WNT signaling plays an essential role in many developmental processes with WNT molecules functioning as directional and differentiation cues. The recapitulation of WNT signaling pathways during embryonic morphogenesis is a promising approach for the development of novel regenerative therapeutics to treat various conditions. Recent work from our lab has demonstrated wnt16, a wnt family member, to regulate zebrafish embryonic myogenesis. We showed that loss of function in wnt16 induced changes in zebrafish muscle morphology. Moreover, myogenic precursors coexpressed wnt16 and pax7, a gene that promotes muscle differentiation and is a marker of satellite cells that support skeletal muscle regeneration. These findings indicate that wnt16 is necessary for muscle morphogenesis, however, its specific function in muscle regeneration remains unclear. I hypothesize that wnt16 influences skeletal muscle regeneration by regulating pax7 in satellite cells. To test this, I am determining the time course of wnt16 expression in pax7+ satellite cells in injured zebrafish muscle. Additionally, I will determine whether wnt16 is necessary for activation of pax7 following muscle injury. Through this study, I expect to establish 1) that wnt16 is upregulated after a muscle injury as an injury response gene, and 2) that wnt16 is necessary for pax7 activation in satellite cells. If the outcomes are as expected, this will show that wnt16 influences skeletal muscle regeneration, mirroring its role in zebrafish myogenesis and that WNT16 may be a target for developing therapeutics to treat muscle injuries.

Oritavancin and dalbavancin are mechanistically similar to vancomycin and daptomycin and are among the longest-acting antimicrobials in clinical use with half-lives up to 14-days. This long duration of action increases antimicrobial exposure but prolongs the mutant selection window: the concentration range that selects for resistant mutants. Previous work shows that these drugs readily select for resistance and cross-resistance to vancomycin and daptomycin in Staphylococcus aureus. We hypothesized a similar cross-resistance selection potential against colonizing opportunistic pathogens including enterococci. While less virulent than S. aureus, enterococci are leading causes of multi-drug resistant infections among elderly and immunocompromised patients. To test this hypothesis, we serially passaged two strains of Enterococcus faecalis and three strains of Enterococcus faecium for 21-days in escalating concentrations of oritavancin or dalbavancin to facilitate the evolution of resistance. Minimum inhibitory concentrations (MICs) for oritavancin, dalbavancin, vancomycin, and daptomycin were performed by broth microdilution on isolates recovered from screening plates. Whole genome sequencing (WGS) was performed to characterize genotypic mechanisms of resistance. Resistance phenotype stability was assessed by passaging isolates on drug-free media and repeated MIC testing. Among dalbavancin-passaged enterococci, only daptomycin cross-resistance emerged. Serial passage in oritavancin selected for cross-resistance to dalbavancin in all strains, and cross-resistance to vancomycin among vancomycin-susceptible enterococci. Cross-resistance to daptomycin was not observed. WGS revealed fewer mutations than expected. Despite elevated MICs, no mutations were detected in E. faecalis. Only one strain of E. faecium acquired mutations in the vanZ and rodA gene. The absence of mutations in our evolved strains requires further investigation, including repeating WGS and screening for gene copy number variants that may have been missed by our initial analysis. Our data suggests that clinicians should exercise caution when using oritavancin to treat enterococcal infections due to the potential for cross-resistance to first-line antibiotics, such as daptomycin and vancomycin.

Electrochemical water splitting, a promising green energy solution, produces O2 and energetically rich H2 gas molecules as products on the anode and cathode, respectively. Attachment of O2 and H2 bubbles on the surface can decrease the overall efficiency of electrochemical water splitting.Therefore, continued investigation of nanobubble characteristics is of utmost importance to improve the technologically relevant electrochemical generation of H2 gas. Nanobubbles can interfere with the efficiency and productivity of industrial processes by blocking electrode surfaces, however, they could also potentially be utilized to catalyze reactions themselves. To better understand these processes, it is important we create consistent samples of nanobubbles that can be formed individually and reproducibly, and gain a deeper understanding of their properties. We have been working to create a procedure that efficiently and reliably produces carbon nanoelectrodes for single nanobubble analysis. To accomplish this, we pull quartz capillaries to a nano-sized tip to create a nanopore, and heat the nanopores while applying a flow of methane in an oxygen-free environment to deposit carbon inside the capillaries, producing nanoelectrodes. We test the electrochemical properties of the nanoelectrodes by measuring the observed current when applying a potential and evaluating whether the cyclic voltammetry graph generated suggests that the nanoelectrode is capable of generating a nanobubble. Certain elements of our current procedure may need to be adjusted to improve the reliability of the nanoelectrodes, but so far our experimentation in nanoelectrode fabrication has allowed for a more reliable process in generating ideal hydrogen nanobubbles. This procedure has helped us gain a better understanding of the impact of nanobubbles on an electrochemical system and provide a better physicochemical description of the bubble. In the future, we plan to apply the knowledge gained through these experiments on theta nanoelectrode fabrication, which is an electrode with a partition in the middle.

During non-REM slow-wave sleep, the thalamus and cortex generate widespread synchronized epochs of action potential firing that repeat at 1 to 7 Hz. This synchronization is thought to be an essential component of healthy sleep. Cortical excitatory synaptic feedback to the thalamus is required to maintain synchronized firing epochs across the thalamus. A major source of cortical to thalamic feedback comes from a subset of layer 5 (L5) pyramidal neurons (PNs) which occur in the largest numbers in the primary motor cortex. There is also evidence that the basal dendrites of those neurons in the motor cortex receive monosynaptic executory inputs from excitatory thalamic neurons but the number and strength of those connections are not known. For our project, we decided to quantify the number of thalamic inputs onto the basal dendrites of those L5 PNs that project back to the thalamus. I used tissue from the motor cortex of thy1 mice which express a yellow florescent protein in the L5 PNs that send axon branches to the thalamus. The tissue was treated with an antibody to the VGLUT2 protein which is selectively expressed in excitatory synaptic terminals from thalamic neurons. The VGLUT2-containing terminals were visualized using florescent immunocytochemical techniques combined with confocal microscopy to count the number of putative thalamic synaptic terminals that were closely opposed to spines (postsynaptic protrusions) on the L5 PN basal dendrites. Preliminary results suggest that between 5-20% of the dendritic spines are closely opposed to VGLUT2-containing presynaptic terminals. The study of this thalamocortical loop will allow for a better understanding of the processes that are necessary for proper sleep and the implications of disrupted neuron synchronization.

