Current models of pain research involve restrictive forms of resident-intruder pairing where experimental mice are involuntarily placed in social situations. These methods have limited application as the research does not account for individual variability and the dynamic social decision-making characteristic of humans. Our research uses a novel volitional social procedure that more accurately represents human behavior in the context of pain. I conducted social self-administration protocols on C57 strain mus musculus to quantify changes in voluntary social interaction before and after neuropathic pain has been induced via spared nerve injury. In addition, I utilized a Von Frey filament test to measure changes in pain sensitivity over this time period. Two social self-administration (SA) experiments were conducted on separate cohorts of C57 mice. In Experiment I, SA was run intermittently at 3-day intervals following neuropathic injury, providing lapses between voluntary social engagement. In Experiment II, SA was run continuously following neuropathic injury. We found that the continuously run SA group experienced a rebound in social interaction to levels matching their pre-surgery states and sham controls, whereas the intermittent group displayed a stark decline in voluntary social interaction that reached statistical significance from sham controls on day 8. Interestingly, tests of allodynia that were conducted to determine prolonged mechanical sensitivity typical of chronic neuropathic pain showed that both groups were experiencing equal levels of increased pain sensitivity throughout behavioral testing. Our results show promise in revealing the dynamic connection between social interaction and pain perception. Research has already identified key areas of interest such as the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) as hubs responsible for regulating social behavior. We aim to further examine the physiological changes that occur in these areas as a result of persistent pain using a variety of sophisticated analytical techniques.

Activins are a class of growth factors belonging to the Transforming Growth Factor-Beta (TGF-β) superfamily that recruit a tetrameric complex of type-I (ACTRI) and type-II (ACTRII) transmembrane serine/threonine kinase receptors to cause downstream signaling in various critical cell-signaling pathways. Inhibition and supplementation of the signaling molecules offer new possibilities in many therapeutic and clinical applications. The overexpression of Activin A has been shown to be a major driver of diseases such as pulmonary hypertension, chronic kidney disease, and cerebrovascular disease. Sotatercept, one of the few existing activin-A-inhibiting therapeutics, is in clinical trials for treating pulmonary arterial hypertension, but, like many ligand traps, it is designed by fusing a large antibody domain to a native activin receptor domain, making it expensive to produce and unstable due to its large size. There is a need for therapeutics that can be produced in bacteria, replicate high efficacy and strong affinity, and minimize side effects. These therapeutics can be developed through de novo protein design, which involves designing proteins from scratch, with a new, unique sequence predicted to fold into its specific corresponding structure or by grafting native protein-protein interfaces onto novel scaffolds. This project aims to use computational de novo protein design methods to develop a therapeutic ligand trap for Activin A. The design pipeline involves scaffolding functional motifs, optimizing protein sequences, predicting protein structures, and filtering designs using metrics calculated by Rosetta, a protein design tool. With this pipeline, we have tested several design strategies, providing knowledge-based guidance and analyzing outputs to inform strategies for future design iterations. Recent designs have passed Rosetta filters, and we have ordered the corresponding genes to express and purify the designed proteins. Once purified, they can be experimentally characterized, testing binding and stability in vitro, and then further optimized to create stable Activin A ligand traps.

Voice assistants (VAs) are transforming how humans interact with technology. While promising, state-of-the-art VAs like Siri and Alexa do not incorporate a user’s spatiotemporal context such as their surrounding objects or gestures, which results in degraded performance and unnatural dialogue. Since pronoun usage is inherent to everyday speech, we expect future VAs to support ambiguous speech queries. We introduce GazePointAR, a wearable augmented reality (AR) system that resolves ambiguity in speech queries using eye gaze, pointing gestures, conversation history, real-time computer vision, and a large language model (OpenAI’s text-davinci-003). With GazePointAR, a user can ask “what’s over there?” or “how do I solve this math problem?” simply by looking and/or pointing. Upon voice activation, GazePointAR listens for the query, takes a screenshot, and narrows the focus by incorporating information from eye gaze, replaces the pronoun in the query with the detected objects and texts, and utilizes a language model to answer the modified query. To assist in this project, Liam and I reviewed relevant literature, brainstormed technical solutions for multimodal integration, constructed user study scenarios, and conducted reflexive thematic coding on qualitative data. To evaluate GazePointAR, we conducted a three-part lab study that compared GazePointAR to two other state-of-the-art query systems (Google Voice Assistant and Google Lens), examined GazePointAR’s pronoun disambiguation for three tasks, and concluded with an open-ended component where users could suggest and try their own queries. Participants appreciated the improved simplicity and human-likeness of context-aware queries; however, they preferred faster response times and better explanations for query results. By combining visual and voice inputs to answer a broader range of questions, GazePointAR provides a foundation for future works of VAs, such as designing a more anthropomorphic VA.

