Recent political administrations have focused on issues surrounding immigration in the U.S., affecting the ways that immigrant communities are seen and treated. This project recognizes that the rhetoric used by political actors influences and perpetuates views about immigration, and asks the question of how rhetoric around immigration in the 21st Century has affected the identities and experiences of Mexican immigrants in the United States. Drawing upon interviews and secondary research sources, including print and social media, we construct an intertextual and diverse series of podcast episodes, which feature candid discussions with members of the immigrant community. These interviews allow us to gain a more personalized understanding of how public rhetoric, as perpetuated by political administrations and the media, have impacted and shaped the lives and identities of real individuals, families, and communities. Through this podcast, we hope to inspire conversation around the intersection between rhetoric, politics, and immigration, while providing more insight into and compassion for individual, real-world experiences of the immigrant community.

 Fascia is a broad term referring to the connective tissue of the body. Fascia has multiple functions: it maintains the shape of different anatomical components, allows cells to convert external mechanical signals into internal biochemical reactions, and distributes the stress of movement and gravity across multiple muscles and joints. The purpose of this study was to gain an in-depth understanding of the physiology of fascia, explore therapeutic practices involving fascial manipulation, apply this knowledge to dance movement and injury prevention, and educate dancers and teachers about the “why” behind these common therapy practices. More specifically, I wanted to understand the mechanistic science behind foam rolling and massage therapy in order to support the hypothesis that these therapies are fascial manipulation and enhance athletic ability by remodeling the fascia on a physiological level. To support this hypothesis, I reviewed literature and scientific studies relating to fascia and athletic ability, participated in an untreated cadaver dissection course that focused on connective tissue, and interviewed professionals in the field of therapeutics. My preliminary results indicate that foam rolling and massage temporarily enhance athletic ability and prevent injury; however, the mechanism behind these results is still unclear. There is some evidence to support that these therapeutic techniques involve fascia, but other mechanistic theories unrelated to fascia may also explain these results. More research on fascia and the “why” behind these therapies is needed to fully uncover the applications of fascia for dance movement and injury prevention. However, it can be concluded that the implementation of these therapies should be considered for dance warmup and cool down practices to help dancers reach their fullest movement potential, maintain their bodies, prevent injuries, and achieve longevity throughout their dancing careers.

Disordered eating, poor body image, and eating disorders are common and ongoing issues in the world of dance. These issues are caused by a complex web of factors, including dance training, perfectionistic tendencies, comments made by friends and family, and social media. This study looks specifically at how the dance classroom environment contributes to the dancer’s relationship with food, eating, and diet. I collected data from three different sources: a survey sent to dance majors and minors at the University of Washington, a survey sent out to one randomly selected dance studio from each U.S. state, and case study interviews. Anticipated results may suggest that the environment created by dance studios and teachers can have long term effects on a dancer’s relationship with nutrition. Dance instruction that places emphasis on dancers’ bodies, as opposed to their movement, can contribute to a more turbulent relationship with nutrition. These findings shed light on the need to analyze and reconsider dance pedagogy, teacher training, and dance studio policies (whether implicit or explicit) surrounding nutrition.

Color guard is a youth performing arts activity usually associated with marching band or drill team. Although the specific context of color guard performances may vary, the central components require performers to manipulate equipment such as flags, wooden rifles, and dull metal sabres, while maintaining a performative, dance-based foundation in relationship to musical accompaniment. The purpose of this study is to determine the role of dance in the world of color guard, and to investigate ways of implementing dance training to enhance color guard educational programs. Interviews were conducted with four seasoned color guard instructors from various geographic locations. Interviews were recorded, transcribed, and analyzed to identify how these veteran teachers view the role of dance in color guard, how they determine success in their programs, and how they have been able to utilize dance to their program’s benefit. Preliminary results indicate that focusing on dance training and body awareness significantly contributes to performers’ success in competition, as well as assists with the social/emotional challenges facing this population. These findings suggest a need for further investigation into teaching practices utilized by instructors in order to illuminate how pedagogical training impacts students.

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.

Specifically addressing proprioception’s relationship to balance control, recent studies have noted how ankle proprioception may be one of the most important components to balance control because of its direct contact relationship with the ground. Dance training has been shown to improve a dancer’s proprioceptive abilities and is one of the fundamental reasons why dancers are able to flawlessly execute complex balance movements on a very small base of support with lights shining in their eyes and while under constant pressure from a judgmental audience. In this research, I aim to explore how proprioception may be improved through dance. My methods are grounded in a literature review from around the world and anecdotal evidence through interviews and video footage of three individuals focusing on improving their static balance. Results from the literature review indicate that dance can be used to improve one's proprioception and balance control in both the youth and elderly population. Video footage analysis and interviews partially support my initial claim that balance can be improved in all individuals. Understanding the kinesiology of balance may contribute to future alternative movement therapies that use targeted dance movements to alleviate symptoms of specific conditions, such as ankle proprioception movements for individuals with Alzheimer's Disease or neck proprioception movements for individuals with chronic neck pain.

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.

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.

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.

Water is universally critical for life on earth, and snowmelt plays an essential role in the hydrologic cycle, contributing up to 75% of water supply in much of the western United States. As a result, estimating the timing and magnitude of snowmelt is an integral water resources challenge; snow surface temperature observations are key to this issue. It is well established that snow melts at 0°C, so frequent snow temperature measurements supply crucial information for evaluating snowmelt. However, few ground observations of snow surface temperature are available, and those that are only represent a small area. By measuring infrared radiation, satellite thermal imaging can remotely determine surface temperature over large areas and time scales where ground observations are sparse or nonexistent. However, this imagery is limited by its coarse spatial resolution, which results in a blurring of temperatures across study regions. This challenge is especially prevalent for mixed pixels, or pixels with varied land surfaces such as a mix of snow and vegetation or changing topography. Thus, this project investigates how well satellite imaging represents snow surface temperature. Airborne thermal imagery were acquired by the UW Applied Physics Laboratory over Yosemite National Park, California, coincident with ASTER satellite imagery on 21 April 2017. I apply methods including data analysis and zonal statistics over the study area in order to compare finer resolution airborne imaging to coarser resolution satellite imagery. Furthermore, I calculate characteristics of land variation to evaluate where satellite imagery can be scaled for more accurate results. This project works towards the implementation of satellite thermal infrared for use in snow models, creating new datasets for hydrologists to more effectively plan our water resources.

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.

Human pluripotent stem cells (hPSCs) are cells capable of self-renewal while differentiating into any cell type in the body. The differentiation into skeletal muscle progenitor cells (SMPCs), results in mature skeletal muscle cells. As the potential of deriving SMPCs from hPSCs has been further researched for medical application, the problem encountered is how to generate fully functional skeletal muscle cells from hPSCs experimentally. Currently, there are many existing protocols (e.g., HX, JC, MS Protocols) for such differentiation, known as myogenesis, but each has problems that need to overcome (e.g., time-consuming, not fully functional cells, resulted in other cell types). Our goal is to figure out what genes and metabolites are involved in the natural process of differentiation (from prenatal skeletal muscle progenitors to satellite cells) and apply the knowledge experimentally. To address this, we present the use of computational methods for the biological network-based integration of transcriptomic and metabolomic data. By analyzing data for both in vivo process and in vitro protocol results, we can find potential genes and metabolites differences, relating from the transcriptomic level to metabolomic level, revealing the inadequacy in the experimental protocols, presenting probable genes to improve the results. This involves RNA sequencing data analysis (e.g., single-cell, microarray) of human and mouse cells from embryonic to postnatal stages, with Seurat (pseudo-time analysis), Monocle, and AFFY, which processes/analyzes the data revealing potential genes. Then further analysis with UKIN, guided network propagation, to rank and identify the most probable genes, and perturb-Met to analyze metabolites involved. This is a time and cost-efficient way to find most probable genes directing the differentiation, which can then be tested and verified experimentally. If successful, it can be further developed into potential treatments much more effective and efficient than available medications and technologies for presently incurable musculoskeletal diseases.

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.

Many human diseases, including cancer metastasis, and developmental disorders, involve sheets of cells moving together. This concerted process, called collective cell migration (CCM), is critical to maintaining the integrity of the sheet of cells during movement. Maintaining cell sheet integrity is dependent on individual cells retaining their shape while still connected to neighboring cells. To study collective cell migration, we use Drosophila melanogaster (fruit fly) embryogenesis. Dorsal closure (DC), an event in Drosophila embryogenesis, is an example of CCM. During DC, two epithelial layers wrap around the embryo. Because CCM’s core mechanics are conserved, studying DC provides an opportunity to indirectly study human diseases/disorders function in a model that has fewer genetic redundancies and is easy to manipulate. Adherens junctions (AJs), are the site of adhesion between individual cells, and what allows for dynamic rearrangements like CCM by strategically increasing or decreasing adhesion. Despite the importance of this process and its relevance to many diseases, CCM is highly complex and how the scaffolding protein Canoe stabilizes adherens junctions under high stress is not entirely understood. We are interested in how junctions between cells are stabilized during a dynamic movement like in CCM. Using a maternally driven Gal4/UAS system, we created moderate canoe knockdowns. We then performed targeted, immunofluorescence antibody staining of embryos during DC. We expect that the loss of Canoe will lead to increases in cell-sheet tension as well as the compensation by other stabilizing proteins like Polychaetoid. Our research improves our understanding of stabilizing proteins at AJs and CCM and may lead to the more effective treatment of disrupted CCM human diseases/disorders like wound healing, palate formation, and neural tube closure.

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.

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 effects lobbying has on Congressional voting has been debated for a long time. Despite scholars’ consistent findings that there is no relationship between campaign contributions and a legislator’s vote, special interest groups continue to routinely contribute money to members of Congress. In this project, I consider the connection between contributions from the finance industry and failed legislative efforts aiming at regulating Wall Street or providing greater financial consumer protections. I employ multivariate regression analysis to examine lobbying contributions to individual members of Congress and their subsequent roll call votes in Congressional sessions during 2009-2018. I test for an explicit connection between the finance industry’s lobbying contributions and the inability for Congress to pass financial reforms post-2008. Since campaign finance plays a vital role in the ability for a politician to be elected to office, I expect to find that a congressperson receiving contributions from the finance industry will be inclined to vote in favor of those interests. The findings from this research contribute to the conversation of the influence of big money in American politics and the influence those interests have on specific legislation.

