, but no study has yet looked at the changes in foraging habits over the course of a year. We set out to answer this question to get a better understanding of raven's seasonal reliance on anthropogenic resources. In addition to overall foraging trends, we also studied how individual identity as well as age, sex, and territorial status affected foraging locations. To monitor foraging, we trapped and attached GSM tags to ravens within the Greater Yellowstone Ecosystem. These tags allowed us to gather location data from the birds as often as every 30 minutes throughout the day across a full year. I identified foraging points as high-density groupings where at least one bird spent one consecutive hour during the day. I determined the land use/cover associated with each point by overlaying them on a satellite image provided by Google Earth. We found a significant shift in the use of anthropogenic and natural resources across seasons. The percentage of foraging points at anthropogenic resources decreases from 88.3% to 21.9% between fall/winter and spring/summer with the most used resource being gutpiles generated from hunting (22.5% in fall/winter). This information, as well as the results from our continued data gathering, will provide wildlife managers with the information required to most efficiently limit large raven populations and mitigate their effects as predators on sensitive species. Currently, ravens are often controlled through lethal methods. Our study informs managers on important food sources that could be managed to reduce the population size instead of directly removing ravens.

Communal roosting, during which two or more individuals of a species gather for the night, is exhibited by a wide range of taxa, including birds. There are both costs and benefits to communal roosting, making it an important behavior to study when investigating the natural history of a species. Many birds in the family Corvidae form communal roosts, including the common raven (Corvus corax). While many short-term, localized studies of raven roosts have been completed, this study is one of the first to use new generation GSM tracking devices, which utilize cell networks to upload location data, to intensively study raven roosts across a large spatio-temporal scale. Using this technology, I am able to identify individual ravens’ roosts by tracking daily movements to and from sleeping sites during winter months. GPS datapoints, time of sunset/sunrise, satellite imagery, and opportunistic observations are used to determine the location of roosts each day. This study of ravens within the Greater Yellowstone Ecosystem aims to answer questions about whether there are demographic differences in roost site fidelity and roost substrate use. I found both territorial and vagrant ravens use a wide variety of roost locations and roost substrate types, including trees, cliffs, buildings, and powerline towers. Native raven populations are dramatically increasing across the western United States, causing human-wildlife conflict and negatively impacting sensitive wildlife species. Further investigation of raven spatial distributions in this ongoing study will add to our knowledge of raven natural history and aid wildlife managers in making effective conservation decisions.

Mycoremediation is a widely researched method using mycelia to clean bacterially contaminated water bodies, such as the Bothell Wetlands, which has been contaminated by crow roosts. This research aims to analyze and quantify the remediative properties of the fungus Stropharia rugosoannulata on the bacteria Escherichia coli, Salmonella enteritidis, Campylobacter jejuni and Klebsiella pneumoniae. Additionally, the remediation of the antibiotic resistance genes (ARG), Tet[A], Tet[B], Tet[M], StrA, StrB, bla_CMY and bla_CTX genes, was examined. One pound rye-seed bags were inoculated with liquid Stropharia. After three weeks, 35g of seeds were used to inoculate 60g of sterile wood chips in four 250mL bottles, for an incubation period of 3 weeks. Including a control bottle containing 60g of wood chips, we added 150mL of wetland water, spiked with 3500 CFU/100mL of the aforementioned bacteria, to each of the 5 bottles that were shaken via rocker. The following water retention times were chosen based on past experiments: 5min, 1hr, 5hrs, and 24hrs. When performing membrane filtration we used duplicate volume of 50ml for both genotypic analysis (qPCR), and for colony counting on selective plates. We analyzed the remediation using colony forming units and quantified using gene copy numbers (qPCR). The remediation of the following ARG was the highest after 1 hour of retention time: StrB (88.6%), Tet[M] (83.5%), and Sul-1 (23.2%), while bla_CMY (100 %) and StrA (73.8%) had the greatest remediation after 24 hours. Results showed no remediation for Tet[A] and Tet[B] for any retention time. The remediation of Campylobacter (83.6%), Salmonella (97.6%) and Klebsiella (93.23%) was the highest after 1hr of retention time, while E.coli (27.61%) showed a greater remediation after 24hrs. The exponential increase in anthropogenic activities promoting bacterial contamination of the ecosystem and the spread of antibiotic resistant bacteria, highlights the urgency to find ways to mitigate them.

It is known that plant-pollinator relationships are central to the proper functioning of agricultural and ecological systems. Of the many navigation pathways pollinators use, floral scent signaling for insects is the most complex yet also the most at-risk from atmospheric human activity. Oenothera pallida, a primrose, interacts with the hawkmoth pollinators Hyles lineata and Manduca sexta, via this scent pathway. Because of their reactivity with floral scent, human-released ozone and NO2 (NOx) are the main perpetrators of scent degradation. To understand the impact of scent degradation on moth responses, I recorded changes of antennal and behavioral responses of these moths to unaltered versus degraded scent, expecting a poorer response to the degraded scent. Moth antennae act as the site of odor reception, bearing sensory hairs that detect odors, allowing the moths to navigate to scent sources. I conducted electroantennographic experiments (EAG) to record the electric signal from the insect antennae in response to each scent blend, with the degraded scent representing the impact of NOx interactions. Following the EAG, I then conducted wind tunnel behavioral experiments to investigate the impact of odor degradation on behavior, and to understand the relationship between antennal and behavioral responses in these moths. I expect that the EAG experiments will have a lower antennal response to NOx degraded scents in comparison to the normal, unaltered scent blend. Likewise, the moths might have reduced behavioral responsiveness to the degraded scent, linking the chemical biology of the scent interaction to the feeding and pollination behavior. This work has broader implications regarding the importance of plant-pollinator relationships, especially when considering environmental and agricultural health as well as the issue of food security in our changing climate.

Recent legalization of cannabis in various states has sparked research on the impact of cannabis use on postnatal outcomes. While previous research has yielded contradictory findings, some studies suggest increased cannabis use is directly associated with increased depression and psychological distress.We assert that prenatal mental health should be an essential consideration in the discourse surrounding prenatal cannabis use, as issues such as untreated maternal depression are risk factors for adverse postnatal outcomes like low birth weight and preterm delivery.However, few studies have considered the relationship between prenatal maternal mental health and cannabis use. Here we question: what is the association between cannabis use and psychological distress in pregnant individuals? For our sample population, we recruited pregnant individuals in the greater Seattle area. 12 individuals reported using cannabis (CB) at least 3 to 5 times a week throughout their first trimester of pregnancy, and 22 were non-cannabis users (n-CB). In early pregnancy, we administered the Brief Symptom Inventory (BSI), a self-reported measure, to evaluate psychological distress levels. We tracked cannabis use with weekly surveys from time of enrollment to birth, evaluating reasons for use and amount consumed, among other variables. We hypothesize that prenatal cannabis use will be associated with elevated BSI T-scores compared to the control group; within the prenatal cannabis use population, we expect cannabis use for mental health reasons to be associated with elevated BSI T-scores. More research is needed on possible causal versus correlational associations between cannabis use and psychological distress, and the role of psychosocial distress as a possible confounder in previous prenatal cannabis use and infant development studies.

Pseudomonas aeruginosa, an opportunistic pathogen that commonly infects cystic fibrosis patients, uses quorum sensing (QS), a form of cell-cell communication, to regulate the expression of virulence factors and public goods based on population density. P. aeruginosa QS consists in part of N-acyl homoserine lactone signal molecules that activate two separate regulatory proteins, LasR and RhlR, which in turn activates the transcription of other target genes in their respective regulons. The las and rhl regulons are hierarchical in lab strains, with LasR activating the transcription of rhlR; however, many pathogenic variants carry nonfunctional alleles of lasR and rely on rhlR as the dominant QS regulator. Two anti-activator proteins, QteE and QslA, restrict the expression of these two QS regulons; however, it is not clear how P. aeruginosa anti-activators function in many pathogenic strains. Identifying how anti-activators regulate QS in pathogenic variants could be crucial in developing therapies that do not rely on antibiotics. To investigate how QteE and QslA modulate QS, we overexpressed each anti-activator in P. aeruginosa and used transcriptional reporters to monitor the activity of rhlA, a RhlR regulated gene. Expression of both genes is reduced significantly in strains with over-expressed anti-activators. However, in a pathogenic variant, only over-expressing qteE delayed QS induction while over-expressing qslA had no effect. These results indicate that QteE can modulate QS by affecting LasR and RhlR levels, while QslA only modulates LasR levels. These experiments lay the foundation for therapeutic strategies centered on inhibiting QS rather than relying on conventional antibiotics.

We have established a head and neck squamous cell carcinoma (HNSCC) cell line resource to facilitate translational research on HNSCC in individuals with Fanconi Anemia (FA). FA is a rare genetic disorder characterized by bone marrow failure and predisposition to leukemia and solid tumors. FA individuals have an extraordinarily high lifetime risk of HNSCC compared to the general population and the pathogenesis of these cancers is not understood. Apart from surgery, effective treatment of these cancers is limited by patient hypersensitivity to standard-of-care therapies that include ionizing radiation and DNA cross-linking drugs. Insight into the mechanistic origins of these cancers and the identification of less toxic, more effective therapies are necessary to improve survival and quality of life. The FA Cancer Cell Line Resource was developed to provide well-characterized, experimentally tractable pre-clinical models to investigate the origins, pathogenesis, treatment and prevention of FA HNSCC. Isogenic cell line pairs or trios included in this Resource were generated from FA patient-derived or sporadic HNSCC cells using CRISPR/Cas9 technology. The biochemical and molecular characteristics of these models were confirmed to verify the presence or absence of FA-associated phenotypes following gene edits. This Resource addresses the critical need for well-characterized and tractable disease models that are FA patient-derived, and versatile enough to permit both in vitro and in vivo analyses. These cell lines are now available at no cost to foster research through the Fanconi Anemia Research Fund-sponsored “Fanconi Anemia Research Materials” repository at Oregon Health and Sciences University (https://apps.ohsu.edu/research/fanconi-anemia/celllines.cfm).

Among the estimated 1.5 million adults and children living with HIV in Kenya, only 68% had suppressed viral loads in 2019. Improving adherence to antiretroviral therapy (ART) and minimizing ART failure is crucial in ensuring viral suppression. New types of viral load (VL) and drug resistance mutation (DRM) testing through point-of-care (POC) assays are potential solutions to optimize viral suppression rates. POC assays have faster turnaround times, can facilitate rapid clinical decision making, and are cost-effective, all of which can facilitate better patient outcomes. In the Opt4Kids and Opt4Mamas studies, children and pregnant/postpartum women either received the intervention, consisting of POC VL testing every three months with targeted DRM testing, or standard-of-care (SOC) testing according to national Kenyan guidelines. As undergraduate researchers, we are responsible for coding and analyzing 68 in-depth interviews collected from key informants and participants, including adolescents, children’s caregivers, and pregnant/postpartum women, to better understand how POC VL and targeted DRM testing influences viral suppression and may be utilized for use nation-wide. We used the socioecological model to identify the individual, interpersonal, organizational, societal/cultural, and structural/policy factors that influence viral suppression in children and pregnant/postpartum women. Through inductive coding and thematic analysis, we discovered six major domains of themes of interest amongst these populations: 1) HIV health literacy, 2) HIV testing and treatment experience, 3) differences between SOC VL testing and POC VL testing, and 4) DRM testing experience, 5) future improvements, and 6) impact of the COVID-19 pandemic on treatment and testing. Our findings will provide insight into ways of optimizing existing protocols and will determine whether our intervention is an improved alternative to current national standards. Ultimately, our research will shape national policies regarding HIV treatment and directly address ways to optimize HIV viral suppression.

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with a recurrence risk of ~40%; however, prognosis for low-risk, stage I disease is excellent with primary surgical management. The role of post-operative radiation therapy (PORT) is controversial as it can cause significant and acute long-term side effects. Here, we assess the efficacy of PORT on local recurrence (LR) rates in patients with pathological stage I MCC with primary tumors on the head/neck (HN) vs. non-head and neck (Non-HN) sites. One hundred forty-seven MCC patients treated from 2006-2020 were identified from an IRB-approved prospective registry who had ‘low-risk’ disease: pathological T1 primary tumor resected with negative margins, negative pathologic node status, and no immunosuppression. LR was defined as tumor recurrence within 2 cm of the primary surgical bed. I led compilation of the cohort, and contributed to discussion of results, and development of the figures and manuscript. Seventy-nine patients received PORT (30 HN, 49 Non-HN), and 68 patients were treated with surgery alone (30 HN, 38 Non-HN). Addition of PORT was associated with a decreased risk of LR across the entire cohort (5-year rate: 9.5% vs. 0%, p=0.004), with 6 LRs in the surgery alone group. The addition of PORT significantly reduced LR rates among HN patients (21% vs. 0%, p=0.034). Conversely, no LRs were observed in Non-HN patients. No significant MCC-specific survival differences were observed. For low-risk MCC of the extremities and trunk, excellent outcomes were achieved with surgery alone. However, HN MCC was a risk factor for LR that was significantly reduced with PORT. Overall, this study demonstrates the importance of primary tumor site location for prognosis and treatment of MCC to determine patients that would benefit from PORT and those that can be spared the toxic side effects of radiotherapy.