The health of Sockeye salmon (Oncorhyncus Nerka) stocks are of high importance to the cultural well-being and sovereignty of Coast Salish tribes. There are multiple ecotypes of Sockeye that include anadromous, potamodromous Kokanee, and resident Sockeye that all carry distinct and significant cultural value for Indigenous communities, as well as distinct ecological roles within the ecosystems they inhabit. Sockeye are impacted by anthropogenic stressors such as habitat degradation and pollution, which can cause shifts in food web dynamics, population declines, and impact commercial and traditional fisheries. To more effectively manage these diverse life histories for conservation, it is crucial to understand the distinct ecological functions that may cause ecotypes to differ in their vulnerability to anthropogenic stressors, thus requiring different management strategies. We will analyze carbon and nitrogen isotopic signatures of Sockeye from Lake Washington to differentiate their ecological niches. Muscle samples were collected from 46 frozen sockeye samples, freeze dried, and then analyzed using mass spectrometry. Preliminary results suggest that there are significant differences between the isotopic signatures of anadromous and potamodromous ecotypes. We hypothesize further interpretation of the results coupled with genetic analysis will identify differing ecological roles in accordance with the diverse life history strategies Lake Washington Sockeye display. This study has been conducted as part of ongoing collaborative efforts with partners at the Snoqualmie Tribe, and the Kokanee Work Group, which aims to restore the Kokanee and Sockeye populations of Lake Sammamish. The results of this work will directly inform management actions taken by our partners to conserve the native Sockeye populations of King County, Washington.

Research, and evolutionary theory, demonstrates that romantic relationships and platonic friendships fulfill different needs and are necessary for survival and critical for reproductive success. These relationships can be highly gendered with different dynamics. Usually, women’s same and cross-sex friendships are more fulfilling and richer. However, there is limited research looking at romantic relationships and platonic friendships and the individual differences and characteristics within these relationships. The current research explores how gender, sexuality, and heteronormativity impact the assessment, comparison, and characteristics between platonic and romantic relationships. We explore which individuals break out of traditional gendered friendship norms and seek similar interactions within their platonic friendships and romantic relationships. 194 Participants answered a questionnaire measuring the different characteristics desired in platonic friendships and romantic relationships. This survey was answered twice, once while thinking about a platonic friend and again for a romantic partner. Relationship characteristics were measured by scales, created by the researchers, that looked at emotional intimacy, physical intimacy, quality time, action affection, verbal assurance, and relationship perception. Romantic relationships had no difference between genders or sexualities for any of the scales. However, there were significant differences between genders and sexualities found in platonic relationships. Non-heterosexual women and non-binary individuals had higher engagement and support and had the highest overall assessment ratings within their platonic relationships. Heterosexual men and non-heterosexual women differed the most in their ratings for all platonic relationship characteristics, heterosexual men having lower scores on average. This supports previous research that men have less fulfilling relationships than women.

Batillaria attramentaria, a species of marine snail, is highly invasive in intertidal habitats along the Pacific coast of North America. B. attramentaria is regularly infected with a parasitic trematode, Cercaria batillariae. Parasitic infection is known to alter both physical characteristics and behavior of snail hosts, in many cases to the benefit of the parasite’s survival and reproductive fitness. I investigated the effects of parasitic infection on B. attramentaria antipredator responses. Snails were sampled from the intertidal area at Penrose Point State Park in Lakebay, Washington, and acclimated to tanks in controlled laboratory conditions. Afterchemical cues derived from a predatory crab were introduced, antipredator responses, including distance moved and reaction latency, were measured for one hour. I hypothesized that snails with parasites would display a reduced antipredator response. If there is a parasitic influence on the host, these behaviors might be advantageous to the parasite cercaria by putting them in more frequent proximity to secondary hosts and increasing the likelihood of completing their life cycle. Enhanced understanding of the parasite-host interaction can give further context to existing studies on the success of B. attramentaria, and provide additional information for current models seeking to understand the influence of this invasive species on native intertidal ecosystems.

Adolescents with prosocial purpose focus on their friends, family and community to develop their identity and maintain positive future expectations. During the COVID-19 pandemic, prosocial purpose may have acted as a source of resilience for teens, especially Peruvian adolescents who faced extreme stress during a uniquely strict lockdown. The aims of this mixed-methods study are to 1) quantitatively investigate gender and school-grade differences in prosocial youth purpose across adolescence and 2) qualitatively describe whether Peruvian adolescents perceive that the pandemic transformed their sense of purpose. Findings show that girls had higher commitment for beyond-the-self goals than boys, there were no school-grade differences in terms of frequency of endorsement of self-oriented goals or beyond-the-self goals. I found that half the students reported that the pandemic had made them shift their goals and perspectives. These findings provide further information on the impacts of COVID-19 on adolescent mental health in underserved populations like Peru.

Developmental language disorder (DLD) is one of the most common language disorders. It interferes with a person’s ability to learn and understand language. Previous studies have found that children with DLD struggle with motor skills more than their typically developing peers. There is also evidence that implicates the striatum (a subcortical brain structure) as a key difference in DLD; one study in in adolescents with DLD found reduced myelin in the striatum. Our study aims to examine how striatal myelination is associated with motor skill differences in adults with DLD. Participants between 18-45 years old will be tested on fine and gross motor skills using the Bruininks-Oseretsky Test of Motor Proficiency. Subtests of these skills include fine motor precision, fine motor integration, manual dexterity, upper limb coordination, bilateral coordination, and balance. In another session, the participants will undergo one hour of MRI scanning with a variety of functional and structural scans. I will analyze one type of MRI data to evaluate myelin markers in the striatum (MTsat and R1 maps). Individual composite scores for fine and motor skills will be correlated with the amount of myelin in the striatum to see if there is a significant relationship between myelin and motor skills in this population. We hypothesize that individuals with DLD will have lower motor skill scores than the control group, as well as a correlation between lower amounts of striatal myelin and motor skill deficiencies in the participants with DLD. We hope that the findings of this study will allow us to have a better understanding of which brain structures and behaviors are affected in adults with DLD. Research on DLD in adults is yet scarce, which consequently leads to limited resources and treatments for individuals with DLD.