Budget policy in the United States is not uniform across agencies, with some budgets subject to cuts year after year. This research looks to answer why some agencies are more affected by budget cuts. I theorize that differences are explained by the politicization of the agency’s function. Specifically, I emphasize the role of neoliberalism, a political ideology which promotes fiscal and moral conservatism, as well as laissez-faire approaches to government. This presentation hypothesizes that subfunctions that have roles which conflict with neoliberal values, such as those pertaining to education, training, and social services, will experience non-incremental budget decreases more frequently than subfunctions that reflect greater neoliberal values. To test this theory, annual appropriations data for 64 subfunctions was collected, resulting in a database of budget outcomes from 1977 to 2018. Preliminary multivariate regression models suggest that while a subfunction’s “neoliberalness” has some effect on the magnitude of budget cuts, specific subfunctions can experience budget patterns that are not explained by neoliberalism alone. This research adds to existing policy literature by exploring the intricacies of budget-making that have previously been dismissed as chaos.

With political polarization in the United States leading to widespread legislative gridlock, the nation is in dire need of instruments to encourage legislators to cooperate with their colleagues regardless of partisan affiliation. This research will investigate how Congress’ ban on earmarks might have affected the tendency of legislators to vote in favor of bills that are not supported by their parties. Earmarks are commonly understood to act as a form of inducement through which legislators can gain electoral benefits within their constituencies in exchange for their cooperation with Congressional leadership. I expected that the presence of more earmarks in a bill would increase the tendency of legislators to back bills that are not supported by their parties. I employ multivariate regression to compare the relationship between the number of earmarks in appropriations bills and the rate at which legislators defected from their party’s position on roll call votes taken. This information is available for bills introduced between 2008 when the rule mandating the declaration of earmark requests went into effect, and 2010 when the ban was enacted. If the data supports this hypothesis then my work will imply that the ban should be repealed to reduce the negative effects of legislative gridlock. If no relationship is apparent; however, it will represent a significant indicator that earmarks in their most recent form provide no benefit beyond the enrichment of the legislators themselves, which would suggest that the ban on earmarks should be maintained.

The United States military is a massive proponent for airstrikes and utilizes them in place of ground forces. Although people expect airstrikes to increase precision and reduce civilian casualties, military conflicts continue to last for years. Nevertheless, the United States continues to rely on airstrikes as a principle strategy despite prolonged military conflicts. It is puzzling why the United States would transform military strategy to include a heavy reliance on airstrikes despite few indicators showing its success as a strategy. This research paper examines whether U.S. airstrikes reduce the magnitude- or the total civilian casualties- resulting from military conflicts. I expect to find that airstrikes increase civilian casualties and theorize that the use of these strikes is more for domestic favorability than any military benefit. I explore this expectation by using multiple regression analyses on wartime data from the Iraq War and the Syrian Conflict. I analyze the United States' use of ground forces during the Iraq War and its use of airstrikes during the Syrian Conflict, all while controlling for other actors and forces within these conflicts. This analysis provides insight into the capabilities of drone strikes as a means of ending conflict. 

 Marguerite Trost is an elite Division I women’s rower, who has competed since the age of fourteen, even attaining unofficial world records in the lightweight class. After a strong freshman year performance at the University of Washington, Marguerite suffered a sacral stress fracture which derailed her sophomore season. Despite a perceived state of full recovery, Marguerite suddenly hit a wall, and it seemed as if she had forgotten how to row. Her body no longer listened to her mind, and she felt as if she was disconnected from the movements that she had found second-nature for so long. After a period of anguish and self-discovery, she began to research the barriers she was now facing, hoping that someone, anyone, may have experienced something similar. And it turns out, they had. This paper discusses the “yips”, an under-researched, yet surprisingly prevalent phenomenon found primarily in sports that require precision to a target, such as baseball or golf. The yips can be defined as a movement disorder, one that has physical and mental etiologies, and entails a seemingly irreversible disconnect between an athlete’s muscle-memory and the motion it produces: a task-specific focal dystonia. There is little to no literature on this phenomenon in rowing specifically. In our paper, we intend to explore and evaluate the convergent factors of rowing “yips” such as injury, the Relative Energy Deficiency in Sports (RED-S), mental health, and societal pressures that all influence an athlete’s experience and ultimate performance. Through interviews with sports medicine professionals, ancillary team staff and impacted athletes in multiple sports, as well as current Division I athletes, we will glean a broader understanding of the contributing factors to develop a framework for further study. Additionally, we expect many directions of neurological, physiological, and sociological research to stem from this beginning.

The University of Washington Bothell (UWB) has a campus emergency response team (CERT) that is not widely known or sustainable. Part of its mission is to train students to participate in emergency response and engage them in other CERT activities. However, UWB CERT struggled in its efforts to recruit students for CERT training and raise awareness of CERT and hazard threats among students. This research project analyzed the successes and best practices of CERT programs in other universities across the Unites States and assessed which practices would be most feasible for implementation at UWB. Research methods included reading case studies about best disaster preparedness practices and conducting interviews of staff at different universities to see what works on each campus. A report centering on recommended best practices will be provided to UWB administrators. This should help UWB develop a plan for the creation of a sustainable CERT program which will greatly increase the number of students trained in emergency preparedness and response skills that they can apply on and off campus, as well as keep the university accountable to student safety.

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.

Due to the COVID-19 outbreak, in-person mental health services became, in many cases, prohibited, increasing the need for telehealth. The present study will investigate clinicians’ perceptions of the effectiveness of delivering cognitive-behavioral therapy (CBT) via telehealth. Telehealth, in this context, refers to remotely delivering psychotherapy via a variety of platforms such as on-line services, video, and/or phone calls. Previous studies found that delivering CBT via telehealth can be as effective as in-person delivery. However, there are barriers to implementing psychotherapy remotely, including lack of training support, organizational and clinician buy-in, and cost. Thus, we aimed to investigate whether clinicians found delivering CBT via telehealth effective, whether it had an impact on engagement and therapeutic relationships, and how well elements of CBT training were provided during telehealth delivery. Data came from a project leveraging a Washington State CBT+ Initiative. Clinicians and supervisors from agencies in WA state participated and got training in CBT for youth depression, anxiety, trauma, and behavioral problems. Clinicians completed pre-and post-training surveys with questions pertaining to how telehealth preparation time compared to in-person sessions, clinicians’ opinions on continued telehealth implementation, clinicians’ ability to deliver effective services via telehealth, and client and caregiver engagement levels over telehealth. We plan to run descriptive analyses to summarize clinicians’ perceptions of telehealth. Preliminary analyses suggest that most clinicians found telehealth to be beneficial and an effective way to deliver CBT to youth. Further, our analyses reveal that clinicians, on average, found engaging younger children in treatment as the most significant barrier associated with telehealth. Harnessing telehealth for the provision of CBTs may be a viable solution that addresses some barriers to access. If telehealth is found effective by clinicians, it could serve as a long-term option for delivering CBTs. Therefore, it is important to understand clinicians’ perspectives.

Diversity training in organizational environments has become commonplace in the 21st century. Most contemporary training seeks to make employees aware of implicit bias and the structural inequities that exist from systematic oppression. However, training is often not grounded in evidence-based practices and past research has shown diversity training may increase stereotyping and bias towards minorities. Additionally, high-status groups (e.g., White people, men) may perceive increased unfairness towards high-status members due to the emphasis on diversity, commonly referred as “reverse racism” or anti-White bias. This study seeks to further investigate how applicants view an organization's approach to diversity. White participants will be asked to apply for a scholarship and subsequently rejected. By investigating anti-White bias after a rejection, this study aims to examine whether Whites rationalize rejection with pro-diversity messages to protect against threat. I am also interested in perceived employability: a principle that has been shown to be linked with emotional regulation, health, and self esteem. I predict the magnitude of perceived qualification for a position moderates the degree of anti-White bias reported. Research on organizational diversity is imperative to decrease turn-over rates and increase satisfaction for minority employees.

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.

As dam removals become more frequent across the United States and globally, understanding kelp forest response to coastal dam removals, in particular, is critical for managing nearshore habitat. Dam removals impact flow and sediment regimes of rivers, both of which can influence coastal kelp forests. The removal of the Elwha River, WA dams released ~19 Mt of sediment into marine ecosystems. This sediment flux dramatically influenced the turbidity of the nearshore water column and permanently increased the amount of sediment released from the river. The objectives of this study were two-fold. First, I developed methods to remotely sense and quantify the abundance of canopy forming kelps from aerial images. Next, I determined how changes in suspended sediment concentration and light attenuation during and following a major dam removal event in the Elwha River impacted the growth of nearshore bull kelp, Nereocystis luetkeana. By incorporating remote sensing to quantify the relative abundance of bull kelp in the area, my study observed that following dam removal, bull kelp abundance declined in the study area. This was an unexpected finding because I calculated that suspended sediment concentration decreased following dam removal, thus improving conditions for kelp growth. Although suspended sediment concentration decreased after the dam removals, other factors, such as an increase in herbivorous predation, could have played a large role in suppressing bull kelp abundance. The supervised classification scheme that I developed for remotely monitoring kelp abundance will make analyzing larger areas feasible in future studies, which may help to better identify regional trends in kelp abundance. Marine aerial monitoring remains an important avenue to better predict and manage future kelp forest response to dramatic changes in the ecosystem.

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.