While Congenital Heart Defects (CHDs) are the most common birth defect in the US, only 20-30% of the genes that contribute to the development of CHDs have been identified. The purpose of this research is to identify the unknown genetic causes of human CHDs through a combination of Protein-Protein Interaction (PPI) network analysis and genetic manipulation of zebrafish using CRISPR. Using the ExAC human genetic database, our lab has identified over 200 new "Candidate" genes for CHDs. For this project, we sought to answer two main questions: (1) Can PPI networks serve to identify new gene interactions potentially involved in the development of CHDs? (2) Can we demonstrate functions and interactions of these new genes using mutant zebrafish embryos? To answer the first question, we utilized STRING—an online database of known and predicted protein-protein interactions—to create PPI networks that predict interactions between our “Candidate” and “Known” genes involved in human CHDs. Through PPI network analysis, we identified the interaction of five proteasome factors (POMP, PSMA6, PSMA7, PSMD3, and PSMD6). The proteasome system has been characterized to be involved in human cardiac disease, but the specific roles of these factors in heart development have not yet been determined. To address our second question, we used CRISPR-Cas9 zebrafish genome editing to preliminarily identify functions of the proteosome factors POMP, PSMA6 and PSMD6 in heart development. We then used CRISPR to create a new POMP mutant zebrafish strain. Our genotypic and phenotypic analyses confirm a critical role for POMP in heart development. This is crucial, as it demonstrates that this cluster of proteasome factors could potentially be identified as a novel grouping of genes that are related to human CHDs. Our results promise to further our understanding of the genetic causes of human CHDs.

Copy number variants (CNVs) are typically a result of chromosomal duplications and deletions, making them a well-known form of genetic diversity and associated with several human disorders. Little is known about CNVs within humans and insight into CNV mechanisms would help scientists better understand, and potentially treat, many genome-based diseases. A particular form of CNV within humans is the inverted triplication of a gene without any chromosomal deletions. A similar phenomenon is observed at the SUL1 gene in Saccharomyces cerevisiae yeast cells, providing a model for studying such CNVs. The Brewer lab proposed a replication error mechanism responsible for this specific amplification described as Origin Dependent Inverted Replication Amplification (ODIRA). What impacts the initiation of this mechanism is unknown, but the proximity of SUL1 to the telomere raises the possibility that properties of the telomere may stimulate replication errors responsible for the triplication. I conducted a literature review analyzing 11 articles discussing various CNV mechanisms and telomeric influence on replication to establish their relationship. Through my review, I found a likely method to test whether the telomere does affect ODIRA. I propose utilizing a CRISPR-Cas9 based method to first circularize and eliminate the telomeres of the chromosome. Subsequently, the chromosome would be linearized at a location distant from the original telomere sites, effectively moving the entire SUL1 site away from potential telomeric influence. This research design allows for a comparison of SUL1 amplification events within the original and the restructured chromosomes and would reveal whether the telomeric region influences inverted SUL1 amplification formation. An observed reduction in rates of SUL1 amplification events with the reconstructed chromosomes would indicate telomeric influence on the amplification mechanism prompting further examinations within that genomic region. Attaining a greater understanding of this CNV mechanism yields information for future implications in genetic disease research.

A plethora of research documents the negative impacts of incarceration and poor nutrition on individual, family, and population health, but little research explores how these seemingly disparate issues may intertwine. Based on the knowledge that children learn to eat and develop lifelong nutritional patterns as part of their families, and that incarceration of a parent breaks family units apart, I investigate the question: How does a parent’s incarceration shape the trajectory of a child’s developmental food environment, including meal structure, staple foods, and changes in routine? Based upon current research on parental incarceration and childhood eating patterns and health outcomes, I predict that parental incarceration will be associated with negative changes in a child’s food environment by disrupting previously held family eating routines and by creating additional barriers to healthfully feeding a family. I conduct semi-structured interviews with formerly incarcerated parents or co-parents whose partners are currently imprisoned. Interviews were coded in NVivo using two rounds. First, I open coded to identify preliminary themes. Then I coded a second-round to refine the themes found and identify patterns between themes. Preliminary coding of the interviews reveals evidence of family eating pattern disruption and constrained choice that limits food options as a result of incarceration. This implies that parental incarceration may be associated with challenges and barriers to feeding a family and disruption of healthy meal patterns for children. It illuminates how large-scale social phenomena such as mass incarceration can patten eating behaviors and habits, therefore directly impacting health outcomes. The conclusion situates this study in the context of Socially Engaged Nutritional Sciences and explores the implications of these findings for children of incarcerated parents and for racial and socioeconomic inequalities in nutrition and beyond.

In the process of uniting Europe, European integration has inadvertently strengthened secessionist movements that seek to fracture the very states it has brought together. Through the creation of the European Single Market, the common travel area, the single currency, and the establishment of the four freedoms of movement for goods, capital, services, and people, many of the benefits a region with a potential desire for independence gains from remaining a part of its host state are transferred to the European level. Currently, nearly all academic analysis of European integration and its impact on secessionist movements has been published around the time of the 2014 Scottish independence referendum and before the UK’s 2016 European Union membership referendum. The current academic consensus argues that European integration has harmed secessionist movements more than it has helped, but in the years since the UKs decision to leave the EU, this argument has become outdated and no longer holds the strength it once did. I conducted a review and analysis of current academic literature on the topic along with statements from current and former European politicians and separatist political parties to explore and compare the cases of secessionist movements in Scotland, Catalonia, and Flanders. My analysis incorporates the events since the UK’s Brexit referendum currently left out of academic literature published to date. I hypothesize that an updated review of European integration’s effect on secessionist movements will show that integration has helped secessionist movements and their arguments. While these cases were chosen for their strength and their diversity in circumstances, these independence movements are united in being transformed from pipedreams to real possibilities thanks to European integration.

Network goodness of fit (GOF), which deals with determining if an observed network could have been drawn from a particular graph model, is a common method to identify network structures and a prerequisite of further network analysis. In this talk, we will present a method that constructs a test statistics using advances in Random Matrix Theory, which traces the limiting behavior of the leading eigenvalue of the observed graph's adjacency matrix to perform network GOF. We show that under many network models - such as the Degree Corrected Stochastic Block model (DCSBM), the Beta model, and the Aggregated Relational Data (ARD) - our method performs well in identifying the correct model with high precision and low computation time. We further extend our method to the popular Latent Space model, and develop an algorithm that fast-and-accurately predicts the underlying dimension of the true model. With precision grows with network sizes, our algorithm enables better estimations for large-scale networks with minimal computational cost.

Systems of non-linear differential equations are often difficult to analytically solve, control, and analyze, whereas systems of linear differential equations are relatively straightforward to solve, control, and analyze because we possess tools to study systems of linear differential equations. Koopman analysis allows us to transform a system of non-linear differential equations into a linear system. The caveat is that sometimes, the resulting linear system is infinite-dimensional, meaning that the mapping between the original space and the Koopman space is infinite-dimensional. This poses a computational challenge because infinite dimensional vector spaces are difficult to computationally work with. Koopman analysis has traditionally been done on one fixed point (equilibrium). Previously, work has been done to study specific examples of systems that have closure i.e. systems with a finite-dimensional Koopman operator. In this project, we looked at how to apply Koopman operators to systems with multiple fixed points. We found effective eigenfunctions that linearize low-dimensional non-linear dynamical systems analytically if possible, computationally otherwise. Under the assumption that the right-hand side of the differential equations are polynomials, we identified appropriate eigenfunctions that linearize the Koopman space with a possible invertible mapping. Previously, we have been able to find a closed-form solution that generates eigenfunctions for one-dimensional systems that have a polynomial form. However, in practice, the resulting integral equation can be difficult to computationally solve with current methods, and edge cases such as singularities and asymptotes are not well understood. Using implicit SINDy (an algorithm that approximates dynamical systems given data), we attempted to find polynomial decompositions that allowed us to describe the eigenfunctions with rational polynomial functions. This work is significant because better understanding dynamical systems allows us to better understand dynamic fields such as natural disaster detection, the firing of neurons, and the spread of pandemics.

Hormones heavily influence feeding behavior and body weight. Estrogen depletion in post-menopause women leads to high risk of obesity, while estrogen replacement therapy suppresses body weight. While previous studies have started to reveal the neural substrate of feeding behavior control, how they are modulated by estrogen action is not well understood. The paraventricular hypothalamus (PVH) is known to control satiety in animals. Thus, in this study, we hypothesized that neurons responding to estrogen in the PVH is related to hormone-dependent modulation of feeding. To evaluate the causal relationship between the activity of PVH estrogen responsive cells and feeding behavior, we virally targeted channel rhodopsin (ChR2) to PVH expressing estrogen receptor beta (Esr2) using Esr2-Cre transgenic mice and Cre-dependent virus. The virus delivers ChR2 mRNA nonspecifically to cells in the PVH. In Esr2-Cre mice, only cells expressing Esr2 will also express the Cre recombinase enzyme to allow successful expression of the virally delivered ChR2. ChR2 is activated by light delivered into the PVH via fiber optics and the feeding behavior of the animal is observed and quantified under on or off lighting conditions. Preliminary results show that the light stimulation of ChR2 expressing neurons decreased feeding behavior, suggesting that the activation of PVHEsr2+ cells are sufficient to suppress feeding. Further studies to reveal the neural basis of hormone-dependent regulation of satiety can shed light on eating disorders in humans, potentially leading to the discovery of new drug targets to treat these disorders.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic event confers neuronal and axonal protection against subsequent ischemic exposure. The cell types responsible for IPC in the brain are unknown. In a novel model of white matter (WM) IPC and ischemic injury, we identified specific innate immune signaling pathways in microglia as required for IPC-mediated axonal protection, leading us to suspect that microglia are required for IPC. The model of WM IPC involves exposing the mouse optic nerve (MON) to a brief ischemic event 72 hours before the MONs are isolated and exposed to oxygen-glucose deprivation. Animals were treated with PLX5622 - a colony stimulating factor-1 receptor (CSF1R) pharmacologic antagonist – to deplete microglia in the central nervous system, including the MON, to test our hypothesis. By recording axonal function, we determined that microglial depletion eliminated IPC-mediated axonal protection in the WM. This study examines the impacts of IPC and ischemia on the nodes of Ranvier, which we hypothesize are protected by preconditioned microglia. The length of the nodes of Ranvier affects conductance and action potential propagation through an axon, with recent publications suggesting elongation of the notes in disease or injury states. We hypothesize the nodes of Ranvier will be shorter in preconditioned MONs than non-preconditioned MONs, which would support IPC-mediated protection. Additionally, we expect the nodes of Ranvier in PLX5622-treated MONS will be of similar length as non-preconditioned control MONs, indicating loss of IPC-mediated protection in animals without microglia. We will use immunofluorescence and confocal microscopy to measure the nodes of Ranvier and correlate these anatomical findings to prior electrophysiology experiments. The results of this research would contextualize a novel understanding of how microglia and ischemia affect the WM in specific regions of the axon, which is vital for advancing the development of neurotherapeutics for stroke.

People who spend early life at high altitude have an increased capacity for aerobic work due to developmental adaptations to hypoxic environments. Since the effects of altitude on aerobic performance became evident after the 1968 Mexico City Olympics, altitude training has been implemented to increase red blood cell carrying capacity and improve performance. However, it is unclear how early development at altitude and current training at altitude may differently advantage endurance athletes. In order to test for possible mechanisms by which altitude enhances endurance performance, this research compares personal records and biomarkers of oxygen carrying capacity among endurance athletes who experienced early development at altitude or sea-level and are currently training at altitude or sea-level. The study aims to determine if the altitude in which athletes developed and are currently training at will be associated with faster gender-adjusted personal records and greater lung capacity. I conducted a cross-sectional observational study with 23 endurance athletes in Seattle, WA and Boulder, Colorado. Participants self-collected chest circumference (CC) at maximum inhalation and completed online questionnaires about running performance, family history, and personal motivations for competing. I devised a gender-adjusted personal record percentile score for each subject’s 5k times based on the top 500 US men’s and women’s 5k times recorded during the 2019 season. This presentation discusses the results on differences in personal records and CC in relation to early development and current training altitude. I examined athlete’s motivation for competing through the qualitative analysis of open-ended interview responses to explore how motivation works synergistically with physiological biomarkers. These findings will be discussed in terms of existing research and consideration for endurance training at altitude and sea-level.