The mammalian target of Rapamycin (mTOR) signaling cascade plays an important role in a variety of cellular processes, such as autophagy, cell proliferation, and protein synthesis. Previous depictions of signaling through the mTOR pathway have suggested linear signal transduction; however, this does not accurately represent the network of interactions between proteins in complexes of this pathway nor their dynamics in response to stimuli. To better characterize mTOR protein interaction network (PIN) dynamics, SEPS lab has developed a panel of antibodies targeting key proteins in mTOR signaling for use in quantitative multiplex co-immunoprecipitation (QMI), a method of detecting changes in protein interactions using flow cytometry. Following QMI of mTOR signaling proteins in serum-starved and serum-refed mouse 3T3 fibroblasts, I validated changes in select interactions from this pool separately via co-immunoprecipitation and western blot analysis. The lab then applied inhibitors of mTOR pathway constituents, including PI3K, AKT, MEK, ERK, and mTOR, to define modules of interactions that comprise the PIN and observe changes in these interactions with stimulation after application of each inhibitor, which I again validated via co-immunoprecipitation and western blot analysis. Finally, to validate antibody specificity in human cells, I prepared human embryonic kidney 293 (HEK293) cells for short interfering RNA (siRNA) transfection and knockdown of mTOR pathway proteins targeted by antibodies from the initial panel. Assuming these HEK293 cells lack any additional proteins with high affinity for these antibodies, I expect flow cytometry data to reflect specificity seen in the 3T3 fibroblasts. Conducting this validation is critical for ensuring the reliability of the PIN changes observed in QMI analysis. These experiments allow us to evaluate coordinated interactions between mTOR pathway proteins and their dynamics during signaling events, which is highly useful in developing treatment strategies for mTOR pathway-associated disorders.

Birth asphyxia is the inability of a newborn to begin and maintain breathing. Twenty-three percent of neonatal deaths globally are caused by birth asphyxia [1]. Birth asphyxia results in a neurological injury called hypoxic ischemic encephalopathy (HIE). Rapid HIE screening within six hours after birth is crucial to identify neonates at risk. Unfortunately, the diagnostic equipment is impractical for low resource settings because it is costly ($20/test and $5,000 for equipment) and requires technical staff, that are in short supply, to operate. We hypothesize that a cost-effective device can be developed for HIE analysis. pHast Cam quickly screens for birth asphyxia and HIE in infants via a paper-based blood pH sensor. The device combines an inexpensive pH sensitive dye, a smartphone camera, and a fixture that controls the imaging environment to quickly identify acidosis that results from HIE. A low-cost paper-based strip is made with a water-soluble resin doped with a pH-sensitive dye, bromothymol blue (BTB), and a membrane to filter out red blood cells. The fixture removes lighting variation. The smartphone camera records the pH indicator image, and an algorithm captures, reduces noise, and accesses color change. pHast Cam incorporates four features: 1) accurate assessment of acidity within 0.05 pH units, 2) require only a few microliters of blood, 3) use electrical hardware and software only from the smartphone, and 4) affordability. At this stage, we have achieved a regressive linear model that predicts buffered solution acidity. In the future, we will transition from measuring buffered solutions to blood-plasma. Ultimately, we expect pHast Cam to screen for HIE by quantifying plasma pH in neonates so that timely therapeutic interventions and plans to address long-term complications may occur. [1] Diaz-Rosello JGP, Niermeyer S, et al. WHO Basic guidelines on new born resuscitation. 2012.

The CandyCollect is a lollipop-inspired saliva collection device initially developed to aid in strep throat testing. The device utilizes open, plasma-treated microfluidic channels to collect pathogens in saliva in a noninvasive manner. The device’s isomalt candy coating aids with saliva production, and acts as a built-in timer to ensure adequate oral sampling time. In our previous studies, multiple institutional review board (IRB) approved and IRB exempt studies have been performed to test the functionality of the CandyCollect devices with human subjects. We have also demonstrated the device’s detection of oral bacteria such as Streptococcus pyogenes and Staphylococcus aureus. We noted throughout these studies that the candy’s intended 3 minute dissolving time was longer than expected due to the coating’s large mass. Thus, we are also developing a 1 and 2 minute version to more accurately match the necessary length of sampling times for various pathogens without being unnecessarily uncomfortable for the user. Our goal for this study is to identify the relationship between candy mass, dimensions, and dissolving time so that we can use the candy as a tunable “timer”. We are modifying the depth, diameter, and mass of the device to develop new CandyCollects targeted to take, on average, 1, 2, and 3 minutes to dissolve. These new CandyCollects are more suitable for specific, higher-concentration oral pathogen collections. By reducing the candy’s mass on the devices, we expect the modified devices will decrease the required dissolving time to make the user experience of oral pathogen collection more pleasant. We plan to recruit participants (>18 years) within the university to test these modified devices, and they will provide feedback on their experience and dissolving time. Using these results, we will be able to improve the CandyCollect device to provide a more comfortable sampling experience.

It is important for us humans to take responsibility for how our actions have exacerbated climate change and the disproportionate effects that climate change has had and continues to have on various minority groups. Through educating students about climate change in a humanizing manner, we can hope that students will view science as a discipline willing to confront inequities to seek change. However, the textbooks commonly used in introductory biology classes fail to accurately communicate the full scope of the inequities behind who is intensifying climate change and who is the most impacted. My research serves to determine if the humanization of climate change is communicated to undergraduate students taking introductory biology through aiming to answer two important questions: To what extent are textbooks acknowledging humans’ responsibilities in the severity of climate change today and to what degree is equity/inequity discussed? In order to investigate these questions, my team and I evaluated six popular introductory biology textbooks in the United States by pulling passages that discussed the connection between climate change and society. Coded passages were evaluated on where their level of humanization of science fell on the following scale: none/scarce, detail, nuance, equity/inequity, and finally justice. Out of the 1351 total “humanizing” passages pulled from the text, only 7.8% of the quotes were categorized as relating to climate change. Of these quotes, 19% were coded as none/scarce, 60% had detail, 6.7% contained nuance, 11% discussed equity/inequity, and 4.8% included justice (sum total > 100% due to two quotes falling under multiple categories of humanization). As climate change will continue to negatively impact humans, it is important to teach students the specific inequities connected to climate change in order to spread awareness and help solve issues of justice in the living world.

Around 7% of the population has developmental language disorder (DLD). DLD is a neurodevelopmental condition that affects learning, understanding, and using spoken language. It can be seen in early childhood but often goes undiagnosed into adulthood. Although DLD is diagnosed based on language differences, children with DLD also show motor differences. It is unknown whether adults with DLD still show motor differences as well. Motor control is the process of regulating the systems needed for voluntary movement and is what allows us to complete everyday tasks such as walking, writing, typing and anything else that includes movement. For this project, I have chosen to assess motor performance using a standardized assessment, the Bruininks-Oseretsky Test of Motor Proficiency Second Edition (BOT-2). Subtests will be used to test fine motor skills (e.g., precision, coordination) and gross motor skills (e.g., balance). For each subtest there are several activities to complete. Fine motor and gross motor are being tested as there is evidence that both are affected in children with DLD. In order to determine if the participants are in the DLD group or the TD group, we use an assessment involving following auditory directions and spelling unusual words. We will test 15 participants in each group. Participants will have learned English under the age of 2, be 18-45 years old, and have no history of speech, voice, hearing, or neurological diagnoses. I will analyze the data using unpaired t-tests on motor performance between the groups, as there are currently no norms for motor performance in this age range. I hypothesize that there will be motor control differences between the two groups, especially in areas such as balance, bilateral coordination, and manual dexterity. Learning more about DLD and motor control in adults will help with diagnosing and understanding how the disorder manifests in older populations.