I am furthering research in growing successful plant life in Martian regolith. The intention of this research is to amend Martian regolith so that it can support edible plant growth. We intend to counteract the characteristics that would impede plant growth and introduce low-mass additives that support plant growth. To simulate Martian ground “MMS-2” is used, this regolith is designed to be over 90% similar in chemical and textural composition to ground cover on Mars.Challenges noted from previous research include inhibited root growth, inefficient nutrient absorption, and impeded water uptake due to clay-like consistency. The additives selected to counteract these challenges include vermiculite to expand the soil and improve water circulation, and Biodyne™ microbes to enhance nutrient uptake. I chose to also incorporate mycorrhizal fungi to support and enhance root development and function. My previous research indicated that mycorrhizal fungi played a significant root development, plant height, and leaf growth. My current research is focusing on reproducing these results with more specimens and clear data points to further evaluate the impact of these additives. Rosemary was selected due to its ability to thrive in harsh, cold, and dry climates. As this plant is notoriously difficult to grow from seed, seeds were pre-sprouted to confirm viability prior to planting in the experimental pots in order to assure more specimens. Experimental regolith are treated with mycorrhizal fungi, Biodyne™, or both receive treatment mixed in water; all regolith and the control are based in a 30/70 earth soil to regolith blend with vermiculite. Plants treated with mycorrhizal fungi are anticipated to have darker and plentiful leaves; plants treated with Biodyne™ are anticipated to show straighter growth, plants treated with both are anticipated to be the most successful with both plentiful dark thick leaves and straight strong growth.

We are expanding previous research which manipulated the composition and chemistry of Mars regolith to better support plant life. The Mars regolith we are using is a 70% regolith 30% compost mix. Our focal plant for this experiment is Salvia rosmarinus (hereinafter rosemary) because of its resilience in cold climates and minimal water needs. Rosemary has been observed to have low germination success in both Earth soil and Mars regolith, to address this we sprouted seeds in water prior to transplant in order to guarantee seeds were viable rather than sowing rosemary directly in Mars regolith. We will ensure cotyledons have developed and stems have reached <5.0mm in length prior to transplanting in regolith to eliminate the variable of low seed viability. We hypothesize the amendments made to Mars regolith along with microbial and mycorrhizal additives will significantly improve rosemary development and growth. Plants we have previously attempted to grow in Mars regolith experienced difficulty establishing roots when transplanting cuttings and when grown from seed due to the clay-like consistency of the regolith. We have adjusted the previous experimental design by adding vermiculite to improve drainage and reduce regolith density, microbes to fixate Nitrogen and assist in nutrient uptake, and mycorrhizal fungi to encourage more effective root establishment and development. Previous experiments with Biodyne™ microbes and mycorrhizal fungi in Mars regolith have shown promising results in kale and rosemary. We anticipate the addition of Biodyne™ and mycorrhizal fungi, both independently and together, will improve root development, plant height, leaf growth, and chlorophyll richness. To test this, we will collect data bi-weekly by noting plant “greenness” and measuring plant height, leaf length, leaf count, and root growth.

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.

Vaccine hesitancy reflects concerns about the decision to vaccinate oneself or one's children. There is a broad range of factors contributing to vaccine hesitancy, including the compulsory nature of vaccines, their coincidental temporal relationships to adverse health outcomes, unfamiliarity with vaccine-preventable diseases, and lack of trust in corporations and public health agencies. In addition, the very success of vaccine programs has seemingly fueled increasing vaccine concern as vaccine-preventable diseases are no longer prevalent in the United States. This decrease in prevalence has led the attention of the public to shift from the necessity of vaccines to the safety of the vaccines themselves. In light of the current COVID-19 pandemic and the heavy economic and public health toll it has taken, it became clear that a vaccine may be the “magic bullet”. In the midst of a pandemic that is taking thousands of lives and has been devastating society, many people will find these uncertainties acceptable. While for others, as with many trade-offs, the benefit may have less emotional resonance than the possibility, no matter how small, of a potential risk. My research seeks to understand the risk perceptions of vaccination in an adult population in the Puget Sound region of Washington state. My hypothesis suggests that social proximity to COVID-19 will influence vaccine decision making & vaccine risk perception. I will be using the annual influenza shot as well as assessing smaller recent outbreaks to build a framework for my thesis. Understanding the factors that are influencing adults, when it comes to vaccine decision making, may help inform strategies for state and public health officials in their multifaceted roles of communicating effectively on the benefits and safety of vaccines to policy makers, media, and the public as a means to better support informed decision making.

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.

Using a large NOAA Storm Prediction Center database covering United States and Canadian tornadoes, our group analyses the traits and tendencies of tornadic activity across North America. With recent improvements in tornadic data collection, there is a surplus of topics to thoroughly explore. Some of our work includes investigating relationships between two parameters such as CAPE (convective available potential energy), and CIN (convective inhibition) or precipitable water and temperature. Furthermore, we created histograms with Gaussian curves and probability plots for the most critical parameters for tornadic development to better understand the distribution of data and how much it deviates from a centered mean. Currently, we are in the process of creating filled contours to visualize the geographic distribution of parameters across the United States. Our goal is to create a more nuanced tornado climatology. In doing so, we hope to further understand tornadoes and their tendencies with hopes of making our interactions with these events much safer.

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.

How do ethnically-oriented mutual support groups like Alcoholics Anonymous (AA) exhibit cultural competence and ensure improved recovery outcomes? Previous research suggests that cultural competence, or the distribution of culturally relevant services within treatment structure, can help ensure successful recovery. However, no prior research has examined to what extent such competencies have been integrated into AA meetings or the effectiveness of them in ensuring recovery from alcoholism. This limitation prevents the understanding of necessary practices of substance abuse treatment that could lead to the successful recovery of ethnic minorities. This study investigates whether (1) addiction focused mutual-support groups like Alcoholics Anonymous exhibit cultural competence in organizational practices of ethnic-specific groups, and (2) the relationship between levels of cultural competence and 12-Step treatment completion. I examined six different AA groups in the greater Seattle area, consisting of both general and ethnically-defined AA groups. Interviews were used to assess the prevalence of cultural competence in ethnic-specific meetings and determine whether there was a link between culturally competent practices and successful treatment outcomes. Utilizing the qualitative data analysis computer software package, NVivo, I looked for themes or language within the interviews that corresponds with the different dimensions of cultural competence. The expected results for this study are that ethnic-specific AA meetings do exhibit cultural competence and that levels of cultural competence are positively associated with successful 12-Step treatment completion. My analysis has practical and theoretical implications. It offers insights for future practices of ethnically-defined AA groups and speaks to the broader importance of cultural competence within substance abuse treatment, especially the successful recovery of ethnic minorities from substance abuse issues. 

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. 

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.

This study evaluates the relationship between collective bargaining agreements (CBAs) and average teachers’ wages with econometric analysis. The literature suggests that teachers’ pay impacts the quality of educational provision, teacher retention, and quality of educational staff. Hence, understanding the determinants of teachers’ salaries is of critical policy importance. Using Washington and Idaho state as the geographic frame for my research, I collected and compiled data from 143 school districts from the National Center for Education Statistics (NCES), the Washington State Report Card, the American Community Survey (ACS), the Washington State Department of Education, and the Idaho State Department of Education. I proposed several regression models and used a CBA dummy variable to explain the variation in teachers’ wages, controlling for other relevant factors including student teacher ratio, expenditure per pupil, etc. The estimation results show that the presence of a CBA predicts a 6.1 to 6.7% increase in average WA/ID teachers’ salaries. This result is consistent with the findings in the literature that a CBA predicts a minor increase in average teachers’ salaries. This study extends the literature by verifying the importance of CBAs in the Washington/Idaho geographic area. The findings could be useful to educational stakeholders in salary negotiations, budget forecasting, and educational administration.

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.

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.

Ovarian reserve, the pool of primary follicles available for recruitment, is a key determinant of female reproductive aging (e.g. age at menopause), but what determines ovarian reserve is not well understood. Telomeres, the non-coding DNA that cap the ends of chromosomes, may provide an explanation. Longer telomere length may establish higher ovarian reserve through three mechanisms: 1) Telomere length may limit the number of mitotic divisions, which determines the number of oogonia available to establish the ovarian reserve. 2) Oogonia with longer telomere length could be more likely to survive and differentiate into primary oocytes. 3) Follicular atresia is more likely to occur when there is an insufficient stock of granulosa cells around the primary oocytes. Telomere length may allow more granulosa cell division, maintaining the ovarian reserve. For these reasons, we expect longer telomere length to be associated with a larger ovarian reserve, which can be proxied by higher Anti-Müllerian Hormone levels. Using data from the Cebu Longitudinal Health and Nutrition Study, we conduct regressions to assess the relationship between blood telomere length and Anti-Müllerian Hormone levels among 298 women (21.70 years ± 0.35). We measure telomere length using the monochrome multiplex quantitative polymerase chain reaction assay and we assay AMH using an enzyme immunoassay. Results and conclusions have yet to be obtained, though we have the data on hand. This is due to our goal of using best practices in science. Blinding ourselves to the data when designing the study may prevent bias and overfitting in our results. We expect our results to match our prediction: that longer telomere length is associated with higher Anti-Müllerian Hormone levels. Understanding the relationship between telomere length and ovarian reserve can provide more insight into the biological basis of reproductive age, menopause, and infertility, which may help guide family planning practices.

 Fluorescence resonance energy transfer (FRET) is a method used to elucidate changes in a protein’s structure (conformational changes). FRET works by measuring distances between amino acids in the presence and absence of ligand. The distance is measured by the highly distance dependent degree of energy transfer between an acceptor and a donor, typically two fluorophores which overlap in absorption and emission spectra. FRET is limited by the range of distances at which it is accurate, incomplete incorporation of acceptors, and its ease of use. The technique we will use to improve FRET is click chemistry. Click chemistry is a set of highly specific reactions that allow for the selective addition of desired functional groups to a target of interest. Our goal is to determine if FRET will be more sensitive to smaller ranges with click chemistry, which would increase measurement accuracy of conformational changes. To determine if click chemistry can be used with FRET, we first isolate maltose binding protein (MBP) with a non-canonical amino acid (ncAA) introduced at our site of interest from cells. The role of the ncAA is to “click”, or undergo a linking reaction with a specific functional group, with our selected fluorophore to ensure its attachment to the site of interest. Our modified protein will be incubated with our fluorophore, and FRET will be done on the protein under different conditions in order to test if our modified FRET works. If this method works, we expect to see distance dependent quenching of the fluorophore which would indicate that 1) the method works and 2) would serve to establish a benchmark for future experiments with different proteins. Combining click chemistry with FRET could be a powerful tool for measuring conformational dynamics in proteins.