Neurons in the brain are dynamical in nature, maintaining constantly changing states. Neurons modulate voltage based on input currents and produce spikes when the voltage exceeds a certain threshold. Additional dynamics after spiking, called evoked after-spike currents, are important for computation and memory over time scales. The diversity of neuronal dynamics and the variability in parameters underlying them give rise to rich and varied dynamics across networks. We hypothesize that the complexity and diversity of biological dynamics in the brain play a critical role in predictive coding of temporally complex systems, and that diverse forms of after-spike currents enable computation over variable timescales. Current artificial neural networks (ANNs), that emulate the structure of biological neural networks, successfully learn relationships between static patterns but have difficulty learning dynamic patterns that change over time. We aim to incorporate complex biological dynamics and diversity in ANNs and thereby systematically explore the function of such dynamics in network computation and learning. To this end, we construct ANNs that express biologically realistic dynamics, developing methods to learn dynamics-generating parameters, such as membrane capacitance and threshold, in individual neurons. Theoretically, diverse dynamics of individual neurons will enable even more complex dynamics when combined in networks and may improve performance on tasks requiring computation over complex timescales, such as determining actions based on temporal patterns of cues. Hence, we hypothesize that when trained on temporally challenging tasks, our networks will learn diverse dynamics across neurons. We present the diversity of parameters learned and the resulting distribution of firing patterns and compare performance between our neural networks and traditional networks that only learn connection weights. This research will inform learning methods for training novel biologically inspired neural networks and will also shed light on the physiological role of diversity in the brain.

New technologies capable of controlling the position-and-spacing of nanostructures have advanced applications such as electronics, sensing, and catalysis. In contrast to conventional top-down approaches such as lithography, biological-macromolecule-templates offer an attractive way to direct the assembly of nanoparticles because their high-information content can be used to direct complex structures over multiple length-scales, just as they encode living structures. Here, we study the use of de novo designed protein-nanofibers to control the assembly of gold nanoparticle chains as a model system. We employ Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, which combines the energy contributions of van-der-Waals-attraction (vdW) and electrostatic-double-layer-repulsion (EDL) to understand the factors governing electrostatic assembly of gold nanoparticles along a protein-nanofiber anchored to a charged substrate, explain observed experimental results, and predict the assembly outcome under varying solution conditions. During the assembly process, as the distance between nanoparticle and protein-functionalized substrate decreases, we expect an increase in the magnitude of vdW and EDL forces. Varying nanoparticle size reveals particle-substrate EDL repulsion limits larger nanoparticles from reaching the protein despite an increase in vdW attraction, while tuning the pH varies EDL particle-protein-attraction and particle-substrate-repulsion, resulting in predictable particle density and binding specificity. We use Python 3.7.2 programming to calculate total system energies at different stages of the assembly process using equations based on the surface-element-integration method. By constructing a virtual representation of the protein-nanofiber as a chain of spheres on a flat plane and a spherical nanoparticle above the fiber at varying distances, we can use DLVO theory to map out the interaction energies for all solution conditions. With the ability to define the energy of a system, we will be able to design new biotemplates, indefinitely predict the solution conditions, and identify potential intervention points that would allow the self-assembly of plasmonic particles for new and/or difficult-to-achieve photonic applications.

Dissolved oxygen (DO) is a vital component of marine ecosystems, providing the key life source for thousands of species of marine vertebrates and invertebrates. Oxygen’s solubility in seawater is influenced by many variables, which can make DO difficult to predict. Estuarine systems experience DO fluctuations, as DO can limit ecosystem reproduction and health. Levels below 4 mg/L induce hypoxic conditions, creating stress for marine organisms, which makes tracking DO levels over time an essential tool for monitoring marine ecosystem health. My research provides Spatial-temporal depth analysis of DO data from the years 2014 through 2021 in the Snohomish River Estuary in Everett, Washington. Temporally, I predicted DO to exhibit a seasonal trend with highs in the winter and lows in the summer and decrease yearly at all depths due to global ocean temperature increase. Spatially, I expected DO to be higher at sites closer to the Snohomish River, and slightly lower at locations further from the river, in the center of the sound. With regard to depths, I predicted DO to be higher near the surface and lower near the bottom, and the oxycline is expected to get closer to the surface over time. Data were collected using an EXO2 Sonde at five different field sites at varying distances from the Snohomish River. I analyzed data using Excel, RStudio, and ArcGIS. Results found that DO is increasing over most sites with seasonal fluctuations of higher DO in the winter, and lower in the summer. There was one hypoxic event in 2016 at Buoy, along with a yearly increase in DO that suggests hypoxic conditions in Possession Sound may not last. Spatially, DO is higher at sites closer to the mainland, contrary to my hypothesis. Continuation of research will include further analysis of Spatial-temporal data in ArcGIS and Rstudio.

The circumgalactic medium (CGM) is a massive reservoir of gas surrounding a galaxy in which density and temperature range several orders of magnitude and contains more mass than the galaxy itself–similar to a cloud engulfing the galaxy. The CGM also plays a substantial role in the life of its galaxy as it will govern the accretion of matter for the galaxy to continue star formation. To better learn about the CGM, many astronomers utilize simulations to test theories by comparing their simulation data with observational data. Yet, current simulations struggle to replicate the CGM and its breadth of properties accurately. This project uses the TEMPEST and Patient0 simulations of Milky Way-type galaxies and a novel analysis method–known as synthetic spectroscopy–to better understand the effects of cosmic rays on altering how the CGM gas is ionized and how cooler CGM gas moves within the cloud. Many simulations tend to omit cosmic ray physics, however, cosmic rays are believed to provide non-thermal pressure support which will change the ionization structure of the CGM. Through our use of synthetic spectroscopy, we extracted column density and velocity information of various ions, such as HI and OVI, from our simulation to generate velocity histograms and plots of column density versus distance from the galaxy. Ultimately, this provided us further insight into the impacts of cosmic rays on setting the ionization and kinematic properties of the CGM which will better inform us on galactic evolution.

NGC 6822 is a barred irregular galaxy located about 1.6 million light years away in the Sagittarius constellation. We are observationally identifying red supergiants (RSGs) in this galaxy to compare with stellar evolutionary models. Stellar evolutionary theory provides us with the expected quantity of RSG populations. The research conducted will allow for a comparison between observational data to theoretical expectations. Here, we propose a new sample of RSG candidates in NGC 6822 that can be utilized as an observational test of such theory. RSG stars are the coolest of the evolved massive stars and have K and M spectral types and temperatures below 4100 K. Typically, they can be up to a thousand times the radius of the Sun and are therefore highly luminous. To find them in NGC 6822, we first used parallax and proper motion values from the GAIA satellite to filter out foreground stars, before using the NIR color-magnitude diagram to eliminate lower-mass asymptotic giant branch star contaminants. Next we transformed the J and K magnitudes to effective temperatures and luminosities to create an HR diagram (HRD), and selected RSGs based on their position on the HRD. Currently, we are comparing our results to previous spectroscopically confirmed RSGs. In combination with population studies done by ourselves and others in the Local Group galaxies IC 10, M31, M33, and the Magellanic Clouds, we can test model predictions across a wide range of metallicities. Additionally, by locating a population of RSGs in NGC 6822, future possibilities for studying these massive stars with direct spectroscopic follow-up are created.

Plants have evolved to represent a diversity of species, characterized by functional traits that dictate their performance in response to changing environments. One such leaf functional trait that relates to plant ecological strategy and is strongly correlated with photosynthetic rates, is minor leaf vein density (LVD). Our study will assess how ecological strategies within plant communities shifted in response to Earth’s most recent major global warming event, the Middle Miocene Climatic Optimum (MMCO) from 17-14 Ma, where rises in both global temperature and atmospheric CO₂ levels occurred. We hypothesize that global warming led to longer growing seasons and ecological strategies that prioritized persistence over productivity became dominant, and more favorable climates increased the diversity of ecological strategies present within the community. We will be traveling to various museums to photograph fossils that have all their minor veins preserved, collected from sites representing before, during, and after the MMCO in the Pacific Northwest region. Our goal will be to include several species per site, and photograph their fossil leaves under a stereo microscope. From there we will measure LVD using the program ImageJ using standard protocols. Once we have that data, we will calculate measures of the community-level distribution of this trait (mean, variance, kurtosis), and then compare those values between sites, and thus across the MMCO. In support of our hypothesis, we predict to see a lower mean and kurtosis, and higher variance of LVD values in MMCO plant communities, relative to those existing before or after the warming event. Overall this research is important to not only understanding how plant communities responded in ancient times to rising temperature but how plant communities could potentially respond to the rising temperatures in the future.

Plants face an enormous number of environmental stressors, including agriculturally important pests and pathogens. To defend against these biotic stressors, plants rely on pattern recognition receptors (PRRs), which are cell surface proteins that recognize conserved, non-self molecules indicative of attack or infection and initiate pattern triggered immunity (PTI) signaling pathways to mount defense responses. Recently, the first PRR involved in immunity against herbivorous pests was discovered in legumes. INR, a leucine-rich repeat receptor-like protein, mediates defense responses to inceptin, a peptide found in the oral secretions of caterpillars. To better understand the structural aspects of INR that are necessary for its ability to bind to inceptin and associate with downstream signaling components, I am developing a reporter system to screen INR variants to identify mutations that affect its function as a PRR. I have generated several different reporter constructs driving luciferase expression with promoter regions of genes found to be upregulated by inceptin when INR is transgenically expressed in the model organism Nicotiana benthamiana. These constructs vary in strength of expression but only one promoter region shows inducibility by inceptin. However, several constructs also show responses to a different immune elicitor, a peptide fragment of bacterial flagellin, which suggests that these constructs could be used as markers of PTI in plants more broadly. Robust reporters of PTI would not only be useful in understanding the structure and function of INR but may also enable further studies that will inform engineering practices to improve crop resistance to pests and pathogens.

Songbirds can learn different song dialects depending on their geographical location. Taxonomically, the American Crow (Corvus brachyrhynchos) is a songbird, but lacks a traditional ‘song’. Despite this, research suggests that crows do possess brain regions necessary for song learning and production. Unlike their songbird relatives, it is not understood whether American Crow populations learn regional call dialects. Using recorded crow calls from the online database, Xeno-Canto, I analyzed the acoustic properties of over 150 audio files across nine ecoregions of North America. Using the bioacoustics program Raven Pro 1.6, I am currently quantifying the lowest frequency, syllable number, syllable duration, and call duration for each call. Geographical differences in call structure, if detected, may indicate that crows learn regional dialects, or the differences may be the result of different selection pressures in each region.

As scientists collect ever larger volumes of data, methods to deal with these data have evolved as well. One of the fields that has thus emerged is the field of network science. Network science has many applications in biological fields as it allows scientists to connect variables of any type in a quantitative way. This versatility makes network science ideal for studies on comorbidity, or the consistent cooccurrence of various diseases in individuals. By analyzing comorbidities, we gain greater insight into interactions between diseases and systems of the body. This helps us understand how potential risk factors such as age, sex, and genotype affect various disease risks as well as the risk of comorbidity. We applied these methods to data on age of diagnosis for over 300 diseases collected from more than 28,000 owner reported surveys through the Dog Aging Project. We constructed comorbidity networks from these data and analyzed these networks using quantitative network statistics which allowed us to compare nodes both in and between networks. We first constructed undirected networks with the nodes representing various diseases to establish and identify pairs of diseases with significantly higher rates of cooccurrence than expected by chance. Using this network, we assigned directions to edges based on temporal data on the relative ages of diagnoses, which allowed us to identify which diseases are precursors to others. We observed how these networks changed through stratifications based on age, sex, and size to identify disease progression through age. In doing so, we hope that we can identify how age affects comorbidity in dogs, which can help researchers identify and develop therapies for lengthening dog lifespans. This knowledge will also provide insight into the mechanisms behind certain disease connections that were previously unknown.

Steller's Jays (Cyanocitta stelleri) emit multiple call types. Variation within and between call types could indicate a sophisticated method of vocal communication. Variation could also indicate different selective geographic pressures or learned regional dialects. It is currently unknown whether Steller's Jays have geographic variation in their calls. In this study, I investigated whether the 'wah call' of Steller's Jays varies between and within geographic regions. I analyzed recordings of 'wah calls' from two geographic regions spanning from Canada to Mexico. I quantified the number of syllables, the syllable duration, the call duration, and the average silence between syllables. 'Wah calls' are highly variable, with durations from 0.41 to 10.00 seconds, one to eleven syllables with durations from 0.26 to 0.71 seconds, and gap (silence between syllable) durations from <.01 seconds to 0.67 seconds. Preliminary analysis shows no significant differences in these acoustic variables between Steller's Jays in the Marine West Coast Forest and Mediterranean California regions. I am currently increasing my sample size, beginning analysis of additional Steller's Jay call types, and expanding data analysis to five geographic regions. The presence of regional variation could indicate that Steller's Jays learn their different vocalizations. Learning vocalizations could be a sign of complex communication within Steller's Jays. 