HIV infection impairs B cell function, in turn, altering immunoglobulin production and function. Immunoglobulins (Igs) exist as isotypes including IgA, IgG, and IgM. Within the IgA and IgG isotypes there are subclasses IgA1-2 and IgG1-4 respectively; all with distinct functions. Previous studies have shown that HIV infection influences Ig isotype and subclass concentrations in serum, but few have explored their concentrations in the breast milk of mothers living with HIV (MLHIV). Widespread use of antiretroviral treatment during pregnancy has led to an increase in the incidence of HIV-exposed and uninfected infants (iHEU). iHEUs have heightened immune activation and inflammation and display high infectious morbidity compared to compared to HIV-unexposed infants. It is plausible that immune factors transferred in breast milk contribute to altered immunity in iHEU. Therefore, knowing whether HIV infection impacts total immunoglobulin concentrations or inflammatory biomarkers in breast milk is important. I used enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of immunoglobulin isotypes and their subclasses and Luminex to profile cytokines and chemokines in breast milk 4 weeks after delivery. Assays were performed according to manufacturers’ instructions and sample values were extrapolated from a standard curve. I compared these factors between MLHIV and uninfected mothers using Mann-Whitney U test. MLHIV had significantly higher mean concentrations of total IgG1 (36.9 ug/mL versus 26.6 ug/mL, p=0.018) and IgG3 (2.9 ug/mL versus 1.3 ug/mL, p=0.0013). There was no difference in concentrations of IgA and IgM between the groups. Furthermore, MCP-1, MIP-1-β, and SDF-F-α were the most abundant chemokines in breast milk in both groups. However, we found no significant difference in concentrations of cytokines and chemokines in breast milk of MLHIV versus controls. Overall, we observed increased breast milk concentrations of IgG1 and IgG3 in MLHIV. Future work will explore implications of these IgG subclasses on iHEU immunity.

Half a million people develop drug resistant tuberculosis (TB) each year. Cases of drug resistant TB often result in poorer outcomes both healthwise and economically for patients, and many populations lack access to the resources needed to treat resistant TB. Increased antibiotic resistance has resulted in an urgent need to develop new, cost-effective drugs that are effective against Mycobacterium tuberculosis, the bacteria responsible for TB. In my research, I am using deep learning methods to design peptides that bind to the enzyme ClpP, a vital protease and known antibiotic target in M. tuberculosis. A class of drugs called Acyldepsipeptides (ADEP) have been shown to bind to ClpP and cause cell death in M. tuberculosis by preventing the formation of the ClpP complex with necessary ATPases, resulting in significantly lower proteolytic activity. We used the structure of ADEP as a basis for the peptide design and employed Rosetta, a macromolecular prediction and design software, to generate cyclic peptides bound to ClpP. I then used a sequence based deep learning tool to generate multiple sequences for each backbone design and computationally validated the resulting structures with AlphaFold, a highly accurate, machine learning based structure prediction tool. The structure of the ClpP binding interface resulted in it being a difficult target to design for with current deep learning methods. One peptide binder was predicted to bind to ClpP in our preliminary design rounds. We will chemically synthesize this binder and test it against ClpP in an enzyme inhibition assay. If the binder inhibits ClpP, it can serve as a basis for an effective and low cost drug that targets the ClpP enzyme in drug resistant TB. We will also expand and refine our design pipeline to produce more binder designs that can serve as viable drug candidates.

Working memory capacity is the active short-term maintenance of information which is essential for our day-to-day functioning. By measuring pupil dilation during a working memory task, we aim to quantify the relationship between pupil dilation and working memory capacity. The goal of the study is to look at how image type and presentation position influences memory for temporal order. Study 1 presented participants with sequences of semantic images (images of known objects) and sequences of abstract images (random line drawings), one by one, in a central position on the screen. Throughout the experiment, the eye-tracker measured pupil dilation. Participants then organized the images in the temporal sequence they appeared. Participants recalled the order of semantic images better than the order of abstract images, supporting the hypothesis that working memory capacity improves when the participant can associate a label with the image. In addition, pupil dilation was greater during the sequences of semantic images compared to abstract images. Study 2 investigates spatial biases in working memory. Participants see the same image sequences as in study 1, except the images are presented from either left-to-right or from right-to-left instead of centrally. We expect participants to show increased working memory capacity with left-to-right sequences compared to right-to-left sequences due to learned patterns in Western cultures. These results will provide information on how the spatial presentation of information influences working memory capacity. More generally, improving our understanding of how working memory capacity processes affect pupil dilation will allow us to use pupil dilation to study working memory capacity in infants and young children.

Many age-related diseases in humans such as Parkinson's and Alzheimer's involve intracellular protein aggregation, but much is still unknown about the molecular mechanisms behind how this occurs. Characterizing these mechanisms is therefore important for developing effective treatments for age-related illnesses. Our work investigates the relationship between cell life span and aggregation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein (RNP) granules that form inside cells experiencing stress and perform several molecular functions that appear to benefit cells experiencing stress. Using GFP-tagged Dcp2 as a P-body marker in S. cerevisiae and microfluidics to study single-cell lifespans, I demonstrated that P-bodies aggregated in aging cells that were not experiencing other forms of stress. P-body aggregation also correlated to the remaining lifespan of any given cell. To investigate this link further, I adjusted cytosol pH and observed a relationship between cytosolic pH and P-body aggregation rate. Slowing of P-body aggregation correlated to extension of cell lifespan. This suggests the need for additional research to determine whether there is a causal link between P-body aggregation and fatal single-cell pathogenesis and if so, whether these pathogenesis mechanisms are conserved in human cells and therefore a possible target for treatment for age-related illnesses.