In response to various stimuli, proteins undergo changes in shape (conformational changes) that are essential to their function in cell signalling, enzyme activity and many other crucial biological processes. Previous studies have shown that intramolecular distance changes associated with the conformational dynamics of proteins can be probed using transition-metal-ion-fluorescence-resonance-energy-transfer (tmFRET), in which a donor fluorophore transfers light energy to an acceptor metal ion in a steeply distance-dependent manner. tmFRET is both highly sensitive and highly specific: each unique donor-acceptor pair can only measure a limited range of distances reliably. Here, we expand the utility of tmFRET by employing new strategies to create a library of unique donor-acceptor pairs each sensitive to a different range of distances.To showcase the accuracy and precision of our measurements, we use a maltose-binding-protein (MBP) model system, which undergoes a well-studied conformational change upon the binding of maltose. In order to label MBP with Cu(II) ions that act as tmFRET acceptors, we utilize macrocyclic chelator-maleimides, which simultaneously coordinate Cu(II) and covalently modify a cysteine introduced into MBP. Using amber codon suppression, we introduce fluorescent non-canonical amino acids (ncAA’s) into MBP to act as tmFRET donors. Our fluorescence measurements show that tmFRET between the ncAA donor and chelated-Cu(II) acceptor accurately reports MBP’s intramolecular distance changes. We also show that by varying the exact structure of the molecule that chelates Cu(II) we can tune the sensitivity of tmFRET to distances ranging from 0.8-1.8 nanometers. Thus, this chelator-maleimide approach to labelling proteins with tmFRET acceptors could prove to be a powerful tool for researchers studying conformational motions of that scale.

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.

Photosynthetic bacteria are ideal systems for the sustainable production of chemicals, but their chemical production is limited by the efficiency with which the bacteria can capture and transfer energy. For this project, I have designed light-harvesting proteins that bind chlorophyll molecules that absorb light of the wavelength reflected by the photosynthetic bacteria Rhodobacter sphaeroides. The LH1 and LH2 light-harvesting complexes found in R. sphaeroides bind chlorophyll molecules in a manner that promotes excitonic coupling and minimizes the energy lost during transfer from the complexes to the reaction centers. The proteins I have engineered are modeled after LH2 and are designed to bind a symmetric ring of pi-stacking chlorophyll dimers, promoting high-efficiency energy transfer between light-harvesting complexes. After characterizing these light-harvesting proteins, they will be further engineered to enable their expression in R. sphaeroides, which will allow these bacteria to absorb a broader range of the solar spectrum and improve their energy transfer efficiency. Incorporating this de novo designed protein into metabolically engineered R. sphaeroides will enhance these bacteria’s ability to convert carbon dioxide to useful chemicals. These engineered light-harvesting proteins could also later be adapted for use in hydrogel-based organic solar cells and forming a 2-D sheet of light-harvesting proteins. In my presentation, I will discuss how I have used the Rosetta protein modeling suite to design hexameric chlorophyll-binding proteins. These proteins are designed to bind twelve chlorophyll molecules, and their cyclical symmetry simplifies the design process. I will present data from characterization experiments such as circular dichroism and fluorescence spectroscopy, which are methods that allow for quantification of the proteins’ binding affinities towards the ZnPPaM chlorophyll, and small-angle x-ray scattering (SAXS) and Size Exclusion Chromatography/multiple-angle light scattering (SEC-MALS), which provide information about the predominant oligomeric states of the designed proteins.

COVID-19 raged through the country, causing immense disruptions within communities as they struggled to support each other, whether for food, safety supplies, health information, or social support. In response to the decrease of in-person social interaction, this study explores the role that public space plays--such as sidewalks, parks, or parking lots--as essential communicative spaces for providing social support during a pandemic. This research focuses on the last ten months of 2020 and how public space can play a crucial role for local communities and neighborhoods to find ways to safely and socially connect and uplift each other. This study asks: What acts/activities did individuals facilitate or engage in to support their local community or neighborhood's social well-being during the year 2020? How was public space utilized or adapted for social support? And lastly, what can be learned from the design of public spaces to better support communities during challenging times? This exploratory study collected hundreds of pandemic-related reports drawing from various local sources, from media to personal accounts. These were intentionally uplifting, socially supportive activities that ranged from signage to performances to art displays to group interactions occurring in public spaces or visible from public spaces in neighborhoods throughout the Puget Sound region. Reports were analyzed using open coding for patterns related to the type of activity, role and number of people involved, messaging themes, and use or adaptation of public space. Some initial findings reveal creative adaptations of public space, such as art performances and motivational messages. Ultimately, such findings from this study will become a resource for cities to better support communities during times of crisis by providing an understanding of how neighborhoods have adapted and used public spaces.

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.

The historically racist relationship between Global South countries and the U.S. has led to misconstrued media representations of the Global South. This breeds ignorance about those countries when privileged U.S. travelers go to them, and a lack of critical consideration leading to limited perspectives of global citizenship and globalization. By intentionally exposing ourselves to proper representation of the Global South, U.S. travelers can begin to decolonize previous understandings of other countries and people. In my research, I analyze portrayals of globalization and global citizenship in the films Sleep Dealer (2008) by Alex Rivera and Ya No Estoy Aqui (2019) by Fernando Frias. I chose these films because they were created approximately a decade apart, which might indicate changes to portrayals of globalization and global citizenship. More importantly, they were created by Latino filmmakers who share the identities of the characters they portray. I explore how these films, when created by filmmakers from the Global South, can aid in efforts to decolonize globalization and global citizenship, and the discourses surrounding them. Such filmmakers push against boundaries set by Western media that offer simplistic, often voyeuristic representations of the Global South, by presenting complex, realistic portrayals that offer more nuance to Western audiences. The films also grapple with issues of immigration; by centering their narratives on undocumented people and their experiences with citizenship, these films challenge the legitimacy of dominant definitions of global citizenship. I found that films like the two I researched here can challenge commonly accepted definitions of globalization and global citizenship and may be successful in changing attitudes of travelers from the U.S. Moreover, these challenges to dominant discourses can breed inclusion for travelers of color who would typically be left out of travel or global citizenship.

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. 

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.

The Large Hadron Collider at CERN is built to accelerate particles to high speeds and make them collide. The curved trajectory of these particles is recorded by detecting electrical charges deposited on multiple layers of tracking equipment as a particle travels through the detector. The resulting patterns are then used to reconstruct the path traveled by the particle. The more the collisions, the higher the number of charge depositions to detect. This exponential increase in data is predicted to be the main issue with the next run of the LHC experiment, where we are set to test higher energy interactions. As traditional tracking algorithms do not scale well with this increase in data, supplementing them with machine learning provides a promising solution. Scaling high-energy particle tracking in the LHC to process petabytes of data is the focus of the Exa.TrkX project, which our study is part of. In our research, we study the robustness of Exa.Trkx models and algorithms against noise and misalignment. Robustness is judged by analyzing performance metrics like purity and efficiency of pairs of charge deposits or “doublets''. Purity is defined as the ratio of true-positives over positives, and efficiency is defined as the ratio of true positives over the number of true values. A true deposit belongs to the same trajectory as the one we are comparing it with. In this presentation, I will discuss how we proved robustness against noise by observing that the change in doublet purity and efficiency was a trivial decrease of 0.3 and 0.2 percent respectively. Our research makes sure that the Exa.TrkX models can be applied to actual LHC data. We do this by proving that the models are not affected by real-life impurities in the data like noise.

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.

This research and public history website explores Jewish immigration experiences and Americanization from the perspectives of young Jewish immigrant women around the turn of the 20th century. I used archival material from Cornell University’s School of Industrial and Labor Relations to highlight these women’s voices, and archives from the Tenement Museum and the Jewish Daily Forward to illustrate Jewish life in this period. Additionally, archives from the New York Times were used to examine how Jewish immigrants were viewed. I searched these archives for primary sources to shed light on how Jewish immigrant women worked and lived, including how life in the Lower East Side’s cramped tenements was photographed and written about by outsiders - White society, and how this propagated the notion of Jewish people as Others. My goal for this research was to tell the stories of these women and listen to their voices. They tell of long hours in crowded and often dangerous garment factories, of unionizing and striking for better labor conditions, and the tragedy of the Triangle Shirtwaist factory fire. These women were often the main breadwinners for their families, but many saved a little for themselves to spend on ‘American’ activities such as seeing shows, frequenting dance halls, and developing an interest in ‘American’ fashion and buying clothes. White society dubbed these women’s fashion as tacky, outlandish and vulgar. Their dresses were described by White society, as well as their own community, as cheap knockoffs of the dresses they made, with too many bows, too much jewelry, and too much makeup. These immigrant women were often called ‘Ghetto Girls,’ as the Lower East Side was called the Jewish Ghetto at this point. This research also brings the present into focus and examines how immigrants are viewed and often judged. 

This paper explores how Dutch wax print has become interconnected within Central and Western African society and culture as well as throughout the diaspora. More than merely a fabric for many, Dutch wax print is an important signifier of African identity and heritage. The fabric also stands as evidence of the complex history of its traveled and cross-cultural past. During the early to mid-nineteenth century, Dutch colonizers began imitating wax resist-dyed batik fabric from Java, Indonesia, with newly developed machine technology. This was done with the intent to infiltrate the local Javanese textile market but did not succeed in doing so. Instead, the imitation batik was rejected by the Javanese market. This disapproval of the fabric in Java eventually led Dutch producers to conduct trade activity with West Africa, where a new market developed. The popularity of Dutch wax print and its association with African identity is linked to the long-standing communications between Dutch wax print distributors and West African women that sold the fabrics. These merchants would receive the fabrics and give them culturally-relevant and significant meanings related to proverbs and local sayings. Through the custom of integrating and redefining imported Dutch wax print, Africans have infused the fabric with deep cultural meaning and importance. For many in the diaspora, Dutch wax print is also a medium for expression. Contemporary artists Njideka Akunyili Crosby and Yinka Shonibare both include the fabrics into their art as a way to communicate ideas about their identity as well as the cultural heritage and multifaceted history of Africans over time. Today, throughout much of Africa and the diaspora, the fabric is a powerful medium for which to communicate ideas and concepts through cultural exchange.