International treaties provide citizens with guaranteed human rights and hold countries to a higher standard of accountability. Through monitoring mechanisms, international organizations can track both the effectiveness and implementation of rights outlined in their treaties. However, this process of human rights protection can be convoluted when involving countries with a rich history of authoritarianism. In this research project, I assess the United Nations Committee Against Torture (CAT), to understand its effectiveness in implementing the Convention Against Torture in the Middle East. The project examines the six Middle Eastern countries that ratified the treaty and evaluates their success in circumventing the violence that it prohibits. To measure this, I have developed an original jurisprudence dataset ranging from 1994 to 2019 that includes all claims brought against Morocco, Tunisia, Algeria, Iraq, Lebanon, and Mauritania. Jurisprudence is broadly defined as the study of law but in this particular instance is the examination of case law. In a historical case law analysis, I code for whether the committee ruled in favor or against a violation to the treaty, participating advocacy groups, and the issue areas most prevalent. Additionally, I analyze annual reports documenting the personal accounts of Middle Eastern countries perceived efficacy in adhering to the treaty. Preliminary findings suggest that the Convention Against Torture is effective on the ground when there is a prominent culture of advocacy present to counteract the durable authoritarian setting it falls beneath. These findings may assist in answering larger questions about the impact of international treaties in safeguarding human rights and holding countries accountable. Importantly, these monitoring mechanisms may progressively inhibit authoritarian regimes from actively denying citizens their guaranteed human rights.

The International Criminal Court (ICC) is responsible for prosecuting the most heinous of crimes, that of genocide, crimes against humanity, war crimes and crimes of aggression. An institution with potentially universal jurisdiction, it was created to promote rule of law globally, not beholden to any one nation and unbiased in its pursuit of justice. However, allegations of an anti-African bias have been made against the ICC by the African Union and others. Is there an inherent bias against Africa or is the ICC merely fulfilling its duties as designed? This research investigates charges of bias through close examination of the ICC’s structure, case history, and the court’s relationship to the United Nation Security Council (UNSC). To fully answer the question of bias, I created a case law dataset cataloging all ICC investigations and cases from its first in 2002 to the present. I have coded each case and investigation according to charges, which referral mechanism triggered ICC jurisdiction, and the current status of the case. Preliminary results indicate a clear focus on Africa by the Court, however, each prosecuted case falls squarely within the jurisdiction entrusted to the Court. While a discernible bias may not be present, the relationship between the ICC and the UNSC does raise questions about impartiality and global separation of power. Future research on possible remedies to charges of impartiality may benefit the legitimacy of the ICC, as well as the Court’s efforts towards international justice.

International human rights law governs and safegaurds individual citizens with the intention of protecting  basic rights, liberties, and freedoms. These international laws are upheld through international courts who act to ensure member state compliance. Acceptance of international court authority and rulings is slow in many transitioning democracies. Yet increasingly, international court decisions are shaping domestic rights protection and the rule of law. In this project, I examine the Inter-American Court of Human Rights (IACHR), an international court governing the Americas, and their case law involving Guatemala to understand how international courts develop and protect human rights. In my analysis, I construct an original dataset that codes all IACHR judgements involving Guatemala between 1996-2020. In particular, the dataset focuses on cases in the area of indigenous rights, children's rights, and the right to liberty, identifying if a violation is found and how this expands human rights protection. Preliminary findings suggest the court has slowly expanded rights in these areas of law, which has led to some strengthening of the domestic legal system. This study illustrates the power of international courts to protect fundamental individual rights, however, it calls for further research exploring the potential limitations of this power in the context of transitioning societies. 

Authoritarian regimes may construct courts to engage in administrative discipline and bolster executive legitimacy. Yet in China, citizens are also increasingly utilizing courts to contest the decisions of local government officials. Litigation is at once a tool of control by the state, but may also suggest an avenue for citizenry power against the state in an otherwise constrained society. In this project, I examine administrative law cases brought to the Supreme People’s Court of China (SPC) to analyze how administrative litigation processes are used to empower citizens or to discipline state bureaucrats. To answer these questions, I developed an original case law dataset of SPC administrative law judgments between the period of 2014 to 2019. Through case law analysis I coded each decision for key identifiers including the districts in which litigation was filed, the type of plaintiff, the issue in dispute, and compensation given by the government. Preliminary findings suggest that administrative litigation is disproportionately in the area of property rights and is most frequently utilized by corporations rather than citizens or public interest bureaus. The patterns hold across a diverse set of localities throughout China with plaintiffs winning in nearly half of the cases in this dataset. These findings bring into question whether courts in authoritarian regimes are merely a pawn of executive power, and instead suggest an avenue for individual and corporate claims making against the administrative state.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that have been engineered into three-dimensional heart tissues (EHTs) are valuable research tools for investigating debilitating genetic diseases that afflict the heart, such as Duchenne muscular dystrophy (DMD). Ensuring iPSC-CMs can be sufficiently matured to model such diseases remains a hurdle in current research, and maturational analysis techniques for iPSC-CMs are either qualitative, manual, or primarily based in two dimensions, leaving much to be desired. In this project, we created a suite of MATLAB image-processing scripts that can quantify the effect of three-dimensional culture and disease-causing DMD mutations on cardiomyocyte structure and maturation state. The iPSC-CMs were differentiated from stem cells, cast into EHTs, stained using immunofluorescence, and imaged using a confocal microscope. Using the scripts to analyze these 3D images of iPSC-CM stains, key maturational features of the cells can be quantified such as nuclei count; cardiomyocyte area; and sarcomere length, orientation, and z-disk width. Analyzing cardiomyocyte area can give key information on cardiomyocyte hypertrophy while examining sarcomere length, orientation, and Z-disk width can provide information on myofibril structure and organization. The suite allows analysis of these maturational features in both 2D and 3D cultures and offers a method for quantitatively assessing maturation in an automated manner. Measuring iPSC-CM maturation will also allow better comparison of existing maturational methods, such as mechanical loading, electrical stimulation, and small molecule treatment. The suite can also create graphical outputs to elegantly display data. Overall, the suite will help improve maturational analysis of EHTs, and hopefully contribute to the discovery of new treatments for diseases that affect the heart.

The spatial distribution and degree of colocalization for two or more proteins, based on their fluorescence intensities, are useful metrics for phenotyping cells and informing on biological function. Yet, most commercial and open-source image analysis tools report global colocalization statistics at the image-level, and offer limited analysis at the individual cell level. To address this, we are developing a python-based image analysis pipeline to quantify robust per-cell metrics of colocalization. Our pipeline stacks image tiff files acquired on high-throughput automated fluorescence microscopes into multichannel 3D image stacks. The images are then restored via deconvolution algorithms, and cropped to remove out-of-focus image planes using Laplacian variance algorithms. For image thresholding and cell segmentation, the pipeline incorporates scripts from the Allen Institute for Cell Sciences to threshold and segment individual cells. Finally, the pipeline calculates per cell colocalization metrics based on the fluorescent intensity of each voxel in each cell. CLARITY was used to quantify the spatial relationships of the peroxisomal biogenesis protein Pex3 with the endoplasmic reticulum protein Sec61 and peroxisomal membrane protein Pmp70. Pex3 colocalized with both Sec61 and Pmp70 and this colocalization could be manipulated by treatment with different kinase inhibitors. In several instances, these differences in localization contributed to a large variance in the measured Pearson’s Correlation Coefficient of cells within the same image. Morphometric analysis showed the volume of peroxisomes per cell negatively correlated with the number of peroxisomes per cell. The automation of per-cell image analysis leveraged in this pipeline will allow for systems-level phenotyping and data mining from fluorescent microscopy images.

Blood clotting plays a heavy contribution to the mortality and morbidity in patients with diabetes mellitus. In blood clotting, the interaction between platelet glycoprotein Ib (GPIb) and blood protein von Willebrand Factor (VWF) is a catch bond, a bond whose lifetime increases under tensile force. Current, identification of this single-molecule behavior of a catch bond is useful but insufficient to understand behavior with multiple molecules known as clusters. It is recognized that patients with diabetes mellitus have higher levels of VWF and GPIb but there is no existing flow assay that accurately demonstrates the difference in thrombosis flow in diabetics. In addition, there is no definitive conclusion on the influences of the amount and geometry of the catch bond between GP1b and VWF in clusters. A recent innovation in the Thomas Lab has developed a DNA origami nanostructure that allows control over the number and spacing of ligands in a cluster, facilitating the study of clusters of catch bonds. I designed a method to quantify the nanostructure-presented ligands on a surface using 96-wellplate reader. This method was used to characterize the effect of cluster size on platelet rolling behavior. The result of the method suggests using quenching low concentration of biotin-4-fluorescein had the highest accuracy and was able to be picked up by the 96 well plate reader. The method allows observation seen in platelet rolling behavior over these surfaces that are cluster-size dependent rather than concentration-dependent. This will introduce a new assay for diabetic studies and further our understand difference in platelets rolling behavior over clusters between diabetic patients and non-diabetic patients.

Gene gun technology uses DNA-coated gold microparticle bombardment to achieve DNA vaccine immunogenicity by effective intradermal transfer. This delivery system results in increased uptake of plasmids by cells, potent cellular and humoral immunity compared to other delivery methods like direct needle injection or electroporation. DNA vaccines are expected to exhibit long term stability, ease of manufacturing, low cost, reduce cold chain requirements, and reduce concern for vector immunity from repeated immunizations. Additionally, gene gun DNA vaccine technology allows for multiple antigens to be added in the same immunization. Beyond the advantages above, genetic adjuvants have the potential to further improve the immunogenicity of DNA vaccines. One strategy to improve the immunogenicity of DNA vaccination would be using chemokines like Xcl1 to recruit dendritic cells for induction of CD8+ and CD4+ T-cells. Xcl1 is a ligand that binds to and specifically activates dermal dendritic cells, which presents Xcr1 and efficiently activates T-cell responses. Two ways to introduce Xcl1 as an adjuvant are fusion and cotransfection, the latter of which is the method used in this project. In cotransfection, Xcl1 cytokines induce localized inflammatory responses which recruit dendritic cells. This allows the antigen of interest to target the dendritic cells, leading to the induction of CD8+ T-cells specific to the antigen of interest. I believe that Xcl1 has the potential to induce antigen-specific effector and memory CD8+ T-cells and enhance proliferation of CD4+ and CD8+ T-cells. I gene gun vaccinated mice with and without Xcl1 DNA and assessed the induction of appropriate T-cell responses to determine whether the addition of Xcl1 as an adjuvant enhances the T-cell and antibody response needed to protect against P. yoelii infection challenge. The results of this ongoing work will be presented. This data will inform whether genetic adjuvants have the ability to increase immunogenicity in DNA malaria vaccines.

The gut microbiome is a central regulator of overall health, and establishing mutually beneficial relationships between the host and resident gut bacteria is important for preventing the later development of pathologies such as ulcerative colitis, metabolic dysregulation, and colon cancer. While microbiota-reactive maternal IgA antibodies in the breastmilk have historically been considered to be a primary regulator of host-microbiota interactions, maternal IgG antibodies have historically been associated with the protection of neonates against pathogens. However, our lab has identified a novel function for microbiota-reactive maternal IgG2b and IgG3 antibodies in preventing dysregulated adaptive immune responses and reinforcing intestinal homeostasis in early life. To investigate how these maternal antibodies function, we created a mouse strain (IgG3^-/-) lacking IgG3 antibodies. Using IgG3^-/- dams, we were able to selectively prevent the transfer of maternal IgG3 antibodies to their pups while maintaining normal transfer of the other antibody isotypes. Compared to the pups born to wild type (i.e. antibody replete) dams, these pups displayed reduced weight gain, increased inflammatory gene expression, dysregulated T cell immunity, and increased susceptibility to intestinal colitis induced by dextran sodium sulfate (DSS). With our data suggesting an integral role for maternal IgG3 antibodies in limiting neonatal immune responses toward gut microbes, my current experiments aim to elucidate the underlying mechanisms by which these antibodies function. Using pups born to C1q deficient and FcγR deficient dams, which lack the ability to activate the complement pathway and FcγRs, respectively, I am aiming to test the roles of these antibody “sensing” pathways in maternal IgG3-mediated suppression of neonatal intestinal immunity. Defining these mechanisms not only gives us a clearer picture of the ways in which neonatal health is regulated; it also advances our ability to manipulate neonatal immunity and early life gut microbiome-targeted treatments to improve human health.

Kaposi’s Sarcoma (KS) is a highly vascularized tumor, which affects AIDS patients worldwide and remains endemic to sub-Saharan Africa. Kaposi’s Sarcoma-associated Herpesvirus (KSHV) is the etiological agent of KS and its latent infection is involved in tumor formation and the induction of angiogenesis in the spindle cell, a cell of endothelial origin and the main proliferating cell type in a KS tumor. Previous RNA-Seq data obtained by our group showed that osteopontin (opn), a secreted protein known to act as a ligand for integrin receptors that activate signaling cascades that promote angiogenesis, is highly upregulated at the transcript level during KSHV latent infection of endothelial cells. To determine whether opn is required for the activation of angiogenesis in KSHV latently-infected endothelial cells, we used CRISPR-lentiviral constructs to knock out opn and evaluate changes in angiogenic phenotypes upon KSHV infection via cell proliferation, tubule formation, and cell migration assays. Preliminary results reveal a significant reduction in tubule formation in opn knockout KSHV-infected endothelial cells. This finding suggests that opn upregulation in such cells is responsible for the activation of this angiogenic phenotype. Future experimentation will include evaluating how KSHV induces the upregulation of opn by infecting wild-type endothelial cells with mutant viruses lacking certain latency protein genes and evaluating differences in opn transcriptional and translation, as well as evaluating the mechanisms by which opn activates tubule formation in KSHV latently-infected endothelial cells. Identifying  the drivers of angiogenesis in KSHV-infected endothelial cells will aid the characterization of therapeutic targets for KS progression in such cells, given that KS tumors are highly angiogenic from its early stages.