Cell division is essential to all living organisms, and microtubules are critical for this process. Microtubules in bundled kinetochore-fibers generate forces to move chromosomes by stochastically switching between assembly and disassembly states. Electron tomographs of mammalian mitotic spindles show that microtubule tips maintain a high level of coordination despite stochastic switching between states. The collective behaviors of microtubule bundles give rise to chromosome oscillation, which promotes the alignment and separation of chromosomes along the spindle equator during metaphase. To understand how stochastically-switching microtubules can produce synchronized motions in vivo, we use a mechanical model that describes the force-dependent dynamics of microtubules. The model reproduces the bistability of microtubule bundles observed in vivo and suggests that this coordination could be explained by sufficiently stiff mechanical coupling. We also propose a new way to characterize microtubule bundle state based on the substates of microtubules within the bundle. We will use this characterization to calculate microtubule bundle switch rates, which are technically difficult to distinguish otherwise, thereby laying the groundwork for future comparisons between bundle switch rates in vivo and in silico. In the future, we plan to explore the assumptions on which this model is based by analyzing the relationship between microtubule velocity and forces. We predict that this result will validate the assumed force-velocity relation of microtubule growth and contribute to a more accurate understanding of kinetochore-fiber behaviors. 

The Hijra is a South Asian community of genderqueer peoples which perform spiritual rituals and give blessings, typically at births and weddings in return for donations. This ancient community, however, has a long history of invisibilization and criminalization in South Asia. I am studying the Hijras’ physical and literary portrayal as it connects with, and unfolds from, their histories of criminality, invisibility, and mobility under imperial and post-imperial rule. Knowing this history is valuable to the field of colonial and gender/queer studies, as it provides a better understanding of how minoritized peoples navigate – and survive – changes in power and status under shifting power. The Hijra is a good interdisciplinary model for this, as they are minoritized at intersections of gender, religion, sexuality, caste, and class. I am using Indian epics, including the Mahabharata and Ramanayana, to theorize the figure of the Hijra as it exists in the dominant South Asian literary tradition. I am pairing this with articles on Hijra identity, which historicize the community’s legal and social statuses under the Mughal and British imperial formations, up into the decades post-South Asian independence. The purpose of tracing this history is two-fold: (1) to attempt the respectful re-tracing of a genealogy which, until now, has been invisible under various dominant empires and (2) to understand the Hijra peoples’ increasing inclusion into both Western research and South Asian democracy. The urgency and relevance of my inquiry has increased as pro-genderqueer legislation is currently being passed at unprecedented rates in South Asia. This includes India’s 2014 “third gender” law, which officially recognizes the Hijra as the nation’s “third gender.” While many celebrate this inclusionary shift, I argue that the change must be critically assessed, as it represents another incoming shift in global power that minoritized communities will have to navigate and survive.  