School segregation is viewed as a problem of the past as schools have become more racially integrated since the Jim Crow Era and the Brown v. Board of Education of Topeka court case. However, a new wave of segregation is still present in our school system as a result of systemic inequalities that stem from continued racial segregation such as socioeconomic inequalities and redlining. The goal of this project is to critically examine the public school system in the Seattle area and expose disparities that exist between the more affluent, predominantly white North Seattle area versus lower income and more racially diverse communities within South Seattle. I conducted qualitative interviews with Black, Indigenous, and People of Color (BIPOC) students from each area to compare how their education differed in higher education preparation. Predicted results are that there are significant disparities in educational experience for BIPOC and other disadvantaged groups in the greater Seattle area as they lack equitable access to academic resources and enrichment classes either from school bias or underfunding. Results may also inform us of how the racial population is disproportionately spread across Seattle as a result of redlining and district dividing lines, with BIPOC taking up more lower income areas with less access to education. By studying the experiences of BIPOC in the Seattle area, I hope to help bring awareness to the still present issue of segregation in our school system. As they speak on their own personal experiences, we can learn how to better support these students and work towards structural change to dismantle the inequities they face in our current education system.

 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.

Manually analyzing neural histopathology is a tedious process, and while there are reference sources available such as the Allen Brain Atlas, one of the primary challenges of using image analysis software to compare neural pathology between animals is the alignment of the rostral-caudal axis. The current study aims to investigate the use of Visiopharm, an image analysis software, and supervised machine learning to automate the assignment of brain sections along the rostral-caudal axis.This research is a part of a larger study conducted to analyze potential neuropathological changes in male mice that have undergone blast-induced mild traumatic brain injuries. Glial fibrillary acidic protein (GFAP), Tyrosine hydroxylase (TH), Tryptophan hydroxylase (TPH), Kappa Opioid Receptor (KOR) and Ionized Calcium-Binding Adapter (IBA) were used to identify histopathology. First, we manually labeled sections in Visiopharm and trained a deep learning algorithm to outline the sections and compute section parameters (e.g., circumference, diameter, convexity). In parallel, we manually assigned each labeled section according to the Allen Brain Atlas (ABA) rostral-caudal axis. Finally, we used python to train a supervised machine learning regressor to predict the assigned ABA slide number based on the section parameters generated by Visiopharm. We have successfully completed workflow for TH labeled sections and are currently working to generalize the algorithms to the other stains. We expect that with additional labeling and training, we will be able to successfully develop an automated process for classifying sections on the rostral-caudal axis.The goal is that after training the app further through the labeling of sections, the app will be able to create parameters for future stains, thus creating a general classification framework.This framework will increase consistency across future labeling, which allows for more reliable and faster classification. This additional time can be devoted to analyzing specific stains and other research questions.

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.

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.

Debates about the causes and consequences of gentrification for urban poor have been active for decades, but inconclusive due to methodological constraints and inconsistent definitions. However, gentrification research in the context of transit oriented development (TOD) allows scholars to overcome these issues, because of its clear definition. While studies generally theorize that populations change through low-income, often non-white, residents moving out of an area, research finds that urban poor are far less mobile than higher income groups. At the same time, metropolitan cities are currently experiencing technology booms and rapid population growth that reveal puzzling racial trends. In Seattle, Black and Hispanic populations are declining, while White populations are increasing in a way not explained by existing theory. The present study attempts to explain this pattern by suggesting that middle and upper income groups are shifting the racial composition of neighborhoods because of their capacity to move. Using descriptive statistics and difference-in-difference estimates, I examine the effect of Light Rail transit on population race and socioeconomic status compositions of Seattle census tracts between 1990-2019 with a unique time-series dataset composed of American Community Survey estimates and Census data. My study has implications for theories of urban demographic change, as it will illuminate how race and class interact in this context. The study is also relevant to urban planning and public policy professionals interested in how local government policy affects existing residents.

The ability to actively reflect on one's own mental processes, or metacognition, plays a significant role in learning and executing complex tasks by interacting with different learning processes. This study examined the influence of metacognition on declarative and reinforcement learning (RL) mechanisms. I collected data from 221 undergraduates (139 female; age from 18 - 22, mean = 19.0) using a within-subjects metacognitive manipulation half way through the stimulus-response (S-R) learning task created by Collins (2018). The metacognitive manipulation aims to activate monitoring by drawing participants' attention to their own learning strategies and task performance. The task consists of learning blocks of differing lengths; the long blocks rely more on RL, while the short blocks can be completed with declarative or working memory processes. Contributions of declarative and RL mechanisms are also assessed through an incidental post-test given after an intervening task. If metacognition differentially affects declarative and RL, we expect a three-way interaction between the task phase (learning/post-test), block type (long/short) and metacognition (before/during). Results showed significant main effects of task phase (F(1,220) = 153.83, p = 6.32e-34), block length (F(1,220) = 527.29, p = 2.79e-102) and metacognition (F(1,220) = 18.00, p = 2.32e-05), with better performance during the learning phase, short blocks, and metacognitive manipulation. A significant phase by metacognition interaction (F(1,220) = 8.21, p = 0.0042) suggested that metacognitive monitoring improved test performance without interfering with learning performance. Future experiments will examine the possible mechanisms by which metacognitive monitoring facilitated long term memory retrieval.

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.

Statistical learning—predictions based on pattern recognition from varied inputs—is critical in language development. People reference previous grammar encounters in their native language to recognize language patterns, and a strong understanding of statistical learning in language could help create interventions that enhance language development and pattern recognition. However, most research studies on statistical language learning have focused on a select group—monolinguals—ignoring the bilingual community that represent 43% of the world’s population. As such, I review current literature on the unique language learning processes of bilinguals and ask the following research questions: Does bilingualism affect language learning speed, and if so, does it increase or decrease it? When subjects first acquired their second language, what key factors (such as age, proficiency, and whether their second language can be considered another native language) are present? Are there advantages or disadvantages associated with bilingualism in regard to statistical learning and language and how does this compare in relation to monolingualism? To answer these questions, I examined 10 articles that focus on languages across numerous learned languages. A preliminary review of the literature shows that bilinguals have different processes of statistical learning in language development compared to that of their monolingual counterparts. They also indicate that age at which a language is acquired further affects these learning processes. Additionally, results could reveal advantages to bilingualism that strengthen these language learning processes. This literature review can inform future research and studies of the effects of more than one language on statistical learning in language development and point to the need for additional research on not only bilinguals, but also trilinguals, etc.

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. 

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.

Alzheimer’s Disease (AD) is a neurodegenerative condition that affects more than 50 million individuals worldwide. The progression of AD is hallmarked by the buildup of beta-amyloid plaques and neurofibrillary tangles, leading to neuronal death on a large scale. In the early stages of AD, the hippocampus is disproportionately affected by this heavy neuronal loss. The genetic elements and pathways contributing to this AD pathology are still poorly understood. Excitatory synapse assembly (ESA) processes have been previously shown to be affected by the pathology of AD, leading us to investigate the expression patterns of genes involved in ESA in different brain regions. A gene ontology (GO) analysis was conducted to isolate individual genes involved in ESA and analyze their relation to beta-amyloid plaque and pTau neurofibrillary tangle severity in patients who presented signs of dementia due to AD. ESA gene expression was shown to be strongly correlated with increasing levels of pTau in all brain regions except for the hippocampus, where there was no correlation, in a linear regression analysis. This implies that the hippocampus has a unique response to AD pathology with regards to ESA gene expression among the different brain regions. The inability of hippocampal cells to express neuronal repair genes in the presence of severe neurofibrillary tangles requires further analysis and could eventually confer a novel target for AD therapeutics.

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.

As high levels of drug use account for thousands of drug related deaths every week, it’s important to investigate the neural mechanisms behind drug escalation as well as develop possible harm reduction strategies for drug taking. Therefore, the purpose of this experiment was to examine the effects of the Kappa Opioid receptor (KOR) on cocaine escalation in the mesolimbic system. Using a preclinical model to examine this hypothesis, CRISPR/SaCas9 was utilized in the ventral tegmental area (VTA) to selectively repress expression of KOR. After four weeks, the rats underwent cocaine self-administration during short access periods and then escalation was tested in long access periods. Lastly we utilized immunohistochemistry to confirm CRISPR/SaCas9 transduction in dopaminergic neurons. We found that decreased expression of the KOR decreased escalation during long access periods. Future research will examine the role of KOR from and in specific brain regions such that KOR expression will be decreased only in dopaminergic projections from the VTA into the NAc.

The primate brain effortlessly segments objects from background stimuli, however, the neuronal underpinnings of this process are largely unknown. Past studies in V1, the primary visual cortex, have demonstrated that neurons encode visual information within the receptive field through edge detection, by which the greatest local contrast evokes the largest response. These studies have typically focused on luminance contrast, but edge detection and object segmentation can rely on a variety of other factors including contrasts in color, texture and blur. The primary goal of my research project is to investigate how variances in contrasts of luminance, color saturation, orientation and blur within a textural image affect neuronal response in primary visual cortex V1 and in subsequent stages V2 and V4. I have created a novel stimulus set that individually incorporates these varying types of contrast. I will record the responses of neurons in the visual cortex of a macaque monkey to these stimuli using Neuropixels, which will allow us to determine whether neurons are selective to object form and whether the strength and latency of form selectivity depends on the stimulus dimensions that define object boundary. We expect that different subgroups of neurons in V1 and V2 may be sensitive to different types of stimulus contrasts, while the same neuron in V4 may be sensitive to object shape regardless of the stimulus modality but the different modalities may be associated with different latencies. Data analysis and discussion of results will be completed and prepared for presentation by May. Our research will build upon and contribute to the greater understanding of visual information processing within the primary visual cortex. 

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. 