The 1972 Marine Mammal Protection Act (MMPA) helps govern human-environment interactions in the United States with public and agency processes. As a result, the 1972 MMPA has accumulated praise and critiques concerning its provisions and implementation. Previous research explored the impacts of the 1972 MMPA, but these publications do not provide a comprehensive assessment based on human-environment interactions and government structures in the Pacific Northwest to determine possible “best practices” for environmental law. The study observes the 1972 MMPA concerning its goals and cultural, environmental, and international implications to determine the 1972 MMPA’s effectiveness and opportunities for improvement in the National Oceanic and Atmospheric Administration’s “West Coast Region.” Through this research, I analyzed literary information and interviews with people from the National Oceanic and Atmospheric Administration, non-governmental organizations, and Indigenous communities to gather a holistic review of the 1972 MMPA as well as reveal pathways for enhancing environmental law in the United States. Available scholarly and media-based literature surrounding the 1972 MMPA suggest accomplishments encompassing the increased abundance of marine species over time, while mitigations for secondary impacts may be lacking. To appropriately address current concerns and perspectives of the 1972 MMPA, the United States public, agencies, and government bodies may collaborate to refine marine mammal securities. Revised implementation of the 1972 MMPA could inform future environmental policies and actions in the United States. Incorporating suggestions and relevant modifications in the 1972 MMPA may provide further effective partnerships in the “West Coast Region” with diverse stakeholder representations and marine conservation between governments and collectives. Consequently, the 1972 MMPA can serve as a case study for current and future environmental law in the United States and the international community.

From the Dakota Access Pipeline demonstrations to increasing popularity around climate-activist-turned-household-names like Greta Thunberg, a resurgence of environmental advocacy has taken the 21st century by storm. The environmental justice movement, a term first coined in the 1980s, was born from the idea that mainstream environmentalism does not examine environmental policy in the intersection of socioeconomic status and race. This literature review explores the history and impacts of environmental racism and the evolution of the environmental movement. It finds that not only have the burdens of environmental degradation been disproportionately distributed across different communities, but that analyses of the ecological impacts of imperialism have created a singular discourse that is paradigmatic of colonial ideologies. It parallels the colonial vision of conservation in North America with the treatment of its Indigenous populations and explores nature as something portrayed as the antithesis of culture. Finally, further examination of the changes from the Trump to Biden administrations is warranted by the construction of a new framework for examining environmental policies, with an emphasis on social equity. The intended outcome of this paper is to create a set of criteria for existing and future environmental legislation.

Code-switching (CS) refers to a multilingual speaker’s alternation between two or more languages in a single context of communicative discourse. It is important to study how children code-switch during narrative production because though there is existing research on code-switching and its prevalence in conversational settings where one may be inclined to code-switch based on conversational needs or factors, there is limited evidence on its prevalence during narrative tasks. Importantly, there is also minimal research on how the occurrence of code-switching may differ with language proficiency. In this study we will answer, (1) In what instances do children enrolled in immersion programs code-switch from Mandarin to English during narrative production? And (2) how are these instances affected by grade level and level of exposure? We hypothesize that as bilingual students progress through the immersion program, prevalence of code-switching during narrative production will decrease as proficiency in Mandarin increases. Thus we expect the number of instances of code switching to decrease as participants’ proficiency scores and their grade level (i.e. 1st, 2nd grade) increases. Participants involved were school-aged (1st-6th grade) children enrolled in a Mandarin-English (ME) immersion program in Oakland, California. Children completed a story-telling task, presented by a native speaker of the language being tested. Mandarin narratives were based on the wordless picture book “Frog Goes to Dinner”. Speech samples were collected and recorded over Zoom and then transcribed on CLAN software. Instances in which individuals of each grade level code-switch during a narrative task were coded from transcripts. We present data on the prevalence of code-switching in a controlled environment. Data collection is complete and analysis is in progress. Code-switching may be indicative of level of proficiency in narrative story telling in a second language (L2) for bilingual students.

Understanding the microbial process in the conversion of food waste into methane is necessary to realize the societal need to convert waste into a sustainable energy source. A critical step in the process is the synergistic breakdown of butyrate to methane. My project Food Waste to Energy allows us to mimic the degradation of food waste to isolate the microbial partners responsible for the production of methane. This process is carried out by a syntroph and a methanogen. Although we do not know the specifications of either organism, identification can allow us to improve the way we utilize bio-reactors dependent on food waste to produce clean energy. Each organism has been grown using media designed to allow for their specific development, doubling methane production within four months on average. We are in the process of conducting a dilution to extinction to reduce the contaminating microbes while selecting for the target majority by close observation of the purity of each sample microscopically. We will sequence the genome of each organism to characterize their respective genetic capabilities and identity which we suspect is novel. Activity and growth characteristics will then be studied to understand the growth kinetics when the organisms grow separately and together. Once the organisms are isolated, we look forward to accelerating methane production by selecting mutants that can catalyze the conversion of butyrate into methane. By identifying the organisms responsible for the conversion of butyrate to methane, we can begin to introduce highly productive bioreactors in urban, rural, and manufacturing settings to combat the use of non renewable energy and create energy from the 40 million tons of food waste produced annually in America.

Bergmann’s rule describes the correlation between increased body size and living at higher latitudes, seen in many endothermic animals. However, there is debate over if Bergmann’s rule is applicable to ectotherms. Using an exhaustive review of the literature, we investigated whether the relationship between body size and latitude is consistent with Bergmann’s rule in >100 species of Alpheus snapping shrimps. We found a significant correlation between body size and latitude (i.e., latitudinal midpoint of the range), using both raw species correlations and correlations corrected for phylogeny (phylogenetic independent contrasts). We discuss multiple hypotheses that may explain adherence to Bergmann’s rule in this group of shrimp, and the broader ecological implications of variation in body size during a time of global change.

The rugged intertidal of the Pacific Northwest serves as a nursery for ecologically and commercially valuable vertebrate and invertebrate species. Climate change is creating a persistent multiple stressor environment that could permanently decrease the survivability of these organisms. Green urchins (Strongylocentrotus droebachiensis) are ideal organisms to examine concurrent abiotic and biotic stressors on intertidal trophic relationships because they are resilient, have long life spans, and their preferred food source (bull kelp, Nereocystis luetkeana), and natural predators (sunflower sea star, Pycnopodia helianthoides) are well studied. In this study, we assessed the independent and interacting effects of abiotic environmental stressors and the presence of a predator on green urchin survival and stress response. Using kelp consumption as a proxy for stress response we designed an experiment consisting of three stressors: (1) elevated water temperatures (+6°C), (2) altered photoperiod (representative of drastic habitat changes due to excess or absence of bull kelp or substrate disruption), and (3) a predator chemical signal (via an isolated sunflower sea star). Urchins were fed a slight excess of bull kelp three times a day to ensure that food was not a limiting factor. Results indicate that exposure to either predator presence or an environmental stressor inhibited survival and increased stress, with the combination of multiple environmental stressors being the most inhibitive. The results imply that green urchins will be unable to fully cope with additional predation pressure in the presence of persistent abiotic stressors. Green urchins mitigate stress by maintaining or disrupting their habitat, and disruption can mask spawning cues causing irreparable loss of biodiversity. Using this information in future studies I will examine a long-range study of stressor response in adult urchins and work to clarify larval recruitment and response to persistent stressors.

Benthic foraminifera are shelled single-celled marine microorganisms which are particularly sensitive to changes in their environment. A census of species found in an area can function as an indicator of the health of a marine ecosystem. My project focused on the distribution in 2018 of foraminiferal assemblages in Budd Inlet, near the city of Olympia, to assess the recent health of the area and the presence of pollutants. I examined nine sediment samples for foraminifera and identified the species. I then compared the abundance and diversity of species found to previous pollution data to pinpoint problems in Budd Inlet. My analysis found a low species diversity overall, as well as a correlation between the distribution of heavy metals and the presence of key species of foraminifera. The strong presence of the species Buccella frigida and Cribroelphidium excavatum and the low species diversity are consistent with a concentration of heavy metal pollutants, and mark Budd Inlet as an area of heavy anthropogenic impact.

Prehistorically, people of Alaska carved household tools from rocks, mostly slate. ‘Ulu’ is an Inuit word that translates as ‘woman’s tool’. It was used for household needs such as meat cutting. The Unangan civilization (Aleuts) in the late Aleutian phase (1000-200 BP) adopted the use of ulus sourced from slate, due to the rock’s durability and ease to sharpen. The paradox is, there is no slate widely present on the Aleutian Islands, yet fine grained volcanic rocks are abundant. In this study, I analyse 5 ulu fragment samples that were excavated on the Tanaxtaxak Spit (UNL_55), Amaknak Island of Unalaska where volcanic rocks and argillite were common. Kodiak Island, the nearest source of sufficient slate, is ~880 km away. The UNL_55 samples in this study were collected from different chrono-stratigraphic layers of the same site.The UNL_55 samples have previously been classified as slate, based solely on visual analysis. This implies possible inaccuracy in identification, since aphyric volcanic rocks and silicified argillite visually resemble slate. Using the Scanning Electron Microscope, I investigate the microtextures and minerals in the ulu fragments to identify whether they are slate, volcanic rock or argillite. Determining the rock type will guide inferences related to inter-island trade and migration of people in the prehistory of the Aleutian Islands. I expect the results to indicate rock composition that is similar to rocks that abundantly exist at the excavation site; volcanic. 

Cachexia is a debilitating condition characterized by the loss of muscle strength and mass. Affecting 50-80% of advanced cancer patients, this condition is associated with weakness, fatigue, poor tolerance to chemotherapy, and decreased quality of life. The mortality rate of patients with cancer-induced cachexia can be as high as 80%, and there is no current effective treatment. Ghrelin has recently been proposed as a therapeutic option for cancer cachexia due to its effects on preventing appetite, muscle, and fat loss. Its orexigenic effects are mediated by the growth hormone secretagogue receptor GHSR-1a, but the extent to which GHSR-1a mediates ghrelin’s effects on preventing muscle mass and function loss is unknown. This study characterizes the pathways involved in muscle mass loss and weakness in the Lewis lung carcinoma (LLC)-induced cachexia model and the effects of ghrelin in wildtype GHSR+/+ and knockout GHSR-/- mice. 5-6 month old male C57GL/6J mice were injected with LLC cells. When the tumor was palpable, tumor-bearing mice were injected with vehicle (saline solution) or ghrelin (0.8 mg/kg). We quantified body mass over 3 weeks and dissected and weighed hindlimb muscles. We analyzed mitochondrial protein concentrations via BCA and measured oxidative phosphorylation markers through Western blotting. Ghrelin attenuated LLC-induced muscle wasting in both genotypes but only prevented the decrease in grip strength in GHSR+/+. There was no significant difference in muscle mass between the two genotypes. The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathways were activated by LLC in both genotypes, and these changes were more pronounced in GHSR -/-. In tumor-bearing mice, ghrelin mitigated the increased UPS markers (atrogin-1, MuRF1) independently of GHSR-1a. Ghrelin only prevented tumor-induced increases in mitophagy markers in GHSR+/+ (p62, Bnip3), and the levels of these mitophagy markers were negatively correlated with muscle strength. In conclusion, GHSR-1a is required for ghrelin’s effects on attenuating LLC-induced loss of muscle strength but not muscle mass, and this is likely due to the alterations in the autophagy-lysosome pathway and impaired mitophagy. By characterizing the preventative role of GHSR-1a in muscle wasting, we hope to improve hormonal therapy options for tumor-induced cachexia patients.

Prior research suggests disproportionate endorsement of pain among Black, Indigenous, and People of Color (BIPOC) compared to White people. Considering the high prevalence of pain in persons with MS, the present study examined potential racial-ethnic differences in pain interference among adults with MS, both before and after participation in one of two telehealth interventions aimed at improving pain, fatigue, and depressive symptoms. A national sample of adults with MS and chronic fatigue, chronic pain and/or moderate depressive symptoms (N=163) participated in a randomized controlled trial of self-management intervention focused on cognitive behavioral therapy (n=75) versus MS education (n=88), both delivered 1:1 via telephone. T-tests explored racial-ethnic differences in pain interference at baseline and posttreatment. Hierarchical multiple regression assessed race/ethnicity as a predictor of posttreatment pain interference controlling for baseline pain interference, and in subsequent blocks explored treatment condition, treatment satisfaction variables and baseline perceived social support as factors accounting for potential racial-ethnic differences and variance in pain interference. Independent sample t-tests revealed significant racial-ethnic differences in pain interference at baseline, t(159)=2.30, p=.023 and posttreatment, t(141)=2.91, p=.004. Specifically, participants who identified as (BIPOC) endorsed greater pain interference at baseline (M=4.66, SD=2.54) and posttreatment (M=4.31, SD=2.56) compared to non-Hispanic White participants (M=3.59, SD=2.32 and M=2.81, SD=2.11, respectively). Hierarchical regression analysis revealed a significant relationship between race and posttreatment pain interference, which became non-significant after controlling for baseline pain interference. Perceived social support emerged as significantly associated with posttreatment pain interference, after controlling for all variables. Ethnicity/race and perceived level of social support accounted for more variance in posttreatment pain interference than treatment type. Future investigations aimed at understanding why BIPOC report higher pain levels posttreatment are necessary to inform and adapt current treatments to address more relevant factors contributing to BIPOC’s experience of pain.