All somatic cells, from heart cells to immune cells, have the same genetic code. Understanding the regulatory processes that allow the same DNA sequence to code for vastly different gene expression patterns is a longstanding goal of biomedical research. To study the regulation of gene expression we examine chromatin accessibility, a measure of the areas of DNA accessible to transcriptional machinery. It’s hypothesized that variation in these accessible regions across different cell states and types enables combinations of Transcription Factors (TFs) to bind and regulate gene expression. This project builds upon the AI-TAC neural network model which predicts chromatin accessibility as measured by ATAC-seq peaks in 81 mouse immune cell types. The trained AI-TAC model was used to identify sequence patterns within regulatory regions that predict cellular differentiation. TFs function through protein-protein interactions with other bound TFs. My recent work found that AI-TAC is unable to sufficiently learn nonlinear TF interactions. I hypothesize that a model trained with higher granularity data to predict base-pair resolution chromatin accessibility will learn the non-linear interactions between TFs encoded in genomic DNA more effectively. I present a model named bpAITAC with a new architecture that predicts base-pair resolution raw ATAC-seq reads. This model will allow us to identify TF interactions important for regulating accessibility. These findings will help us better understand immune cell differentiation. Future iterations of this model will be trained on human immune cell data, and will be able to identify rare disease-associated gene variants from patient DNA sequences. This will allow us to develop personalized therapeutics to address the disease-related effects of an individual’s genetic variations.

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.

Treatment of individuals with HIV using antiretroviral therapy (ART) is highly effective, but effective clinical management depends on maintaining therapeutic drug concentrations. Antiretroviral (ARV) drug concentrations in patients with HIV can vary due to differences in drug metabolism, medication adherence, or interactions between multiple drugs. These individuals may have subtherapeutic or supratherapeutic drug concentrations, putting them at risk of treatment failure, acquisition of drug resistance, and risk of hospitalization or death. Current measurement of ARV concentration is done through liquid chromatography tandem mass spectrometry, which requires expensive equipment and requires a labor-intensive protocol. This restricts accessibility to specialized laboratories, making it difficult for persons with HIV to have routine measurements of ARV drug concentrations. The goal of the project is to develop an assay that is simple to perform and uses standard equipment to increase access to routine clinic-based drug level monitoring to improve HIV care. We designed an assay using a 2-step process of DNA strand transfer and quantitative polymerase chain reaction (qPCR) to quantify integrase strand transfer inhibitors (INSTIs). We tested for dolutegravir (DTG) and cabotegravir (CAB) in both buffer and plasma -- the latter to simulate patient blood samples. We were able to demonstrate that the assay could quantify clinically relevant drug concentrations of DTG and CAB. By developing an assay that can be readily integrated into most clinical laboratories, we will contribute to increasing access to routine HIV drug level monitoring to improve clinical HIV care and maintaining viral suppression in persons with HIV.

Sexual assault is pervasive (1 in 4 women experience assault in their lifetime; RAINN, 2019) and holds the highest risk for posttraumatic stress disorder (PTSD) of any type of traumatic event. However, Asian women are underrepresented in media representations and research regarding the prevalence of assault and the etiology of PTSD. The purpose of this study was to examine the frequencies of lifetime occurrences of sexual harassment, unwanted sexual touching, and penetrative sexual assault (i.e., rape) in Asian women and compare these rates to their white peers. College-aged women (N = 201) completed an online version of the Sexual Experiences Survey - Victimization (SES-SFV). 60.86% of Asian and 74.65% of white women reported experiencing sexual harassment, 28.70% of Asian and 39.43% of white women reported experiencing unwanted sexual touching, and 20.00% of Asian and 21.13% of white women reported experiencing rape. Three independent samples t-tests compared rates of sexual harassment, unwanted sexual touching and rape in white women (n = 69) compared to Asian women (n = 101). Results showed no significant differences between occurrences of sexual harassment and rape. However, white women (M = .75, SD = .43) experienced significantly more unwanted sexual touching compared to Asian women (M = .57, SD = .50), t(168) = -2.4, p = .016. These findings indicate that Asian women encounter similar rates of sexual harassment and rape compared to white women. The parity found between Asian women and white women is underrepresented in media and research and may have negative consequences on the believability of and support for Asian women who have experienced harassment or rape. Further research is necessary to understand the unique mental and physical health impact of sexual assault on Asian women. 

The process of learning new words is a significant developmental task where children learn through social interactions with others. However, children with neurodevelopmental differences such as autism spectrum disorder are known to have distinct manners of social engagement. The current study examines one potential factor that might relate to how easily autistic children learn new words, and how success in that manner of social interaction predicts future vocabulary development. Participants (N = 29) were drawn from a larger study of 2- to 11-year-olds with autism with a focus on language and development. In that sample, nonverbal IQ scores ranged from 34 to 133 (M = 98.29; SD = 21.48) and receptive vocabulary standard scores ranged from 20 to 138 (M = 91.41; SD = 29.02). In a laboratory task, an examiner presented several novel objects with novel labels (i.e., nonwords) to the child. Dyadic coding of the child's verbal interactions with the examiner during exposure to the nonwords was completed for recordings of those sessions. Coding procedures included marking instances of vocalizations and overlapping speech. Overlapping speech was divided into three categories: overlapping speech during the prompt teaching the nonword- "Look it's a... It's a great...," overlapping speech during the nonword- "heypaum," and overlapping speech at any other point. Overlapping speech is of particular relevance as the instances might indicate uncoordinated turn-taking and moments where the child may be unable to completely process and learn from their social partner's input. In the current sample, 14 of 29 participants had at least one instance of overlapping speech during the nonword. We hypothesize that coordinated turn-taking supports vocabulary development, such that fewer instances of overlapping speech will be associated with learning more nonwords. This research on children's vocabulary development can guide understanding and optimizing of teaching strategies to support language learning.

Ocean acidification leads to the decrease in availability of dissolved oxygen. Carbon enters the atmosphere from a wide variety of sources, most notably the burning of fossil fuels, and this atmospheric carbon is absorbed by the ocean. Algae need carbon, and as more carbon enters the water, algae can reproduce rapidly. Dissolved oxygen is taken in as this algae blooms as well as decomposers breaking down the algae once it dies, leading to a severe decrease in the oxygen content of the water. A severe lack of dissolved oxygen in water is called hypoxia. Because of ocean acidification, hypoxic events are increasing in and around the Puget Sound, an area notable for its aquaculture, specifically of the pacific oyster. Triploid oysters are a rarely occurring genotype of oyster that are larger and sterile than most other oysters. These traits can be cultivated, either by selective breeding or chemical alteration, and are essential for oyster aquaculture. However, the effect of hypoxia on oysters, especially on triploid oysters. is relatively unknown. How does varying oxygen levels affect diploid, chemically induced and “natural” triploid pacific oyster mass? We placed thirty of each type of oyster in tanks of 100, 80, 60, 40 and 20 ppm tanks, a total of three tanks of each treatment. The oysters were fed regularly. We recorded the average oyster mass of each oyster before and after one month of each treatment. This shows the change in oyster mass depending on each treatment over time. We expect to see the mass of chemically induced and selectively bred triploid oyster will decrease with reduced dissolved oxygen concentration because there will be less oxygen because they will reduce their metabolic rate to cope, hindering growth. As oceans acidity increases, it is important for oyster fisheries to know how  oysters react to decreased oxygen content to be better prepared for the future, especially when choosing which kinds of oysters will tolerate these changes best. 