Why do some American social movements enjoy Congressional attention while others do not? By contextualizing this question in existing social movement and policy elite preferences theory, this paper proposes a novel theory identifying social movement threat as a key causal mechanism underlying recognition from Congress. To operationalize this, “social movement threat” was measured by analyzing the size, frequency, and violence of protests organized under a corresponding social movement in a given year, while “Congressional attention” was measured by the amount of proposed legislation addressing a corresponding social movement in a given year. The project focuses on the actions of formal social movement organizations as a unit of analysis and hypothesizes that a continuous trend of threatening protest tactics is key to sparking Congressional attention. Preliminary multivariate regression analysis of three distinct social movements and over 300 protests in the 20th century indicates that more threatening social movements may have garnered greater Congressional attention in the form of proposed bills but did not necessarily enjoy a significantly higher amount of passed laws. Further, while Congressional attention did not increase with movement threat, it does appear that it increased over the lifespan of a social movement. The implication of these preliminary findings is that protests do little to influence Congressional decision-making, but that some other characteristic of an ongoing social movement may spark increased Congressional attention.

The Fermilab Muon g-2 experiment seeks to measure the anomalous magnetic moment of the muon to 140 ppb. A highly purified beam of muons is delivered to a magnetic storage ring in bursts of ~15,000 muons called fills. The rate of change of the angle between a muon’s momentum and spin while orbiting in the storage ring is the anomalous precession frequency, which is directly proportional to the anomalous magnetic moment. During each fill, muons orbit in the storage ring until they decay into positrons which spiral into electromagnetic calorimeters stationed around the ring. Positrons which impact the calorimeter deposit their energy in the calorimeters as Cherenkov radiation. The time dependance of the positron energy spectrum is used to extract the anomalous precession frequency of muons in the storage ring. “Early to late effects” are a class of systematic uncertainty in the experiment which result from coherent changes of experimental conditions within each fill. These effects can directly bias the measured anomalous precession frequency. One such effect arose from malfunctioning resistors in the ring’s electrostatic quadrupoles, resulting in non-ideal vertical focusing of the muon beam. This led to coherent downward motion of the beam during each fill. This directly couples into one of the largest systematic effects, as the calorimeter acceptance depends in part on the beam's vertical position. Using data from the calorimeters, I quantified early to late change in the beam’s vertical position and vertical distribution. These results were used to cross-check results from simulation programs. If the Fermilab Muon g-2 experiment retains the same central value as the previous generation measurement but with 140 ppb precision it will be in greater than 5-sigma tension with standard model calculations. Results from Run 1 of the experiment are expected to be published in early 2021.

Accurate calibration of the Laser Interferometer Gravitational-wave Observatory(LIGO) is vital to detecting gravitational waves and interpreting astrophysical events. Gravitational wave measurements have prompted extensive studies in coalescing binary systems, early formation of the universe, and other areas of cosmology. The current calibration method relies on radiation pressure to induce a calibration force. However, a new method of calibration, the Newtonian Calibrator, uses gravitational attraction to inject a calibration force. We have developed two simulations to model this injection: a finite element analysis and multipole-based calculation. These simulations allowed for the accurate prediction of the injected force, yielding a precise absolute calibration. The accuracy of this calibration is crucial for cross-checking LIGO's current calibration techniques and making future cosmological predictions. 

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. 

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 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.

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. 

DNA is often correctly referred to as one of the building blocks of life. Understanding the combination of adenine, thymine, guanine, and cytosine that ultimately defines our characteristics is crucial to many genomic applications ranging from synthetic biology applications to our understanding of health and diseases. Modeling and understanding the causes and consequences of these base pair level interactions can impact the development of DNA origami and our understanding of genome stability in different health/disease states. DNA is widely known for its complex structures and uncovering, modeling, and predicting the structure and formation of DNA can provide valuable insights into various aspects of genomics including visualization and probe design. Modeling DNA’s inter-strand and intra-strand interactions has been the core focus of genomics for some time. Specifically, understanding intra-strand secondary structure formation across genomes has largely been unexplored due to computational limits. To further our understanding of DNA, I present the use of thermodynamic partition functions to model and predict intra-strand behavior of secondary structure formation. I have efficiently calculated secondary structure formation across hg38 at three relevant thermodynamic conditions and their corresponding length scales. With this information, I examine the distribution of the entire genome while also cross examining this information against specific regions with known biological features such as genic vs non-genic regions. Additionally, I apply the same predictive model to fluorescent in situ hybridization (FISH) probe design and provide additional quantitative metrics and insights in the development of optimal probe design. This research has the potential to further our understanding of the causes and consequences of non-random genes. Having additional information about the distribution of secondary structure formation of certain regions can be vital information about learning more about the selection of base pair sequences and its macro effects.

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.

 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.

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.

Tuberculosis (TB) is one of the top ten causes of death globally. Although it is mostly curable, the antibiotic treatment is long, challenging, and carries dire consequences if discontinued. Patients face many known barriers to treatment adherence, such as traveling to TB clinics, lack of TB knowledge, stigma, and the medication's adverse effects. In response, the TB Treatment Support Tools (TB-TSTs) intervention has been iteratively developed with patients and TB teams to improve treatment outcomes. TB-TSTs is a two-part system. The patient-facing mobile application allows patients to report taking the medication daily, potential medication side effects, submitting a photo of a test strip that detects a TB drug metabolite, asking questions, and interacting with a treatment supporter. The other part of the system is for treatment supporters who can be, for example, social workers, physicians, or nurses to review the daily reports, encourage and support patients through treatment. As part of this larger study, the aim is to improve the treatment supporter interface by analyzing and synthesizing interview transcripts with treatment supporters to provide recommendations for the design team. Interviews were conducted with actual or potential Argentinian treatment supporters and transcribed for qualitative analysis of main themes using Atlas.Ti. In collaboration with research team members, we iteratively developed a codebook. The team identified five main themes and seven sub-themes. Main themes include limitations of the system, what works, design recommendations/features to improve, and subthemes include usability/UI feedback and cultural adaptation. Expanding on patient events (e.g., appointments) and quickly visualize adverse effects are the main recommendations. The main findings were presented to the design team to influence the treatment supporter interface of the TB-TSTs for the next stage of testing. This study will improve the TB-TST, and the larger project presents the usability of future treatment supporter tools for chronic diseases.

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.

Currently there are few qualitative research studies that examine the experience of advanced-stage breast cancer from both points of view in a couple. My research bridges that gap by focusing specifically on how patients and their spouse caregivers view their lived experience with advanced-stage breast cancer. My research utilized interview transcripts from a descriptive study of discovery regarding women with advanced-stage breast cancer and their spouse-caregivers. These interviews were completed in between the years 1999 – 2000, but they offer rich descriptions of the challenges from both patient and spouse caregiver perspectives and provide insight into couples’ lived experiences of advanced breast cancer. The goal of my research project is to compare the patient and spouse caregiver experience in their own words. I performed qualitative research methods that involved coding the data through inductive content analysis and creating a subsequent conceptual framework for studying couples and spouse caregivers. I addressed four research aims: 1. Describe the spouse caregiver’s experience with his wife’s breast cancer, in his own words. 2. Describe the patient’s experience with her breast cancer, in her own words. 3. Compare the two experiences by dyad and by total sample (compare between women’s perspectives, compare between spouse caregiver perspectives). 4. Generate implications for clinical services, based on comparison findings. My preliminary results show there exists a disconnect in views between the spouse and patient in the relationship, but similar views are held within the collective roles (i.e. the patients as a whole had similar views as did the spouse caregivers).

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.

Humans are incapable of regenerating a majority of their major tissues following traumatic injury. Xenopus tadpoles have the ability to regenerate a variety of complex tissues quickly following tail amputation, but lose this regenerative competency during metamorphosis. Though tadpoles have been extensively used to study regeneration, we do not yet understand the roles that stress signals from injury play in directing regenerative gene expression. We have found that inhibition of the stress responsive transcription factor Hypoxia Inducible Factor 1α (Hif1α) with the Hif1α inhibitors 2-methoxyestradiol (2ME) and Echinomycin (Ech) prevents regeneration. In particular, inhibition of Hif1α decreases Wnt mediated gene expression. Wnt is known to be one of the primary signaling processes necessary for proper Xenopus regeneration, specifically tail regeneration. While we have shown that Hif1α and Wnt regulate expression of similar gene programs in regeneration, we predicted that there are unique processes that Hif1α regulates to facilitate growth. To investigate how Hif1α and Wnt regulate regeneration, we utilized the two Hif1α antagonists, as well as the Wnt antagonist, IWR-1 (IWR) which I have previously shown inhibits regeneration. To determine Hif1α and Wnt regulated genes, we performed RNA-sequencing 24 hours post amputation. I then identified genes downregulated in Hif1α inhibited tadpoles, which are likely Hif1α dependent. I then removed genes downregulated by Wnt in order to isolate genes uniquely regulated by Hif1α. With the 250 genes uniquely regulated by Hif1α, I used PANTHER to perform gene set enrichment based on Gene Ontology terms. The main biological process of interest found to be regulated by Hif1α during regeneration was DNA replication. By determining how Hif1α uniquely regulates DNA replication during regeneration, we will continue to enhance our understanding of the roles that stress signals play in directing how regenerating cells meet increased proliferative demands post traumatic injury.

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.

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.

In synthetic chemistry, the direct functionalization of C-H bonds with oxygen-containing groups is a powerful strategy to efficiently synthesize molecules used in materials and therapeutics. However, current methods to accomplish such reactions suffer from limited substrate scope, selectivity, and tolerance towards other functional groups. Iron-dependent enzymes represent a promising solution to this problem, as they are known to mediate a plethora of complex oxygenation reactions in a highly selective fashion while using inexpensive and earth-abundant reagents. In prior work, we found that Fe(II) 2-oxoglutarate dependent hydroxylases (Fe(II)/2OGs) exhibit non-native oxyfunctionalization activity on olefinic amino acids. Here, we explore the ability of Fe(II)/2OGs to catalyze non-native asymmetric oxyfunctionalizations. We plan to evaluate the ability of our library of Fe(II)/2OGs to catalyze oxyfunctionalization of non-native substrates with various functional groups. Subsequently, we will optimize activity using directed evolution to arrive at a highly active and enantioselective enzyme capable of this chemistry.

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.

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.