The pupillary light reflex (PLR) curve is an important point-of-care biomarker for the diagnosis of traumatic brain injury (TBI). Using PLR, first responders can determine the severity of TBI in the field and direct patients to a trauma center where staff can continually assess PLR to monitor TBI severity. Manual pupillometry, the most commonly available method for first responders and most clinicians wishing to assess PLR, is qualitative and often inaccurate. The current gold-standard device for PLR measurement is digital infrared pupillometry, but such devices are fragile and expensive. Our research team has developed a smartphone-based pupillometer (PupilScreen) with the ability to assess PLR using a standard iPhone, assisted by a cloud-based neural network. To demonstrate the feasibility of using PupilScreen in a realistic clinical setting and compare the accuracy of the device to the current clinical gold-standard, we have built an annotated dataset of the PLR in n=120 patients with TBI who are hospitalized in a neurological intensive care unit. Pupillometry is performed using the mobile device and the gold-standard digital infrared pupillometer. Pupil videos are manually annotated and used in the further training of our machine learning algorithm that generates a PLR curve for each patient. We anticipate that our technology will demonstrate accuracy in assessing the PLR that exceeds that of manual pupillometry and is at least equivalent to the gold-standard digital pupillometer. This technology has the potential to alleviate the current undertreatment of many TBI patients in the United States and abroad that results from a lack of accurate and cost-effective pupillometry equipment.

Disruptive mutations to DYRK1A, located in the Down Syndrome critical region of chromosome 21, are associated with autism spectrum disorder and medical comorbidities. Previous literature suggests facial anomalies in children with DYRK1A mutations, and studies of DYRK1A’s regulatory functions confirm its role in the expression of several morphology-affecting genes, particularly DCAF7. This study attempted to determine if quantitative differences in facial features exist between children with DYRK1A mutations and the general population, including unaffected parents. From a sample of 28 children with de novo DYRK1A mutations, analyses focused on nine white non-Hispanic children (M age = 11.33 years, 77.78% male) whose data were collected using a 3dMDhead System through an ongoing genetics-first study. Measurements between facial landmarks were later calculated using 3dMDvultus. FaceBase’s 3D Facial Norms for European Caucasians were used as a control group, and Z-scores were calculated for all complete measures. Six measures—intercanthal width, outercanthal width, palpebral fissure lengths, cranial base width, and philtrum width–were selected for analysis based on previous clinical findings. Wilcoxon sign-rank tests compared Z-scores between probands and each biological parent, assessing familial genetic influence on observed dysmorphologies. Outercanthal width in probands significantly differed from both biological parents and was significantly below population average. Additionally, palpebral fissure lengths significantly differed between probands and fathers and fell below population averages for probands. These findings bolster the link between DYRK1A and genes that code for craniofacial development and suggest the facial phenotype associated with DYRK1A mutations may be more variable and nuanced than expected, presenting challenges for clinical assessment. Additional research should examine how DYRK1A interacts with genes that code for eye regions, facial phenotypes in non-white participants, and possible differences in dysmorphology between sexes.

The nucleus accumbens (NAc) is a midbrain region associated with addiction-related behaviors. The NAc consists of medium spiny neurons (MSNs) that project to the substantia nigra (SN) or the ventral pallidum (VP) forming the direct (Go) and indirect (No-Go) pathways, respectively. The Go and No-Go pathways are mostly dichotomous in their expression of distinct receptors and neuropeptides. Dopamine receptor (D1) and adenosine receptor (A2a) expression corresponds to the Go and No-Go pathways, respectively. My goal is to investigate whether collateralization exists (i.e., single neuron projecting to both the VP and SN). This will be assessed by injecting retrograde tracers into the output regions and quantifying expression in MSNs. I used D1-iCre and A2a-iCre transgenic rats that express codon-improved Cre recombinase (iCre) in neurons expressing D1 or A2a, respectively. When the iCre expressing neurons are infected with a canine adeno-associated virus (CAV) containing a double-floxed and inverted (DIO) copy of a fluorescent protein, this anatomical marker protein is inverted and expressed specifically in these cells. I bilaterally injected four D1-iCre and four A2a-iCre males with CAV-DIO-TdTomato into the VP and CAV-DIO-ZsGreen into the SN. D1 and A2a neurons projecting to the viral injection site will uptake the virus and retrogradely label D1- or A2a-expressing MSNs in the NAc. The expression of these fluorescent proteins within the NAc will be quantified to investigate the projections’ dichotomy and collateralization. The dichotomy could be validated if the tracers expressed in exclusive populations of the NAc. Based on mouse studies, I hypothesize D1-Cre rats may have minimal colocalization in NAc MSNs but A2a-Cre rats may only express TdTomato in the NAc. Addiction continues to affect millions of individuals. We aim to elucidate anatomical differences to gain a better understanding of these midbrain pathways, which could be critical for the future of clinical treatment.

Induced pluripotent stem cells (iPSCs) can be directed to create 3D mini retinas in vitro known as retinal organoids. Retinal organoids recapitulate the developmental timeline of the human fetal retina and have the potential to serve as disease models for a multitude of retinopathies. However, we have observed that retinal organoids contain a disorganized inner nuclear layer and discontinuous lamination hindering their ability to be considered as a comprehensive disease model. The protein β-catenin is produced from the canonical WNT signaling pathway and has been shown to be essential for retinal lamination in mice due to its role in cellular adhesion. I investigated the effects of a canonical WNT signaling pathway agonist, CHIR,  in order to improve the disorganization and lamination of retinal organoids so that we can develop a comprehensive disease model for different retinopathies. We have used three different stem cell lines to construct retinal organoids that I cultured with the addition of the WNT pathway agonist, CHIR. I performed immunohistochemistry staining followed by microscopy analysis and have obtained data that shows an increase in lamination and cellular organization with the addition of the WNT signaling pathway agonist. Our data suggests that the WNT signaling pathway plays a role in maintaining organization and lamination in the developing human fetal retina. 

Skin can detect a wide range of stimuli through the touch system, which is mediated by specific cells and structures. Merkel cells, one of these specialized types of skin cells, sense gentle touch and texture. In mammals, Merkel cells are identified by the expression of the transcription factors Sox2 and Atoh1. Recently, the Rasmussen lab identified Merkel cells in the zebrafish skin that share many characteristics of mammalian Merkel cells, including expression of Sox2 and Atoh1a. Interestingly, a paper by Konig in 2018 also described a novel cell type that expresses serotonin (5-HT), calretinin, and synaptic vesicle glycoprotein 2 (SV2), termed “HCS” cells, within the zebrafish skin. These researchers proposed that HCS cells are a different sensory population than Merkel cells, because of differing visual characteristics between these cells. However, the paper did not present any conclusive evidence, making the relationship between HCS cells and Merkel cells still uncertain. I hypothesized that Merkel cells and HCS cells are actually the same population of cells. I tested my hypothesis using antibody staining and confocal imaging of zebrafish skin. I found that Atoh1a-positive Merkel cells express serotonin and SV2, demonstrating that Merkel cells in zebrafish are the same cells as HCS cells. Overall, my results resolve the identity of recently described sensory cell types in the zebrafish skin. In future research, I hope to use zebrafish Merkel cells as a promising model to better understand touch system development and regeneration.

Human adenoviruses (AdVs) infect and cause disease in multiple organ systems, and certain human AdV serotypes are associated with particular diseases; however, the basis for AdV tissue tropism is unknown. To better understand this problem, I am using mouse adenoviruses (MAdV). Like human AdVs, MAdV serotypes are associated with distinct tissue tropisms: MAdV-1 infects macrophages whereas MAdV-2 infects intestinal epithelial cells. By swapping genes between MAdV-1 and MAdV-2, I hope to uncover the genetic basis of tissue tropism in MAdVs. Importantly, these studies will be aided by the availability of a cell culture system that recapitulates the cellularity of the intestinal epithelium and supports MAdV-2 replication, but not MAdV-1 replication. Due to genetic conservation, principles of MAdV tissue tropism are likely to apply to HAdVs. A major determinant of viral cell tropism is receptor usage, which may also play a primary role in tissue tropism. For both MAdV species, the trimeric fiber protein that extends from the icosahedral capsid is thought to be the viral attachment protein that binds to a host receptor. Although the MAdV receptors are not completely known, they are distinct for MAdV-1 and MAdV-2. To gain insight into tissue tropism, I have created a chimeric MAdV-1-M2f virus, where the fiber gene of MAdV-2 has been inserted in place of the native fiber gene in the MAdV-1 genome. A prior student in the Smith lab created the inverse chimera. If replication is fiber dependent, the chimeric MAdV-1-M2f virus should be capable of infection. By studying these chimeric viruses, I will uncover whether the fiber/receptor interaction plays a central role in determining tissue tropism.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide and has been established as a risk factor for neurodegenerative diseases such as Alzheimer’s disease (AD). The progression of AD is characterized by intracellular aggregates of phosphorylated tau protein, which is mainly found in neurons and plays an important role in the stabilization of microtubules. One of the mechanisms that may contribute to tau aggregation is decreased tau clearance by the glymphatic system, a pathway that clears solutes from the brain. This fluid movement is facilitated by the astrocytic water channel aquaporin-4 (AQP4) which is primarily localized to the astrocytic endfeet that line perivascular channels surrounding the brain vasculature. Prior studies demonstrate that solute clearance along these pathways is slowed following TBI, and that there is a loss of perivascular localization of AQP4. Based on these findings we hypothesized the loss of perivascular localization of AQP4 may impair interstitial tau clearance and promote neurodegeneration. We first tested this hypothesis by examining whether loss of perivascular AQP4 following TBI promotes tau pathology in a transgenic PS19 mouse that spontaneously develops tau pathology. We then evaluated whether deletion of perivascular AQP4 in an alpha-syntrophin knock-out mouse promotes tau pathology both in the presence and absence of TBI, and when crossed with a PS19 tauopathy mouse. Alpha-syntrophin is a protein that anchors AQP4 and is important in perivascular localization; therefore, deletion of alpha-syntrophin results in loss of localization of AQP4 and impairment of clearance. We assessed levels of pathological tau using histology on the transgenic mice and crosses both with and without TBI. If validated, our findings may suggest that loss of perivascular AQP4 may increase the brain’s vulnerability to tau aggregation and neurodegeneration following TBI and provide the basis for potential treatment to prevent the development of post-traumatic neurodegeneration.

Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive compound found in Cannabis sativa. The psychoactive and cannabimimetic behaviors associated with THC have been well described as being dependent on the partial agonist activity of THC at the endogenous cannabinoid 1 receptor (CB₁R). We are investigating the direct action of THC on the medial prefrontal cortex (mPFC, a brain region primarily responsible for executive function), and the effects of adolescent THC exposure on µ-opioid receptor (MOR) expression in adult periaqueductal grey (PAG, a brain region involved in opioid-mediated pain inhibition). To increase our understanding of the cannabimimetic behavioral effects of THC, and its direct pharmacological action in the brain, it is important to map the neuro-anatomical expression of target proteins. We examined expression patterns of CB₁R and MOR in the mPFC and the PAG, respectively. To do this, we utilized a form of in situ hybridization, RNAscope. We leveraged RNAscope by preparing tissue samples from brain regions of interest for treatment with mRNA-specific probes, allowing us to target CB₁R and MOR mRNA. After a series of washes and incubations, these fluorescent probes hybridize to our target mRNAs and allow us to visualize their expression under a confocal microscope. Analysis of mRNA expression informs us on the localization of the CB1R/MOR and known neuron types within our brain regions of interest. After imaging, we are able to utilize HALO software to analyze the levels of expression and co-localization of CB₁R/MORs with neuronal markers for glutamatergic and GABAergic neuron types. By creating and optimizing a workflow for extraction, preparation, hybridization, and analysis, we determined CB₁R mRNA is primarily co-localized with glutamatergic neurons in the mPFC. Moving forward, we are utilizing this RNAscope technique to investigate differential CB₁R expression GABA interneuron subpopulations in the mPFC. (Funded by DA051558)

Amyloid β (Aβ) plaques are a hallmark of Alzheimer’s disease (AD), the most common form of dementia that afflicts over 5 million Americans. Soluble proteins, including Aβ, are cleared from the brain by the glymphatic system, a brain wide network of perivascular spaces that facilitates the intermixing of cerebrospinal fluid and interstitial fluid. Prior studies report that aquaporin-4 (AQP4), a water channel polarized to perivascular astrocyte endfeet, supports glymphatic clearance of soluble proteins from the brain. In the aging brain, glymphatic clearance becomes impaired and AQP4 becomes depolarized from astrocytic endfeet. Such loss of perivascular AQP4 localization is correlated with AD status and Aβ plaque burden in the human brain. In the present study, we test whether such AQP4 depolarization promotes Aβ plaque formation. AQP4 is anchored to astrocytic endfeet via the dystrophin protein complex that includes the adaptor protein α-syntrophin (α-Syn). We crossed the α-Syn knockout mouse, which lacks perivascular AQP4 localization, with the 5XFAD mouse line which spontaneously develops Aβ plaques. Our preliminary analysis suggests that loss of perivascular AQP4 localization with α-Syn knockout increases Aβ burden relative to controls. These findings demonstrate that loss of perivascular AQP4 localization, such as occurs in the human brain in the setting of AD, contributes to the development of Aβ pathology. In the future, it may be possible that targeting the localization of AQP4 may be the basis for new therapeutics that can slow or even reverse AD pathology.