The ecological impacts of nutrients in dry deposition on lake ecosystems is under-researched and the relative effect may be undervalued because this influx is usually estimated from a few published studies rather than measured directly. Dry deposition is particulate matter from atmospheric or nearby sources carried aerially. Disturbance to the landscape and added pollution from sources in agricultural, industrial, and urban areas have the potential to contribute phosphorus and nitrogen in the form of dry deposition which has potential to significantly increase nutrient loading to the lake and promote harmful algae blooms. Spirit Lake, located at the base of Mount St. Helens, has been the focus of ongoing study to elucidate chemical, biological, and ecological changes following its re-creation during the 1980 eruption of the volcano. A nutrient budget was constructed for Spirit Lake in the last decade but the researchers found a significant imbalance between inputs and outputs, with outputs greater than inputs. All fluxes were directly measured except for wet and dry deposition, which utilized published regional values instead. We hypothesize that dry deposition may be considerably higher than average regional values and significantly contribute nutrient inputs to Spirit Lake. In this study, we deployed passive samplers on hummock islands and attached them to embedded logs to capture dry deposition falling onto the lake. We collected monthly samples from 10 sites over the summer and analyzed total nitrogen (TN) and phosphorus (TP) concentrations in each sample to estimate the average summertime inputs of N and P in dry deposition. We found total measured summertime deposition was 1,326 kg N higher than the previously estimated value, and conversely measured P deposition was 18 kg P lower than previous estimates. As the disturbed landscape surrounding Spirit Lake is subject to greater movement of pollen, soil, and insects we can see there is a greater influx of nitrogen than estimated by regional data.

The period around birth is when neonates are at the highest risk of neurological injury or death. A common neonatal neurological injury is hypoxic-ischemic encephalopathy (HIE), which occurs after the brain does not receive enough oxygen or blood flow. There is a large disparity in the severity and long-term neurodevelopmental outcomes of HIE between high-income countries (HICs) and low-and-middle income countries (LMICs). In HICs, HIE occurs in 1-4 neonates per 1,000 births. In LMICs, the instance of HIE is at least 2-3 times higher. Furthermore, cases of HIE seen in LMICs suggest a different type of injury - a more prolonged intermittent injury resulting in white matter injury - compared to HIE in high-income countries that is more acute and affects the deep grey matter. Therapeutic hypothermia (TH) has been the standard of care for HIE in HICs; however, TH is not an effective treatment for HIE in LMICs. Thus, the creation of alternative and accessible therapies for HIE in LMICs is crucial. This study will seek to model HIE as seen in LMICs through an in vitro ferret model that may be used to pilot therapies before applying them to in vivo models. Organotypic brain slices from postnatal day (P) 21 ferrets, equivalent to a term neonate, will be cultured and randomized to receive increasing intervals of oxygen glucose deprivation (OGD), with and without serum deprivation. Serum deprivation is defined as culturing in 2.5% serum as opposed to the standard 5% to mimic certain aspects of malnutrition that may be more common in LMICs. Cell death and white matter injury will be assessed 24 hours after OGD. We hypothesize that slices with more rounds of intermittent OGD and serum deprivation will display relatively more cell death and white matter injury, thus serving as a model of HIE in LMICs.

Emotional obstacles affecting those living with chronic Inflammatory Bowel Disease (IBD) are a pain point that often lacks support. Emotional obstacles include feelings of depression, anxiety, body image issues, experiencing isolation, or feeling unheard, which can impact one’s quality of life. Support systems, or individuals who provide emotional or physical support, can help people manage the effects of these obstacles to support illness self-management. Research on IBD and emotional support demonstrate that many people do not know how to best support their loved ones with IBD. Poor understanding of patients’ needs often results in ineffective support that is not perceived by IBD patients as beneficial; support system members are perceived as being overly worried, being hyper-fixated on physical IBD symptoms, or trying to distract the patient from emotional pain. These strategies carry the risk of IBD patients suppressing their emotional obstacles, withdrawing from their support system, and struggling on their own. I want to improve social support systems for IBD patients. As a first step, I administered online surveys asking people with IBD what emotional obstacles they face, and how these burdens affect their daily life. To date, respondents (n = 57) reported experiencing body image issues (57%), anxiety (68%), feeling hindered from their potential (51%), depression (66%), and social isolation (61%). Respondents stated that their emotional obstacles inhibit their IBD self-management (73%), ability to follow medical advice (38%), and ability to follow their medication regime (40%). These findings characterize common emotional obstacles and key impacts on self-management, a principal factor in disease remission. As we continue to survey people with IBD, we are conducting follow-up interviews to understand their experience and support needs in greater depth to inform improvements to social support systems.

In recent years, gene editing has been growing rapidly, showing seemingly unrivaled potential experimentally and clinically. My research utilizes promising gene editing technologies in tandem with cardiomyocyte generation in vitro as part of greater efforts aimed at developing safe and efficacious methods of in vivo gene editing. My objective is to test the efficiency and side effects of a novel gene editing procedure, developed by Dr. Vo and Dr. Freedman, in cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs). This procedure uses a non-viral delivery method of the CRISPR-Cas9 RNA-protein complex. I differentiated hiPSCs into beating cardiomyocytes using a specialized cell line containing a transgene for a red fluorescent protein, tdTomato, which is expressed when the cells are edited. The detection of tdTomato under immunofluorescence microscopy indicated successful gene editing that allowed us to measure its efficiency. I fixed and stained the cardiomyocytes with a cardiomyocyte-specific protein marker, MLC2A, and imaged them. The efficiency was measured through the colocalization of the MLC2A and tdTomato signals. There were three different groups of cardiomyocytes: 1) gene-edited, 2) negative control using a scrambled guide RNA sequence, and 3) untreated. The gene edited group showed an efficiency of approximately 11% while the untreated and negative control groups showed no editing. I also live-imaged the different groups of cardiomyocytes before fixing to compare their beating frequency to test our hypothesis that our editing procedure does not impact structure and function. This project has demonstrated the success of our gene editing methodology in in vitro cardiomyocytes, bringing us one step closer to safe and efficacious in vivo editing. However, it is important to recognize that this methodology must still be optimized. Our current editing efficiency is too low for any clinical applications, so I am working on improving its efficiency, targeting a 50% editing rate.