Refugee resettlement job training programs do not necessarily offer positive long-term economic outcomes. I have discovered a gap in peer-reviewed articles about the flaws within the existing resettlement employment services. The problem is that there are two types of employable refugees with distinct needs: (1) Refugees who already have professional credentials who need their credentials officially recognized so they may continue their chosen profession and (2) Refugees who need professional development that will connect them to sustainable paid employment. I have performed a systematic literature review that focuses on factors that contribute to the economic stability of the adult refugee population during their resettlement period in the United States. The selection criteria materials have been retrieved from the University of Washington Library Search and Web of Science including search terms related to refugee resettlement services that focus on the refugee population in the United States with referenced English language peer-reviewed scholarly articles published within the last five years. The overall findings show that refugees are to acquire basic job skills within the first six months of their resettlement as they are provided funding and housing during this short window of time. My research reveals that established programs only offer short-term solutions thus the significance of this problem is that the current system focuses refugees on survival employment with low paying jobs that do not necessarily match their qualifications. Refugees who are highly skilled and overqualified face insurmountable obstacles for credential recognition in the United States and are grossly underemployed, unable to sustain a livable wage. The implications of this have prevented both sets of refugees from reaching sustainable economic equity and have generational impacts on the livelihood of refugees in the country.

Management of muscle-invasive urothelial carcinoma is extremely limited with Anti-FGFR3 (Erdafitinib) being the only kinase-targeting therapy FDA-approved for treating advanced bladder cancer patients. Kinases represent an important family of proteins for drug development due to their well-characterized role in tumor growth and disease progression. Until the last decade, the process of identifying essential proteins was carried out by individually depleting their production using siRNA or shRNA knockdown. To expedite the process of discovery, we’ve applied a machine-learning model that predicts the activity of 428 kinase inhibitors and identifies the most essential kinases associated with promoting viability for five bladder cancer cell lines used in this study (COCAB1, SW780, COCAB11, UMUC5, & SCABER). This technique, known as polypharmacology, leverages the use of non-specific kinase inhibitors that target multiple individual kinases. We have validated the results of this computational method by measuring the effect on cell viability for 7 kinase inhibitors representing strong, moderate, and weak predicted impacts. Using live-cell imaging we quantified relative cell growth over 72 hours and found a positive correlation between predicted and observed effects on viability for all cell lines. The regression identified PTK5 (FRK) and VEGFR2 (KDR) as a common top essential kinase across all five cell lines. Currently, we are exploring validating the essentiality of these kinases by siRNA knockdown. Complete validation of this polypharmacology method would suggest continuing the evaluation of PTK5 and VEGFR2 as candidate novel therapeutic targets to treat advanced bladder cancer patients.

Many standard oxidants and reductants are non-reusable and toxic, so it is important to pursue cleaner alternatives. In this project, we have synthesized and characterized two metal-bipyridyl supramolecular cages and have studied their application as catalysts for the electrochemical reduction of organic substrates. Supramolecular cages are formed from the self-assembly of organic ligands and metal ions in solution, and they contain internal cavities with unique electronic microenvironments, similar to the interior of enzymes. While these polyhedral structures have been investigated as catalysts for traditional synthetic pathways, their role in electrosynthesis remains underexplored. Electrosynthesis involves the transfer of electrons to and from substrates using an applied potential, rather than chemical redox agents. This method is often hindered by a high kinetic barrier at the electrode-substrate interface, but catalysts can lower this barrier. We hypothesize that redox-active supramolecular cages – cages that can readily interconvert between charge states – can serve as effective electrocatalysts by encapsulating and transferring charge to substrates. To understand the effect of ligand geometry on electrocatalysis, I have synthesized two redox-active ligands with bipyridyl chelating groups. One contains a highly conjugated perylene core, while the other contains a compact core formed from pyromellitic dianhydride. We have metalated these ligands with iron ions to form two tetrahedral supramolecular cages. We then utilized cyclic voltammetry to assess cage-facilitated charge transfer to vicinal dihalide substrates. We observed that the reduction of multiple substrates, including 1,2-dibromo-1,2-diphenylethane, occurred at milder voltages in the presence of the cages, indicating a reduced kinetic barrier. For these substrates, we then performed bulk electrolysis, from which we determined that the percent conversion to the desired product was significantly higher when a cage was present, supporting our hypothesis. Ultimately, we aim to use these cages to enable electrosynthesis of organic feedstocks at lower voltages and with fewer byproducts.