 Mu-opioid receptors (MOR) are expressed on populations of neurons within the brain. Exogenous activation of these receptors by drugs of abuse, such as heroin and fentanyl, causes feelings of euphoria and can be highly addictive. During natural behavior, MORs in the brain are activated by the endogenous ligand enkephalin. Spiny projection neurons (SPN) in the nucleus accumbens (NAc) are known to express enkephalin and likely release it during neural activity. It is known that these enkephalin SPNs can be either excited or inhibited while animals consume natural rewards. However, it remains unclear whether these functionally distinct enkephalin SPN populations are anatomically intermixed or are anatomically separated within NAc. In this study, we used 2-photon calcium imaging through endoscopic lenses to examine the neural activity of enkephalin SPNs in NAc while mice consumed sucrose rewards. We characterized the reward-excitations or reward-inhibitions of individual enkephalin SPNs over multiple imaging sessions. Through precise post-mortem histological examination, we then verified the anatomical placements of our endoscopic lenses and associated the relative anatomical location of neuronal populations to their reward-related neural activity. We found that enkephalin SPNs in anterior NAc were consistently reward-inhibited while enkephalin SPNs in posterior NAc were reward-excited. This is the first demonstration of anterior-posterior axis differences in the reward-related modulation of enkephalin SPNs and is a key step to understanding how opioidergic neurons function in natural reward behavior.

Alzheimer’s Disease (AD) is a neurodegenerative disease, characterized by amyloid-ß plaque deposition in the brain, that affects more than 5 million Americans. The glymphatic system is a network of perivascular spaces that facilitates fluid movement and solute clearance from the brain, and its dysfunction in aging has been implicated in the development of AD. The water channel aquaporin-4 (AQP4), located in astrocytic endfeet bordering the perivascular spaces, supports glymphatic function. In the aging rodent and human AD brain, loss of perivascular AQP4 localization is associated with impaired glymphatic function and increased amyloid-ß deposition. Yet the molecular basis for this loss of perivascular AQP4 localization is unknown. Aquaporin-4ex (AQP4ex) is a novel translational readthrough variant of AQP4. Selective deletion of AQP4ex results in the mislocalization of AQP4 all over the astrocytic membrane, indicating that AQP4ex is a crucial element in the perivascular localization of AQP4. In this study, we quantitatively analyze the expression and localization of AQP4ex to determine whether changes in AQP4ex associate with aging, AD status, or AD pathology. Using immunofluorescent double-labeling, confocal microscopy, and custom digital image analysis techniques, we define AQP4ex expression and localization between young and aged mice, and compare these changes between wild-type animals and transgenic animals that spontaneously form amyloid-ß plaques. Using a case series of post mortem human frontal cortical tissue, we compare AQP4ex expression between healthy young adults, cognitively intact aged subjects, and aged subjects with an AD diagnosis. This is the first characterization of AQP4ex expression in the murine brain and in a human case series, and these data will contribute to the small but growing body of research on AQP4ex and its relationship with AQP4 localization, creating opportunities to identify a new novel mechanism and novel target in AD pathology.

Environmental stress and threat influence feeding behavior in animals, but how that interaction occurs is still largely unclear. Neurons in the bed nucleus of the stria terminalis (BNST), part of the extended amygdala, have dense projections to the parabrachial nucleus (PBN) in the brainstem. We have uncovered projections in these neural circuits that link the modulation of feeding and threat assessment in mice. This project aims to investigate and characterize these previously unrecognized neural circuits with the incorporation of a variety of optogenetic, surgical, and histological techniques. We used Cre-dependent anterograde and retrograde viral tracers in order to trace the anatomy of these neural circuits, and found functional projections from inhibitory (GABA) and excitatory (glutamate) populations in the BNST to neurons in the PBN. We also used translating ribosome affinity purification (TRAP) to isolate the mRNA of these projections. This proved useful in separating and identifying the molecular expression profile of different GABAergic and glutamatergic subpopulations. Furthermore, we used a variety of behavioral assays to determine the BNST-PBN circuits’ role in feeding and threat-response behavior. We used fiber photometry to track the activity of GABAergic (vGAT) and glutamatergic (vGLUT2) populations during these behaviors, and found that vGAT and vGLUT2 populations have differing roles in threat and feeding behaviors. vGAT neurons increase their activity during feeding and decrease in response to threat, while vGLUT2 neurons decrease their activity during feeding and increase in response to threat. We also used optogenetic activation of these neurons to determine their causal role in behavior. With activation, vGAT populations drive place preference, operant positive reinforcement, and increased feeding. Conversely, vGLUT2 populations drive place aversion, operant negative reinforcement, and reduced feeding. These findings characterize the distinct nature of BNST-PBN neural circuits and the mechanism behind the evaluation of threatful stimuli and the integration of feeding.

Plant hormones are necessary for their growth, development, and overall function. Gibberellins (GAs) are a class of plant hormones that are used for cell elongation and growth. The GA pathway has been genetically manipulated in many crops to enhance agricultural yields. Research has also found that under elevated CO2 concentration conditions, GA dwarf plants revert to their less productive wild-type forms. This is very problematic for the agriculture industry because this reversion can potentially reduce their yields by half. Through my research, I hope to test whether modulation of GA signaling pathways can create crops that are more productive and better adapted to climate change. Interventions like this are needed because climate change is occurring more rapidly than plants are able to adapt to. Rewiring of the GA pathway is a potentially significant solution to this problem because we can modify plants in a manner that is likely transferable across many species. By using a novel genetic tool called a GA-sensitive Hormone Activated Cas9-based Repressor (GA HACR), we can modulate targeted genes in the GA hormone response pathway to turn down their transcriptional activity. The GA HACR targets specific genes through guide RNAs to repress the gene with complementary DNA sequence. The GA HACR is degraded in the presence of GA, which allows them to have a natural response to the activating hormone signal. We hypothesize that by targeting the HACRs to genes involved in GA biosynthesis (GA20 oxidase) and GA response (GID1 genes), we can modulate root growth of these plants. We are currently growing plants with ideal dwarf GA phenotypes in a CO2-enriched growth chamber to simulate future climate conditions and test how their growth responds. In this way, I will determine whether genetic intervention targeting the GA is a feasible strategy.

 Phytoliths are microscopic silica structures produced in plant tissue, and palms (family Arecaceae) are prolific producers of them. The stability of these structures contributes to the abundance of palms within the fossil record, as these structures can fossilize in sediments where leaves cannot, providing a wider range of evidence of the past palm geographic distribution. Palms generally grow in warmer and wetter environments, but certain species can live in more extreme climates (i.e., cold, dry). However, because 90% of palms are currently distributed around the equator and tropical rainforests, they are often used as an indicator of warmer environments in the fossil record. The current understanding of the taxonomic groups below the family level is limited, and it is unclear whether palm phytolith shape can be used to identify different palm groups in the fossil record. The Palm Project hopes to address this gap by testing phytolith classification using a morphometric approach. Over 100 species of known modern Arecaceae (~30 images/species) phytoliths have been imaged with a confocal microscope, and a semi-automated script in ImageJ quantifies the overall shape (globular or hat), density, and size of the phytolith ornamentations. Next, multivariate analysis on the morphometric data attempts to distinguish the phytoliths of various modern subfamilies based on distinct morphological traits, forming a quantitative baseline to describe and classify phytoliths. When combined with the collected ecological data, this could potentially identify signals between the environment of modern palm species and the phytoliths they form. Once the morphological differences between groups of modern palms are quantified, the same analysis can be applied to fossil phytoliths of unknown origins. This could reveal which taxonomic and ecological groups they belong to based on the most similar modern phytolith, giving insight into the phylogeny of Arecaceae and the paleoenvironment the fossils came from.

Reactive oxygen species (ROS) such as superoxide (O2–) and hydrogen peroxide (H2O2) are partially reduced oxygen molecules produced during cellular metabolism in all organisms. Plant chloroplasts and mitochondria are major ROS sources because of photosynthesis and aerobic respiration in these organelles. We previously found that the levels of oxidants were higher and antioxidants lower in maize (Zea mays) seedlings grown in the light than in the dark; these changes were accompanied by greater oxidative damage to the DNA in both chloroplasts and mitochondria in light-grown than dark-grown plants. In my research project, we will measure both H2O2 and antioxidants (catalase and peroxidase) after exposing dark-grown plants to light for increasing periods of time. Maize seedlings were grown in continuous darkness followed by 2, 4, 6, 12, and 24 hours of light. We then measured the levels of H2O2 and antioxidants in the chloroplasts and mitochondria isolated from leaf and stalk tissues during seedling development. Our data show the time course of ROS accumulation after the transfer from dark to light growth conditions and should provide a better understanding of the role of light in plant oxidative stress. Specifically, oxidative stress by ROS can be linked to DNA (mitochondria, plastid and organismal genomes) damage and by looking at the gradient of light exposure, it can open doors to discover how plants physiologically limit genetic impairment. This research can be used to assess more cancer-related topics in regards to ROS levels and how scientists can possibly find a solution in plant evolutionary-engineered genetics.

Despite recent advancements in cancer treatment, the 5-year survival rate for glioblastoma is only 22%. The greatest challenge in brain cancer treatment is the blood-brain barrier (BBB), a physical barrier that protects the central nervous system (CNS) from circulating solutes in the blood, but in cancer, prevents therapeutics from entering the brain space. While surgery is the gold-standard treatment, this procedure is high-risk. Thus, we are investigating injectable therapies to treat brain tumors by crossing the BBB. To do this, we are developing nanoparticles (NPs) to cross the BBB via receptor-mediated transcytosis (RMT). In RMT, ligands (keys) bind to receptors (locks) expressed by the BBB to gain entrance into the CNS. By attaching the ligand transferrin, we can trick the BBB into granting our NPs access to the brain. Prior to testing particles in vivo, I am developing a cellular model of the BBB to assess the ability of different NP formulations to cross the BBB in vitro. The transwell model comprises two main compartments: the donor and acceptor compartments, separated by a cellular monolayer that only allows transport between compartments via RMT. Here, I developed a co-culture consisting of brain endothelial cells and astrocytes. This co-culture model is more representative of the BBB and provides higher monolayer integrity than single-cell models. In parallel, I am investigating the use of acidification inhibitors to enhance the ability of NPs to cross the BBB. If successful, these inhibitors will be incorporated into a new NP design. Through this project, I am (i) developing a more representative in vitro model of the BBB and (ii) exploring alternative mechanisms to enhance NP transport through the BBB. Ultimately, these two aims will enable us to better direct NP behavior in vivo and cross the BBB.

Though renewed efforts in tuberculosis (TB) research have facilitated massive strides towards treating Mycobacterium tuberculosis (M.tb), TB remains a global health problem with an estimated 10 million infections and 1.4 million deaths in 2019. The ability of the pathogen to hide itself inside a granuloma, a mass of immune cells whose precise mechanism of regulation is unknown, prevents both the study of M.tb pathogenesis and the development of drugs or treatments. Current in vivo models have been established to study TB infection using animal models or tissues, limiting its biological relevance as it relates to human disease while current in vitro models lack components of the complex lung microenvironment during infection. We present the creation of a novel microscale infection model, which uses open and suspended microfluidic principles to enable spatial and temporal manipulation of cultures in suspended hydrogel plugs. By stacking together two devices, we demonstrate the ability of a suspended model granuloma consisting of M.bovis BCG (Mycobacterium bovis bacille Calmette-Guérin) and monocyte-derived macrophages to interact with a model vasculature layer consisting of endothelial cells. Analysis of soluble factors for proinflammatory cytokines and characterization of infection-dependent angiogenesis in the vasculature layer are used to verify communication between cultures. In the future, we envision this model expanding to contain multiple immune cell types and to incorporate additional aspects of the lung anatomy to approach a more accurate pathophysiological model as a tool for other researchers.