Purple sea urchins (Strongylocentrotus purpuratus) pose a serious threat to the environment by consuming entire kelp forests that serve ecological functions like carbon sequestration. We compared the preference of purple sea urchins on various kelp species to understand how feeding habits changed across different environments: temperature (ambient 13 °C, high 20 °C) and salinity (ambient 30 PSU, low 22 PSU). Urchins were collected from the subtidal zone in Friday Harbor, San Juan Island, WA. At the University of Washington Friday Harbor Labs, urchins were exposed to either ambient (6.8L), high temperature and ambient salinity (6.8L), low salinity and ambient temperature (6.8L), or high temperature and low salinity water (3.1L). Within these treatments urchins were given Bull Kelp (Nereocystis luetkeana), Ribbon Kelp (Alaria marginata), and Fringed Sieve Kelp (Neoagarum fimbrata) for 24 hours. To track kelp consumed, we weighed the kelp before and after each trial. Results showed that Bull Kelp was consumed the most in every environment except high temperature, low salinity. In low salinity and high temperature urchin feeding was significantly different and lower from the ambient environment, as most urchins ate nothing over 24 hours (Kruskal Wallis and Dunn’s test, p-value > 0.05). Our findings suggest that in areas with lower salinity and higher temperatures, urchins may be a smaller threat to kelp. In many environments, bull kelp is most vulnerable to urchin feeding, making it an important species for conservation efforts.

We are doing research that supplements a larger archaeological study on the impacts of contact with 19th-century Russian colonists on the Indigenous Sugpiaq community of the Kodiak Archipelago, Alaska. We are experimenting with dendrochronological methods to determine the most appropriate strategies for collecting data from wood samples, and whether the results will improve chronological resolution at the Ingyuq archaeological site that straddles Russian conquest of the region. Dendrochronology is a dating method that compares patterns in tree ring growth between living wood samples and wood found in the archaeological record. In the lab, we first measured the tree ring growth patterns on dwarf willow, birch and alder samples collected from living trees in the region around the site. Patterns were estimated using a microscope, magnifying glass, and photographs to count annual rings and measure their relative thickness. We then compared ring thickness across samples to determine if there is a shared growth history among trees around this site. This project forms the first and foundational piece in a longer project that will overlap growth patterns in living wood samples to those found in the archaeological record at this site. At the end of the project, we hope to find patterns across charcoal samples excavated from this site that allow us to develop a firmer understanding of the sequence of and temporal relationships between individual stratigraphic layers to better focus research on broader questions about the impact of Russian colonization on the Sugpiaq people.

Kānaka Maoli (Native Hawaiian) health practices have been significantly impacted by colonialism, beginning with the illegalization of practices like hula, lā’au lapa’au, ‘ÅŒlelo Hawai’i (Hawaiian language), and the growing inaccessibility of cultural foods. Increasing attention has been paid to creating culturally-grounded interventions that address these disparities, which have proven to be effective in increasing health outcomes in Indigenous communities. This study aims to understand the potential benefits of Papa and Pohaku, a culturally-grounded family intervention created and led by esteemed elder Uncle Earl Kawa’a through Keiki O Ka ‘Ä€ina (KKA). KKA is a non-profit organization built to perpetuate Kānaka Maoli culture for ‘ohana (families) and keiki (children). Uncle Earl Kawa'a, a respected kupuna (elder), leads courses on creating a papa (pounding board) and pohaku ku’i ‘ai (stone pounder) to promote healing and wellbeing through grounding participants in cultural practices. To understand the impacts of the intervention, two focus groups were conducted with participants and KKA staff. Questions explored the benefits of participating in Papa and Pohaku, specifically its impact on relationships and traditional Hawaiian knowledge. Our team used Collaborative Qualitative Analysis, which is a structured and rigorous method of conducting inductive thematic analysis. All research team members identified as Indigenous, consisting of one faculty advisor, one doctoral student, and two undergraduate researchers. Respondents reported the intervention positively impacted the following: 1) participants’ pilina (relationships) with their ‘ohana, partners, and with other participants; 2) appreciation for the huaka’i (journey), or process, and commitment to future growth; and 3) understanding of and connection to mo’oemeheu (Kānaka culture). The findings of this study indicate the various benefits of culturally-grounded family-based interventions and a greater need for the availability of culturally-grounded interventions for Indigenous communities.

The New Zealand mudsnail (NZMS), introduced to the United States in the 1980s, is a priority invasive species in Washington State. Its presence was first detected in 2015 in Spirit Lake, located north of Mount St. Helens, where its population is quickly increasing. Studies of this highly invasive species have revealed no ecological benefit to Spirit Lake, with fish species eating, but not digesting or assimilating snail tissues. Rather NZMS may be affecting the dietary intake of rainbow trout, the only fish species in the lake, and ultimately causing a trophic cascade due to its lack of natural predators, voracious herbivory, and interspecies competition with native snail species. In order to track the spread of NZMS and its ecological impacts on Spirit Lake, temporal sampling of vegetation and sediment was carried out throughout the lake from July-September 2022. Snail counts were used to calculate snail population density (NZMS and native species), spatial and temporal variability, substrate preference, presence in sediments, and interspecies competition. Results provide evidence of interspecies competition with both NZMS and native snail species showing habitat overlap in vegetation samples. However, all species were found to have vegetation preferences with NZMS favoring stonewort (order Charales), as well as uniquely showing a utilization of sediments. In addition, NZMS were found to remain spatially isolated along the south shore of Spirit Lake with possible expansion towards the outlet tunnel at very low densities. These results reiterate the threat of NZMS to the Spirit Lake ecosystem and surrounding waterbodies. Their spread should be of immediate concern with ongoing construction plans by the US Forest Service related to the Spirit Lake tunnel intake gate, including the construction of a shoreline staging area in the heart of NZMS-infested waters. If this moves forward this project could pose a serious threat of spreading NZMS into the Toutle River, Cowlitz River, and Columbia River drainages while causing irreparable damage to the ecosystem.