Edentulism, or the loss of all natural teeth, is a condition typically resulting from dental caries and periodontal disease, described as the ultimate manifestation of oral health disease burden. Edentulism has significant effects on masticatory function, such as chewing, swallowing, and speaking, as well as mental health and overall quality of life. Food insecurity has been associated with adverse oral health outcomes. However, this relationship has been insufficiently studied among low-income older adults, who are disproportionately impacted by oral health disparities and more likely to be edentulous. We hypothesized that food insecurity would be associated with increased prevalence of edentulism among low-income older adults in Washington state. To test this hypothesis, we collected data at seven community-based sites across Washington, including the Multi-Service Center in Federal Way and the Pike Market Senior Center. We administered a survey containing a 10-item measure of food insecurity to 218 participants aged 50 years or older. We also conducted dental screenings that involved examination of the mouth, gums, and individual teeth. 45% of participants (n=98) had marginal, low, or very low food security. 7.8% of participants (n=17) had zero teeth. We utilized logistic regression models to examine the association between food security and edentulism. Our preliminary analysis indicates that older adults with low or very low food security had 2.44 times the odds of being edentulous compared to those with high food security, after adjusting for age, sex, and race. Further analysis can help inform the development of feasible and effective interventions to address oral health inequities among low-income older adults.

In humans, the KRAS protein normally acts as a switch to regulate cell growth. Acquisition of certain mutations result in constant activation of the protein, leading to uncontrollable cell growth. The most frequent such mutations are at the glycine-12 residue of the protein, including changes to valine (G12V). Cells with this mutation can be recognized and eliminated by T cells engineered to express an antigen-specific receptor (TCR), but posttranslational modifications, such as methylation, can interfere with the ability of such engineered T cells to recognize G12V mutant KRAS. The objective of my project is to identify and eventually disrupt mechanisms that lead to methylation of KRAS mutant antigens. I hypothesize specific enzymes are responsible for methylation of mutant KRAS. To accomplish this, I will generate CRISPR-Cas9 gene knockout libraries to screen for potentially responsible enzymes. The CRISPR library will target human genes encoding methyltransferase and demethylase enzymes. I will co-culture these CRISPR-Cas9 knockout cancer cells with T cells engineered with a TCR targeting the unmethylated KRAS mutant antigen. From the tumor cells that have survived this coculture, I will sequence genomic DNA to determine which knockouts are enriched/depleted after the co-culture. Preliminary results show certain gene knockouts are significantly enriched/depleted in co-culture compared to baseline. The sgRNAs that appear from the high-throughput knockout library most likely to be involved in the KRAS methylation pathway will be individually evaluated in additional co-culture experiments. This should allow me to assess the proliferation/susceptibility of these cancer cells in more detail. For confirmation of mechanism, I will evaluate the methylation status of the KRAS mutant antigen in cancer cells rendered susceptible. My goal is to determine the process leading to methylation of KRAS antigen and then target it to allow for more effective targeting of KRAS-driven cancers using TCR-T cell immunotherapy.

Microchimerism (Mc) occurs when a small amount of genetically different cells (or DNA) is acquired from another individual. Mc is acquired naturally during pregnancy due to exchange between the mother and child and can be detected decades later. Maternal microchimerism (MMc) is when a person harbors Mc from their mother. MMc is frequently detected in healthy adults but is increased in individuals with some autoimmune diseases, including scleroderma. Few studies have investigated MMc, especially whether it changes in a woman after her own pregnancies. One study tested MMc in peripheral blood of women during the time they were pregnant and occasionally detected MMc, but not if the woman had preeclampsia. No study has addressed whether MMc prevalence and quantities change in healthy women according to the time since the woman’s own childbirths or number of childbirths. This study addresses this knowledge gap. MMc was assayed using a panel of polymorphism-specific real-time polymerase chain reaction (qPCR) assays on DNA from peripheral blood. Most assays employed human leukocyte antigens (HLA)-specific primers and fluorogenic probes targeting non-inherited, non-shared HLA sequences. Each woman and her mother were HLA typed to identify an appropriate target. In total, 142 women were tested, and 266 qPCR experiments were run. Preliminary analysis found evidence of MMc in 59 of the 266 samples and a trend of MMc prevalence being highest within the first year postpartum and ten years after childbirth. Prevalence and quantities of MMc are being analyzed in collaboration with a biostatistician. Pregnancy and childbirth are known to affect some autoimmune diseases and cancer risk. Addressing the knowledge gap about MMc according to the time since birth and the number of births in women could provide further insights about some autoimmune diseases and cancers.