 Stroke is the leading cause of long-term disability in the United States. Disabilities can range from a loss of sensory function like touch to motor functions like controlling arm movements due to the damage to the brain’s network. Despite the prevalence of stroke, the underlying network dynamics that lead to functional deficits are not well understood. Due to the physiological similarities between non-human primate (NHP) and human brains, an NHP model is essential for studying the effects of stroke and developing therapies. Here we used the photothrombotic (PT) stroke technique to study network dynamics in the NHP sensorimotor cortex following an ischemic lesion. Using the PT stroke technique, we induced a focal ischemic lesion on the NHP sensorimotor cortex. We collected local field potentials from both hemispheres using an electrocorticographic  array on the cortical surface. As a measure of neural activity, we calculated ipsilesional power in the low gamma band. Channels were then organized into three clusters based on their net change in power (increase, decrease, no change). As expected, the cluster with an overall decrease in power corresponded to the lesion's physical location. We also studied the network connectivity by calculating pair-wise coherence across different frequency bands: theta, beta, low gamma, and high gamma. Overall, we saw that low frequencies were associated with decreases in coherence, while higher frequencies were associated with increases following stroke. Preliminary results from the contralesional hemisphere show similar changes. In this study, we observed local neurophysiological changes up to three hours following an ischemic lesion. The observed increases in power in the perilesional region and coherence at high frequencies suggest compensatory mechanisms immediately following an injury. We can use this study's results to guide future developments in stimulation-based therapy to alleviate the functional deficits from a stroke.

Glioblastoma (GBM) is a cancer originating in glial cells in the brain that accounts for more than 60% of all brain tumors in adults. The low survival rate can be attributed to high resistance to radiotherapy due to the hypoxic tumor environment which induces signaling networks in cancer cells that lead to the epithelial to mesenchymal transition (EMT). EMT gives rise to mesenchymal cancer stem cells (MSC) with a highly invasive phenotype which resists traditional means of therapy. Phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides, has been shown to regulate pathways that prevent ferroptosis, a unique iron dependent form of cell death initiated by an increase in reactive oxygen species. Disrupting the GPX4 pathway by siRNA-induced gene knockdown induces ferroptosis. Therefore, NPs as a vector for gene therapy may be able to eliminate mesenchymal state stem cells for a more effective treatment. Using hypoxia to induce EMT to develop a cell model for this work, preliminary results from quantitative real time PCR showed a correlation between GPX4 and EMT markers of human glioblastoma cells in hypoxia. GPX4 siRNA were evaluated using commercially available transfection agents on hypoxic and normoxic cells as proof of concept in vitro over a period of ten days. NP mediated delivery of validated siRNA were optimized using different ratios of NP and siRNA. Incubation time was also optimized. Finally, dual therapy of siRNA knockdown and radiotherapy were performed to evaluate sensitization of cells. The capabilities of NPs, along with concurrent radiation therapy, may provide a means to overcome radioresistance in GBM therapy.

The focus of this project is to study two newly discovered gene fusions that involve the Anaplastic Tyrosine Kinase (ALK) gene and define their role in Acute Myeloid Leukemia (AML) and potential for utility as therapeutic targets. ALK is a Receptor Tyrosine Kinase that contributes to brain development, regulation of cellular functions, and is linked to cell growth. Mutations and other genetic alterations are identified in various cancers. We recently discovered novel ALK fusions within a small set of AML patients in individuals with Monosomy7 AML (Loss of chromosome 7) and one of the most refractory subtypes of AML, making this particular group of patients high-risk. Additionally, ALK is otherwise not expressed in normal bone marrow making this a potential target. The current focus has been to verify if the ALK fusions are viable gene targets for chemotherapies that are already approved for clinical use, specifically Crizotinib, a chemotherapy drug and Tyrosine Kinase Inhibitor; by focusing on an FDA-approved targeted chemotherapy drug, this eliminates much of the process and time it would take to be able to treat AML in these high-risk patients. Crizotinib is successful in treating abnormal ALK expression seen in Anaplastic Lymphoma, Neuroblastoma, and Non-Small Cell Lung Cancer (NSCLC), and functions by preventing critical cell signals. We have engineered AML cell lines to express these fusions of interest in order to test Crizotinib in the context of AML.

 Cancer recurrence and migration remains a persisting challenge in oncology. As such, accurate models of tumor growth in the laboratory (in vitro) are critical to our continued understanding and treatment of the disease. This project aims to use one such model—the 3-dimensional spheroid—to measure the effects of novel targeted therapy combinations on different cell lines. Two cell lines were explored in this experiment: UM-SCC74A (a tongue squamous cell carcinoma) and HCT116 (a colorectal carcinoma). Spheroids were formed by culturing cells in ultra-low attachment well plates for 24 hours, allowing the cells to coalesce and form a 3-dimensional sphere. An extracellular matrix was added in some trials to further improve the accuracy of the model. Spheroids were cultured in the presence of drug for 48 hours, then allowed to grow in drug-free media following washout. Spheroid size and viability were assessed via imaging and Cell-Titer Glo assays, respectively. The results from both cell lines echoed those obtained in 2-dimensional assays and validated the efficacy of our drug combinations in a 3-dimensional setting. These models could provide an intermediate step between 2-dimensional in vitro experiments and those conducted in live organisms (in vivo). The success of this model in supporting different cell lines affirms the robustness of this approach and its potential for use with a wide variety of cancers.

The female reproductive tract must maintain normal reproductive functions while also being able to elicit immune responses to sexually transmitted microbes and viral pathogens. In response to virus exposure, the balance of these two functions can determine the outcome of infection and disease through alteration of vaginal mucosa integrity. Recent in vivo research in our laboratory revealed that vaginal infection by Zika virus (ZIKV), an emerging mosquito-transmitted flavivirus that is also sexually transmitted among humans, induces epithelial cell-specific innate immune response that accelerates a homeostatic form of cell death known as cornification. We hypothesize that specific processes in vaginal epithelial cells mediate cornification in response to ZIKV infection, and that this cornification may alter barrier properties, innate-adaptive immune crosstalk, and ultimately, the degree to which ZIKV disseminates from the female reproductive tract. We therefore analyzed and compared vaginal epithelial cell infection by both African and Asian lineage ZIKV strains using traditional 2-dimensional cell culture and organotypic 3-dimensional culture infection models. Differences in viral infection and replication kinetics, and innate immune response were characterized through RT-qPCR, immunoblot analysis, cell imaging, and viral plaque assay. Ongoing analyses are expected to reveal the application of organotypic 3-dimensional cultures in capturing in vivo qualities of vaginal epithelial cell/ZIKV infection and response compared to 2-dimension cultures. These studies will provide insights for application of vaginal epithelial cell culture models of ZIKV infection that encapsulates the complex functional and structural aspects present in vivo.

Tuft cells are a lineage of chemosensing epithelial cells that contribute to intestinal “type 2” immune responses during parasitic worm (helminth) infection. Tuft cells are thought to monitor the intestinal lumen, looking for type 2 agonists. While rare at steady state, during a helminth infection the number of tuft cells in the gut increases greatly (a phenomenon called hyperplasia), directly proportionate to the underlying immune response. Nippostrongylus brasiliensis is a common model of hookworm infection. This parasite is injected under the skin, migrates to the intestine, and can be cleared within 10 days, with a tuft cell response mediated by the cytokine interleukin (IL) 25 and largely independent of the IL33 receptor, ST2. In contrast, the roundworm Heligmosomoides polygyrus is ingested orally and has a lifecycle in which it matures inside the intestinal tissue rather than in the lumen, then re-enters the lumen and establishes chronic infection. Given their distinct lifecycles, we hypothesized that N. brasiliensis and H. polygyrus are sensed differently by tuft cells. Since the kinetics of tuft cell hyperplasia during N. brasiliensis are well defined, we created a comprehensive timeline of tuft cell hyperplasia in wild type mice infected with H. polygyrus for comparison. Intestinal tissue was collected from three mice on every other day of infection, until 20 days post infection. We used immunofluorescent staining to quantify the tuft cells on each harvest day and discovered distinct waves of tuft cell hyperplasia that align with the two separate times that H. polygyrus worms enter the lumen. This timecourse highlighted two peak timepoints of hyperplasia, which we studied further to test the hypothesis that IL33 is critical in the H. polygyrus immune response. Through this work, we strive to better understand how and why our bodies respond to the various type 2 agonists, and how this response can be most effective.

Cystic fibrosis (CF) is a genetic disorder characterized by chronic lung infections involving various organisms, including the gram-positive pathogen Staphylococcus aureus. Antibiotics, such as trimethoprim-sulfamethoxazole (SMX), play key roles in treating CF infections; these drugs inhibit bacterial growth by disrupting important bacterial metabolic processes. SMX specifically inhibits folate metabolism, causing DNA damage that results in bacterial cell death. However, S. aureus is able to persist in CF pulmonary infections despite treatment with antibiotics, and evidence suggests that S. aureus does so through adaptive mutations. Our goal is to identify the adaptive mutations of S. aureus during SMX exposure in vitro, to understand how this pathogen persists and to prevent the emergence of resistance. We grew S. aureus in the presence of super-inhibitory SMX concentrations for 24 hours in Luria Bertani broth. We sampled the culture at specific timepoints and measured viable bacterial counts on chocolate agar; we evaluated all resulting colonies for SMX susceptibility and associated genetic changes. Surprisingly, we identified mutants that survived SMX treatment carrying diverse adaptive changes not associated with folate metabolism or DNA repair, suggesting previously-unknown lethal effects of SMX against S. aureus. These mutants carried mutations predicted to decrease production of reactive oxygen species (ROS) - toxic compounds produced by all cells during aerobic respiration and in response to stress. Our results indicate ROS may play a role in SMX-mediated S. aureus cell death, suggesting that treatments that augment the effects of ROS could improve antibiotic efficacy. We are now exploring the involvement of ROS in S. aureus killing by SMX using engineered S. aureus strains with knockout and overproducing mutations in ROS detoxification genes. This study will help us better understand SMX’s mechanism of action and S. aureus’ response to this drug, in order to improve the treatment of diverse infections caused by this pathogen.

Skin cancer is the most prevalent cancer in the United States, and its annual incidence exceeds all other cancers combined. There is thus a pressing need to develop novel approaches to inhibit skin cancer. Inactivation of tumor suppressor genes is a frequent event in carcinogenesis. In skin cancer, the CDKN2A gene, which encodes the p16^INK4A tumor suppressor protein, is often silenced by epigenetic abnormalities such as promoter DNA methylation and histone deacetylation at this genomic locus. The p16^INK4A tumor suppressor functions as a cell cycle regulator and plays an important role in tumor growth and metastasis. Unlike gene loss, gene silencing by aberrant epigenetic modifications can be reversed by small-molecule compounds such as DNA methyltransferase inhibitors and histone deacetylase inhibitors. However, these small-molecule inhibitors are nonspecific, affecting epigenetic modifications globally. Here we use CRISPR-Cas9-based epigenome editing tools for targeted histone acetylation at specific genomic loci. To induce histone acetylation at the CDKN2A promoter, nuclease-deactivated Cas9 (dCas9) is fused to histone acetyltransferase p300, and the dCas9-p300 fusion protein can be recruited to the target site by using guide RNA (gRNA) that binds to the CDKN2A promoter. After dCas9-p300 with gRNA was introduced into the A431 skin cancer cell line to target the CDKN2A promoter, RT-qPCR revealed that p16^INK4A mRNA expression levels increased by approximately 30-fold. Cell proliferation assays further revealed that proliferation of A431 cells decreased by approximately 20%. To fully inhibit proliferation of skin cancer cells, further investigations are needed to evaluate the combined effects of targeted histone acetylation and DNA demethylation at the CDKN2A promoter. This study provides insight into how epigenetic abnormalities can be targeted by novel epigenome editing tools in order to reactivate dormant tumor suppressors and inhibit cancer phenotypes.

Over the past fifty years, survival rates for most cancers have risen as innovative treatments have been developed. However, Diffuse Intrinsic Pontine Glioma (DIPG), a rare pediatric brainstem tumor, has seen no such improvement and remains one of the deadliest cancers. DIPGs are genetically distinguished from adult gliomas by a lysine-to-methionine mutation in a variant of histone H3 called H3.3 (H3.3K27M), found in 80% of DIPG tumors. This mutation is absent in canonical H3.1 and H3.2, yet it triggers a global reduction in the levels of polycomb repressive complex 2 (PRC2) mediated H3K27me3 (tri-methylation) marks, which is traditionally considered a driving event in tumorigenesis. However, genome-wide studies of H3K27me3 marks in DIPG cells have revealed that this global loss of H3.3K27me3 is accompanied by a sharp increase in H3K27me3 repressive marks at certain genes. These trimethylation spikes represent regions with high transcriptional repression and may be key in understanding and treating DIPG. Our lab has previously described the inhibition of PRC2 using a computationally designed protein EBdCas9. Using a complementary guide-RNA tiled to the promoter region of a gene, I can target EBdCas9 to that specific region on the genome, remove existing H3K27me3 marks, and increase gene transcription. Identification of candidate genes which are repressed in DIPG cells under these H3K27me3 spikes remains an ongoing project that I am working on. Results of one target, tumor-suppressor gene p16, demonstrate that I can transfect primary DIPG cells with EBdCas9 plasmid and a p16-specific gRNA to trigger a 20-fold increase in p16 expression. I hypothesize that restoration of critical cell-cycle genes like p16 will reduce DIPG viability and offer potential therapeutic targets. I’m currently working to improve transfection efficiency in order to increase p16 expression, and future steps include in-vivo testing of this system, either via mouse model or a brainstem organoid.