 Motor neuroprosthetics are brain-computer interfaces (BCIs) that record neural signals from an individual, use a decoding algorithm to predict the individual's intended movement, and perform that movement using a prosthetic device. As the neural code for movements is still being uncovered, current devices are still limited in their ability to create naturalistic movements. Development of motor decoding algorithms that can predict more naturalistic movement may enhance these prosthetics. In this project, we aim to implement and elaborate on newly published motor decoding algorithms and then compare alternative approaches to predicting the direction of movement in upper body joints (initially just the wrist) based on electrocorticography (ECoG) recordings from the surface of the human brain. Our dataset uses neural data collected during one week of continuous ECoG recordings from four subjects undergoing chronic, in-patient seizure monitoring, and includes hours of naturalistic movement, previously mapped from concurrent video to provide joint tracking of the head, shoulder, elbow and wrist. Carrying out this study on existing ECoG recordings provides a basis for extending our decoding work to future, real-time neural recordings. We have identified electrodes that correspond to hand-related areas in motor and sensory cortices for our initial focus on motor decoding of wrist movement. The goal of our project is to explore motor decoding of dimensionally-reduced spectral features using various neural network architectures. This will allow for more accurate responses in motor neuroprosthetics since they rely on motor decoding of naturalistic movements.

Internet censorship is accomplished through technological means, it is sometimes enforced via automated keyword filtering with a keyword list, implemented on a platform or state level. Individual users who discuss “sensitive” issues online may find their social media posts are made invisible or removed and may even have their accounts shut down. In this project, I experimented with using images as carrier of text data to send sensitive inforamtion over a censored network and made a publicly available data-hiding web tool for this project. A PRNG-based, spatial domain embedding method was used to embed the text data, the carrier image is then sent over a network where a keyword-based censor was detected. The result of the experiment shows that steganography can be used to combat keyword-based internet censorship, which promotes the free exchange of information. 

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.

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.

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.

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).

Cancer is an evolutionary process driven by mutagenesis and selection for malignant phenotypes. Correspondingly, mutations that elevate mutation rates, producing a “mutator phenotype,” accelerate tumor formation. Heterozygous mutator alleles affecting the catalytic subunit of DNA polymerase (Pol) epsilon (encoded by the POLE gene) or bi-allelic mutations affecting mismatch repair (MMR) components, such as MSH2, arise frequently in a subset of colorectal and endometrial cancers. In some tumors, mutator alleles from both classes occur together and synergistically cause tremendous genetic instability. A key unanswered question is whether the severe mutation accumulation compromises tumor cell fitness and imposes a selection for “antimutator” phenotypes that allow the tumor to escape extinction. Evidence supporting the selection for antimutator alleles in strong mutator cells have been obtained using budding yeast, whose DNA replication and repair machinery are highly conserved with their mammalian counterparts. Our initial studies in haploid yeast show that mutations in the pol2 mutator alleles represent a major class of antimutator alleles. However, mutator suppression in diploid cells remains understudied. The strongest mutator allele known in human disease is POLE-P286R, which corresponds to pol2-P301R in yeast. We previously evolved diploid pol2-P301R/POL2 msh2Δ/msh2Δ mutator yeast strains to identify candidate antimutators that may arise in diploid cells. We also identified putative human antimutator alleles from The Cancer Genome Atlas database. Here, we test whether these alleles do indeed exert antimutator phenotypes by re-engineering them into diploid yeast. Our findings will provide a direct test of the relevance of antimutators for tumor evolution and define likely antimutator candidates for further study.

Charcot-Marie-Tooth (CMT) is a group of inherited nerve disorders resulting in muscle weakness in which the motor and/or sensory peripheral nerves are affected; hereditary neuralgic amyotrophy (HNA) is a rare genetic disorder characterized by severe pain and muscle wasting in the arms. Using whole exome sequencing (WES), we aimed to find the causative mutation in a five-generation family with CMT and single-generation family with HNA. WES is an efficient method to identify possible disease-causing mutations by searching for variations in the protein-coding region of any gene. After excluding variants from a known list of genes to be causative of CMT, we used stepwise criteria to filter and prioritize the candidate variants in the exome data from two patients in this family: heterozygosity, alternative splicing, gnomAD frequency, CADD score, function of the gene, and its expression. In CMT, out of the 26 candidate variants, 10 were INDEL mutations and 16 were single nucleotide polymorphisms (SNPs). Each variant was Sanger sequenced to verify the exome variant; the verified variants were further evaluated for co-segregation with the disease by Sanger sequencing affected and unaffected individuals of the family. The results from these analyses did not identify any candidate variant as a possible candidate, concluding the CMT WES study without yielding a causative gene. Our next steps for this project will be to use whole genomic sequencing (WGS) to reveal DNA variation outside the exome or copy number variant or repeat expansion that may alter protein production to identify potential causative mutations. Currently, we are using the same methodology to screen for candidate variants in three exomes from the family with HNA. Out of 22 candidate variants, we have screened 2 INDEL mutations and 2 SNPs. We are actively screening the remaining variants in search of a causative mutation for HNA in this family.

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.

Although fertilization is a crucial process for sexually reproducing organisms, the molecular mechanisms mediating this process in humans and other animals remain unknown. In part, this is due to reproductive proteins evolving rapidly between closely related species. Sequence diversification and gene duplication makes establishing gene orthology (the same genetic locus in different species) difficult. The step of fertilization that I study is the sperm binding to the egg coat. ZP3 is an egg-coat protein and ZP3r is its sperm binding partner in mice. My research has shown that the human ortholog of mouse ZP3r has been misidentified as C4BPA and is instead the pseudogene C4BPAP1. This misidentification is likely due to difficulties in determining orthology when the gene is surrounded by duplicates. C4BPAP1 shows testes-specific expression despite being a pseudogene, consistent with a function in fertilization. However, pseudogenizing mutations in the C4BPAP1 locus have accumulated in humans and other great apes, indicating this gene may no longer play a role in fertilization in these species. Phylogenetic analysis of loci performed by maximum likelihood (Phylip) and synteny analysis shows that C4BPAP1 is the human ortholog of mouse ZP3r. By comparing protein sequences across species, we have found that ZP3r/C4BPAP1 varies in the number of CCP domains. The ancestral form of this gene in mammals contains 8 CCP domains, meanwhile mice contain 7. During my next two quarters, we will examine the evolution of the ZP3r gene family, look at ZP3r’s conservation across mammals, and determine whether ZP3r is undergoing positive selection, a characteristic common to functional fertilization genes. Our research is refining the identification of genetic loci implicated in mammalian fertilization. The precise identification of these loci is important for the development of novel contraceptives and for studies of infertility.
 

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.

STS-121 is a NASA space shuttle mission to the International Space Station (ISS). The ISS is a habitable satellite (Space station) in a low Earth orbit. We employ calculus-based methods to analyze and study the flightpaths, altitude, velocity, and acceleration profiles of the STS121 data reported by NASA as it travelled through outer space. Our studies unravel information about the critical points, local maxima and minima, concavity, and inflection points in the altitude data. The velocity profiles were fitted to polynomial functions using least square data fitting using linear algebra-based methods. The acceleration data involve piecewise functions which is related to the time scales involving burning of the propellent and separation of the external propellant tank as the space shuttle gets ready to move into orbit. We estimated the work done in transferring a load from Earth to the International Space station. We used optimization methods to design an optimal solar panel geometry for a satellite by minimizing the surface area. This research provides novel applications of the fundamental theorems of calculus to study motion in outer space and involves mathematical modeling, optimization, curve fitting, data analysis and data visualization.

Comets are cosmic snowballs of frozen gases, rock and dust that orbit the Sun. Isaac Newton suspected that comets were the origin of the life-supporting component of air and a key source for water replenishment in planetary interiors. A close-up view of comet Hartley 2 was taken by NASA's EPOXI mission during its flyby of the comet, using the spacecraft's medium resolution instrument. Comet Hartley has a novel asymmetric dumbbell-like shape. We employed mathematical models to study comet Hartley. Using calculus-based methods, we estimated various static properties, including the arc lengths (outer boundary length), surface area, and volume of Comet Hartley. Assuming a constant density, we also estimated the mass, center of mass, and moments of inertia for Comet Hartley using triple integration methods using cylindrical coordinates. The center of mass, moments of inertia, and radius of gyration form key inputs for studying the orbital mechanics of the comet in outer space. This research project provides excellent opportunities for hands-on explorations using multivariable calculus studies for engineering and space sciences applications. This research is important as studies of comets unravel secrets about the formation of the solar system.

Gliders are robotic vehicles used in the air and underwater to collect and transmit real-time data. Studies using gliders have important applications in oceanography, engineering, and remote sensing. The goal of this project was to model and identify aspects of a glider’s flight using vector-calculus and matrix-algebra based methods. We employed mathematical models to study the flightpath of a glider using vector valued functions and calculated the osculating plane of the glider. The model parameters were optimized to minimize turbulence. We studied the kinematics of underwater gliders using GPS data reported from gliders deployed by Rutgers University and the University of Washington. We analyzed the reported glider velocity data and applied vector-calculus based methods to calculate the instantaneous and average velocities and acceleration vectors. Additionally, we applied matrix-algebra based methods to translate and rotate the glider to position it at appropriate coordinates underwater for gathering data. This research provided insight into mathematical modeling of real-world data and involved applied optimization and data visualization. These studies provide novel avenues for hands on exploration and application of key mathematical concepts.

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.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that is estimated to infect around 23 million people per year and there are not any approved vaccines that are therapeutic (that act in infected subjects) or preventative (that would prevent an infection). Most vaccines that we use today rely on injecting antigens subcutaneously or intramuscularly in order to elicit an adaptive immune response. However, with mucosal pathogens, mucosal immunity might be preferential because having local immunity in the site of the first pathogen exposure has the best chance at preventing the spread of infection beyond the pathogen portal of entry. My hypothesis is that a mucosal immunization would prime memory cells to reside in vaginal tissues and provide better protection for HSV-2 than other routes of immunization. From preliminary data, intranasal immunization was found to be effective in protecting mice from an HSV-2 infection. I have also characterized the role of CD8+ T cells in preventing infection in order to provide insight into the role of mucosal immunization for HSV-2. This work contributes to the efforts to make an effective vaccine to prevent HSV-2 mediated disease. 