Hemagglutinin (HA) is a glycoprotein found on the surface of the influenza virus. HA binds to sialic acid receptors on host cells and mediates membrane fusion, allowing the virus to enter the cell. This project investigates how mutations associated with species crossover affect viral fusion mechanisms in influenza HA. HA from the past outbreaks of H5N1 influenza strains in Vietnam in 2004 (VN04) and Indonesia in 2005 (IN05), are being compared to an HA from an on-going avian influenza outbreak using a strain isolated in Colorado in 2022 (CO22). Through site-directed mutagenesis, mutations that affect acid stability and affinity for the human receptor were added to HA based on the VN04 and IN05 strains; we are studying these effects in recombinant protein rather than on infectious virus. These mutations are believed to enable the virus to increase transmissibility among mammals including humans. To compare how HA from these H5 isolates with and without the adaptive mutations behave, hydrogen-deuterium exchange mass spectrometry (HDX-MS) is being used to measure changes in deuterium incorporation on the protein backbone for specific peptide segments, giving a profile of local dynamics and structure throughout the HAs. By comparing the dynamic profiles for each as pH is lowered, mimicking acid-activation in host cell endosomes, we can probe how their structure in important fusion and human receptor-binding regions change as the fusion protein becomes activated. By comparing the structural dynamic changes of the WT and mammal-adapted, mutated IN05 and VN04 HA to the new CO22, we will be able to increase our understanding of the effect of the mutations that are associated with species crossover and hopefully be able to gain insight into the potential of this new avian influenza strain’s ability to become transmissible among humans.

Over the past 20 years, the capital city of Indonesia has sunk four meters into the ocean and continues at a rate of approximately 11 inches per year. At this rate, more than 95% of Jakarta, the capital city, is predicted to be completely submerged by 2050, inevitably drowning the city if stricter policies are not set in place for groundwater extraction. Due to the population’s poor access to piped water, private businesses and the public have resorted to illegal groundwater extraction, which has caused extensive land subsidence. The combined effects of regulated and unregulated groundwater extraction and the lack of equitable water access to the population have worsened the situation. Jakarta is not alone; other countries like China, Thailand, Vietnam, and the United States are also experiencing land subsidence due to extensive groundwater extraction. My literature review will demonstrate the effects of groundwater extraction and land subsidence in Indonesia by using a comparative public policy analysis on the different policies and management approaches of countries in Southeast Asia and North America. I will be comparing the policies in use by these different countries to determine the necessary policy reforms to create a more effective approach to solving the groundwater issue in Indonesia. I establish that the Indonesian government’s short-term plans to overcome land subsidence is not a sustainable solution to the damage that has been done to the city of Jakarta. Rather, the policies set in place need to be reformed to ensure that equitable water is supplied throughout the population and groundwater extraction is done safely and in moderation. This work is important not only to prevent the further sinking of Jakarta but also to overcome the long-term environmental impacts of groundwater extraction in Indonesia and other countries.

In long term data collection, sampling is a key parameter that can dictate the amount of data collected and also influence the available conclusions to be drawn. In the context of passive acoustic monitoring, recording an area’s soundscape at intervals (i.e., subsampling based on duty cycles) can alleviate data management costs, and has been widely investigated in the bird monitoring literature. However, the influence of duty cycle-based subsampling in passive acoustic monitoring of bats has not been thoroughly studied. Here, we discuss the effects of subsampling on ultrasonic recordings collected using AudioMoth recorders in the Union Bay Natural Area in 2022. We recorded continuously over the summer and then computationally applied duty cycles onto our data to generate subsampled data. The subsampled data was then fed into multiple bat call detection algorithms to understand the influence of different subsampling schemes. Our results show that subsampling schemes can impact the trends and activities that can be acoustically observed from echolocating bats. The results also show how the subsampling parameters may be tuned to collect valuable information while keeping data management costs low. We anticipate that this detailed investigation will aid in the design of efficient, long-term bat acoustic monitoring projects.