Venezuelan Equine Encephalitis Virus (VEEV) is an arthropod-borne virus spread by mosquitos. It causes a range of acute disease from mild flu-like symptoms to more severe illnesses such as encephalitis. Certain species of rodents serve as a reservoir host for VEEV and display no symptoms, whereas horses, as amplification hosts, often exhibit lethal symptoms, suggesting that differential immune responses in these hosts contribute to differences in pathogenesis. Because reservoir and amplification hosts exhibit distinct pathogenesis profiles, this suggests VEEV replicates differently in these hosts and may be able to better evade the equine innate immune system. We have shown that changes in 3’ untranslated region (UTR) sequence and structure affect the ability of VEEV to evade host innate immune sensors such as IFIT2 and RIG-I. We generated mutant viruses which encode 3’-UTR sequences from either pathogenic or attenuated strains of VEEV and are using these to investigate the role of RNA structures in the 3’ UTR in limiting VEEV replication in equine cells. By stimulating the interferon response in equine fibroblast cells (NBL-6 cells) using a synthetic double-stranded RNA analog, Poly I:C, we predict equine antiviral responses will inhibit VEEV replication. Furthermore, we hypothesize pathogenic chimeras of VEEV will replicate more efficiently in Poly I:C-treated cells than attenuated chimeras. These studies will expand our fundamental understanding of the molecular mechanism of how RNA structure in the VEEV genome contributes to host innate immune evasion and has implications in future antiviral therapeutics and vaccination strategies.

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.

A mesoporous silica (MPS) membrane is an ultrathin permeable material characterized by numerous and uniform embedded pores whose sizes are on the order of 2-3 nanometers. MPS membranes are widely used in a number of research and industrial applications such as biomedicine for the isolation and characterization of macromolecules including DNAs and proteins. Such membranes can be synthesized in a variety of ways including electrodeposition. In our research, we have been developing an electrochemistry-based method for the preparation of ultrathin MPS membranes ranging from 50 to 150 nm in thickness. These membranes are synthesized on an electrode by a novel pulse deposition process and can be transferred onto other solid supports. A highly sensitive single-molecule analysis platform is being developed based on the use of such MPS membranes. We anticipate that our MPS membranes will find extensive use in future applications ranging from single-molecule analysis to high efficiency purification of macromolecules and other small biomolecules of interest.

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.

Lightning is an intensely energetic atmospheric phenomenon that causes significant loss of life and property, ignites wildfires, and sustains the natural cleansing power of the atmosphere. Lightning is also an indicator of storm intensity and of the microphysical properties of cloud precipitate. Thornton et al. (2017) showed that lightning frequency over two of the world’s busiest oceanic shipping lanes is on average double that over neighboring ocean areas. Fuel combustion by ships emits sub-micrometer particles which alter cloud microphysics and possibly storm intensity, providing a potential explanation for the increased lightning density over the shipping lanes. Starting January 2020, the International Maritime Organization imposed a seven-fold reduction in the amount of sulfur in shipping fuel. Fuel sulfur is a significant contributor to particulate matter in ship exhaust. In addition, there was a significant temporary reduction in maritime ship traffic related to economic impacts of COVID-19. We use lightning frequency observations from the World-Wide Lightning Location Network (WWLLN) to analyze how these changes in shipping activity and emissions affect lightning enhancements over major shipping lanes in 2020 compared to the past decade. We find that the lightning enhancements over shipping lanes in 2020 were significantly lower compared to the past decade, with more than 100% reduction in the lightning enhancement, temporarily, as well as sustained, but smaller reductions in over half of the seasonally active months. Together, these changes suggest both the temporary COVID-19 reductions in ship traffic and prolonged reduction in ship exhaust emissions may have significantly altered lightning activity in major oceanic shipping lanes.

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.

Disease is a major threat to tropical coral reefs which can be made worse by local stressors like overfishing and nutrient pollution and global stressors such as climate change. However, not all coral species suffer disease at equal rates. It has been hypothesized that these differences may be due to differences in coral innate immune strategies, biogeography, or the symbiotic associations between corals and protective microorganisms (the ‘coral probiotic hypothesis’). This project seeks to test if there are properties of coral microbiomes that correlate with differences in disease susceptibility. We’ve tested this using 1272 coral 16S rRNA gene amplicon libraries and three long-term coral disease datasets. Establishing a general picture of coral disease susceptibility requires integrating data from multiple regional disease monitoring projects. This is challenging because these projects use different methodologies, monitor different species and occasionally use different terminology for the same diseases. I’ve merged three long-term coral disease datasets: the Florida Reef Resilience Project, the Hawai’i Coral Disease database, and an extensive unpublished dataset from Dr. Joleah Lamb. This combined dataset consists of 141 different coral taxa and 31 unique categories of diseases and stressors. This combined disease resource allows for both investigation of the evolutionary history of coral disease susceptibility and comparison against our microbiome data. Our results so far identify coral groups especially susceptible to certain diseases (e.g., Acropora and Skeletal Eroding Band). So far, phylomorphospace analysis indicates an intriguing potential association between microbiome complexity and disease susceptibility. Preliminary results also indicate a strong correlation between microbiome richness and skeletal eroding band disease over more than 450 million years of coral evolution. This new evidence, if confirmed would support the coral probiotic hypothesis.

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.

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.

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.

Mycobacterium abscessus (Mabsc) infections occur in more than 5% of patients with cystic fibrosis (CF), increasing over the last decade. Antibiotic treatment for those with the infection can take many months and often are not successful at eradicating the organism, requiring long-term maintenance therapy. Currently, there is not an effective and evidence-based drug regimen for treating Mabsc infections in patients with CF. The conventional recommended treatment consists of 3-4 drugs, which together have an eradication rate as low as 20% in Mabsc subspecies abscessus. The goals of this project were: to collect experimental data to train the INDIGO-MABSC, an in silico model for predicting synergistic and antagonistic antibiotic interactions to treat M. abscessus infections; and to identify possible synergistic interactions between antibiotic combinations. I treated liquid cultures with 40 different antibiotics for 6 hours and then isolated genomic ribonucleic acid (RNA). The RNA was then sequenced to determine gene expression changes which can be used for model construction. Using broth microdilution, I set up checkerboard assays to observe the interactions between two different drugs and assess the combination’s efficiency in inhibiting the growth of Mabsc type strain ATCC19977 qualitatively by eye. I calculated the fractional inhibitory combination for each combination. Preliminary results show putative synergistic interactions between drug pairs Cefoxitin-Rifampin, Imipenem-Cefoxitin, Imipenem-Clarithromycin, and Imipenem-Linezolid. From sequencing results, we saw that Rifampin, Imipenem, Clofazimine, Rifabutin, and Cefoxitin did not induce sufficient gene changes. Future directions include additional checkerboard testing for combinations that were inconclusive due to no minimum inhibitory concentration recorded. Additionally, I will work optimize drug treatment for antibiotics that did not induce enough gene expression changes, and also work to collect RNA from cultures treated with novel drugs and antibiotic combinations that are commercially available.

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.

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.

Perchlorate is a water-soluble endocrine-disrupting contaminant that is ubiquitous in our environment and is known to be toxic to humans, however it is not currently regulated in our drinking water. Daphnia magna, an aquatic microcrustacean and established model for ecotoxicology, will be used to study the interactive effects of perchlorate and hypoxia (low oxygen) on the invertebrate endocrine system. Exposure of D. magna to hypoxia is known to increase the expression of the protein hemoglobin, and results in an observable phenotypic shift in color from clear to pink. Another characteristic of D. magna that will allow us to identify environmental stress is the juvenile hormone present in the endocrine system of this species. Juvenile hormones play a role in modulating the switch between asexual (when environment is stable) to sexual (when environment is stressed) modes of reproduction. The regulation of juvenile hormones may be disrupted by some environmental endocrine-disrupting contaminants, like perchlorate. In this study, I will test the hypothesis that D. magna respond to hypoxia and perchlorate through transcriptional regulation of hemoglobin and phenotypic plasticity. Specifically, D.magna will be chronically co-exposed to different concentrations of perchlorate and different levels of hypoxia using a factorial design. Gene expression changes will be evaluated using quantitative real-time PCR of hemoglobin genes (dhb1, dhb2, and dhb3). Phenotypic plasticity will be assessed using microscopy, with an emphasis on quantifying morphological characteristics and mode of reproduction. I anticipate that perchlorate and hypoxia exposure will cause the reproductive modality of D.magna to be altered due to disruption of the juvenile hormone by perchlorate, and additionally, that hemoglobin genes will be transcriptionally upregulated. The data we generate from these studies is critical for future policy related to perchlorate regulation, which will ultimately help protect human and environmental health.

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.

 Alzheimer’s disease (AD) is a complex and common neurodegenerative disease in humans. It is diagnosed by the presence of A beta amyloid plaques and tau tangles in the brain during autopsy, decades after neuropathology has developed. Early diagnosis is still not definitive and contributes to the inability to successfully treat the disease at an early stage. Generally, animal models are not reliable for preclinical studies since lower mammals, including laboratory rodents and nonhuman primates, pet dogs and domestic livestock do not naturally develop AD. The reason is based on conformational differences in amyloid precursor and tau proteins. We have recently observed one exception to this list of mammals- the aging pet cat. Formalin fixed paraffin embedded brain sections from ten 16 to18-year-old pet cats obtained from a regional veterinary teaching hospital were stained with antibodies 6E10 and AT8 specific for A beta and phosphorylated tau using standard immunohistochemistry procedures for diagnosing human AD. One cat showed robust tau tangles and moderate amyloid plaques, while a second cat showed moderate tau tangles and mild amyloid plaques. Brains from additional cats need to be examined for AD neuropathology but this preliminary observation suggests old pet cats develop AD. Since pet cats share the same environment as humans, clinical studies to search for ways of early diagnosis and treatment of this naturally occurring disease in cats will have a huge impact on the translational implications for early diagnosis and intervention studies in human AD.

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.

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.

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.