Genetically encoded fluorescent indicators (GEFI) change fluorescence level under microscope following a conformational change when bound to a target molecule, and can be used to visualize spatiotemporally specific biological processes involving a targeted molecule. Various imaging analysis tools exist to analyze the non-temporal fluorescent cell data, however there was no industry standard for the pipeline used to analyze molecular dynamics when imaged with GEFI in time-series experiments. This project aimed to develop a computational pipeline that analyzed the fluorescent readout of single cells in spatiotemporal experiments that utilized GEFI. The pipeline included both segmentation of cells, utilizing Cellpose, an existing deep learning-based generalizable and highly efficient segmentation program, and tracking of single cells across all frames. I personally contributed to the design, implementation, and testing of the tracking component of the pipeline. The tracking algorithm was designed using unsupervised machine learning, specifically k-means clustering with convolutional neural network feature extraction techniques. The pipeline was implemented using Python and made available and open source, accessible through Google Colaboratory for a more user friendly version, as well as Github for more thorough documentation and generalizability. Ultimately, this project aimed to minimize bias to result in more accurate and efficient high-throughput investigation of molecular dynamics when using fluorescent probes for dynamic cell imaging. Preliminary results demonstrated the effectiveness of the pipeline in tracking cells across various time points and provided a foundation for future optimizations and applications.

Colorectal cancer (CRC) is a prevalent and often lethal form of cancer, with complex underlying biology and a wide range of associated risk factors. CRC is a significant global public health challenge, it is the second leading cause of cancer-related deaths in the United States. A growing body of research suggests that activin can stimulate the MAPK pathway which plays a critical role in the development and progression of CRC. In the context of colorectal tumors, innate immune cells such as macrophages, dendritic cells, and natural killer (NK) cells can both promote and suppress tumor progression. Macrophages, for example, can either promote tumor growth by secreting growth factors that stimulate angiogenesis and immune suppression, or they can suppress tumor growth by producing cytokines that activate T cells and other immune cells. In this study, our aim is to examine the relationship between activin and the MAPK signaling pathway in macrophages and to determine the role of macrophages in producing and responding to activin. We will be using RAW264.7 macrophage cell line to study the role of macrophages in cancer and Western blot to detect changes in specific proteins in macrophages stimulated with activin. We predict to see an increase in MAPK activation in activin-stimulated macrophages which will enhance tumor elimination. Our findings will also improve the understanding of the innate immune response to colorectal tumors and how it can be modulated by targeting activin signaling. This research will provide new insights into the complex interplay between the immune system and colorectal tumors and may inform the development of new diagnostic methods and more effective treatments.

Colorectal cancer (CRC) is the third most common cancer globally, and the second leading cause of cancer deaths in the US due to metastasis. The five year mortality rate of Stage IV CRC patients remains around 90%, and most treatments for stage IV CRC are palliative. Activin A, a cytokine that regulates proliferation and apoptosis, is known to induce metastatic phenotypes in CRC cells primarily through the PI3K/ AKT pathway. Glycogen synthase kinase-3 beta (GSK3β) is an enzyme downstream of AKT that regulates energy metabolism and apoptosis. An inactive form, phosphorylated GSK3β (pGSK3β), is unable to inhibit β-catenin activity, which promotes proliferation and epithelial to mesenchymal transition. Very few connections between activin A signaling in colorectal adenocarcinoma cells and GSK3β phosphorylation have been established in scientific literature. Our project aims to study the correlation between activin A signaling and pGSK3β by testing the hypothesis that activin A signaling induces phosphorylation of GSK3β in CRC tumor cells, promoting the attenuation of cell cycle arrest. We will perform Western blot analyses on FET cells treated with a vehicle control, activin A, or TGF-β, a cytokine that is activin-dependent in the context of CRC, to measure the levels of pGSK3β relative to GSK3β. We expect to see the highest levels of pGSK3β relative to GSK3β in FET cells treated with activin A and the lowest relative pGSK3β levels in cells treated with the vehicle control. The findings of this project can contribute to identifying biomarkers useful for risk stratification in metastatic CRC to provide more individualized treatments for patients.