Postpartum depression (PPD), with prevalence rates in East Africa ranging from 17% to 24%, is associated with adverse health outcomes among offspring of affected mothers including emotional and developmental delays and poor growth. The 2022 World Health Organization (WHO) maternal and newborn care recommendations call for routine PPD screening using a validated screening tool. In Rwanda, a low-income country severely impacted by the 1994 genocide, routine PPD screening has not been implemented. This study was conducted to describe the prevalence of PPD among new mothers and determine sociodemographic characteristics and health factors associated with PPD and recent suicidal ideation. Postpartum mothers delivering a live birth at the Kabutare District Hospital (KDH) in Huye, Rwanda between August and September 2022 were recruited for this study. This study was a cross-sectional survey administered via face-to-face interviews conducted in Kinyarwanda. Following written consent, the mothers responded to sociodemographic, and maternal/ infant health questions, and completed the Edinburgh Postnatal Depression Scale (EPDS). Postpartum depression was defined as an EPDS score of > 10. Data collection was approved by the KDH Ethics Committee. Our study population consisted of 66 Kinyarwanda-speaking mothers. Over half (52%) had PPD, and 26% had suicidal thoughts in the past 7 days. Many reported a history of depression (39%), PPD (18%), or anxiety (29%). Mothers with a history of depression, anxiety, or PPD were more likely to have PPD and recent suicidal ideation. There was a greater prevalence of PPD among mothers reporting pregnancy-related complications or a history of mental illness compared to their counterparts (70% vs. 44%, p < 0.05; 67% vs. 36%, p < 0.05). Mothers at particularly high risk for PPD are those with pregnancy-related complications and a history of mental illness. These findings demonstrate a need for routine PPD screening among new mothers, as recommended by WHO.

Pesticides are an important tool for protecting the food supply. The safe use of these chemicals are important for workers and community members, especially in rural areas. The goal of this project is to understand the best available estimates of mass applied for the most common agricultural pesticides in the Pacific Northwest. Environmental statistics and biostatistics are key tools for visualizing data in public health research. I processed data from the Pesticide National Synthesis Project, a large repository of agricultural pesticide use estimates maintained by the United States Geological Survey (USGS). I focused on three states in the Pacific Northwest--Washington, Oregon, and Idaho--and produced low- and high-end pesticide use estimates by state. I used R to compute and analyze this data and prepare a final report that visualizes pesticide use in the Northwest. We hypothesized that the ten most commonly used pesticides would be similar across the three states and higher in agricultural regions. For Yakima County, Washington, the ten most commonly used pesticides by high-end mass estimates were petroleum oil, sulfur, calcium polysulfide, kaolin clay, glyphosate, metam, copper, dichloropropene, chlorpyrifos, and copper sulfate. We found that many of these chemicals were commonly used in orchards and vineyards. We are applying our knowledge from this approach for one county to more fully understand use patterns across all three states. This work will give me the chance to return these results to interested stakeholders and help me explore future research possibilities.

Mutations in the genetic sequences of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) has played a major part of the pandemic. This is evidenced by the increasing number of distinct strains that have appeared. Evaluating these mutations and their frequency within genetic sequences offers the opportunity to identify patterns that aid in increased virility for SARS CoV-2. We identified prevalent SARS CoV-2 strains in GISAID and downloaded genetic sequences from the NCBI nucleotide database. We used MAFFT (Multiple Alignment using Fast Fourier Transform) in Seaview to align SARS CoV-2 strains to the reference genome. We also built a custom python script to identify locations of mutations, and their potential effect on the proteins’ amino acid sequence. Preliminary work identified a mutation in the ORF1ab gene of the omicron strain. Part of this gene codes the typically conserved NSP-16, associated with the product 2’-O-ribose methyltransferase, an enzyme that catalyzes the transfer of a methyl group from a methyl donor molecule. The modification could affect the stability, localization, and function of RNA such as RNA splicing and post-transcriptional modification. We then generated a phylogenetic tree using BEAST/BEAuti to estimate the frequency and history of mutation across different strains. Our analysis identifies mutations accumulated over the course of the pandemic. Studying the effects of these mutations offers insights into SARS CoV-2 virology. These insights can be used to build a predictive model to aid in effective and efficient vaccine development.

The human genome has a great deal of variation. Variants larger than 100 base pairs (bp) are termed structural variants (SV), which can be pathogenic and linked to rare diseases. By studying SVs in patients, particularly SVs that are not inherited, or de novo, we can learn a great deal about the underlying DNA repair mechanisms involved, which can then be applied to other important areas of study, such as cancer formation. We focused on a patient with tetralogy of Fallot, short stature and multiple congenital anomalies. This patient has an SV on chromosome 2. Using array comparative genomic hybridization, we identified a 3.2 megabases (Mb) copy number variant (CNV) on chromosome 2q31, which means the DNA in this area of the chromosome is copied several times. Analysis of the parental DNA indicates this SV is de novo. Optical genome mapping gave a picture of the general structure of the CNV, while detailed analysis of the short-read whole genome-sequencing allowed us to identify exact coordinates where the start and end of the extra DNA copies are located. This has allowed us to determine the size of the CNV to single nucleotide resolution, and the genetic location of the CNV relative to the reference sequence. Using these combined methods, I will be able to solve the overall structure of the rearrangement. This can lead us to an understanding of how complex changes in genetic structure alter the physiology of the patient. We can also use the information from the breakpoint junctions to investigate the underlying mechanism that formed the CNV. This project contributes to our understanding of processes causing genetic rearrangements, and potentially informs how other types of DNA alteration, such as in cancer cells, takes place.

Music, both vocal and instrumental, captures the attention of children and facilitates language learning. Infants have been shown to prefer infant-directed song over infant-directed speech. Furthermore, education professionals and researchers agree that music supports language acquisition. In early childhood, children are exposed to a great deal of auditory input, including music, in a variety of settings. Much of this exposure occurs in the home, which presents difficulties when attempting to capture data that is truly representative of a child's auditory environment. Thus, the main aim of Tots and Tunes is to quantify and describe the music and speech that infants hear in their home environment during first two years of life. To achieve this, I have randomly extracted 12,000 10-second audio segments from a longitudinal dataset of audio recordings taken of children at ages 6, 10, 14, 18, and 24 months of age by the Language Environment Analysis (LENA) system. This system uses a small device worn by a child throughout their day to record their naturalistic linguistic environment. My team is using a novel citizen-science approach to outsource the annotation of the segments by naive listeners via a platform called Zooniverse. For each segment, we ask participants to listen and subsequently identify whether there is speech and/or music present, whether the speech and/or music is directed to the child wearing the LENA device, and if the speech and/or music is in-person, through an electronic device, or both. In the span of a month, 20% of the segments recorded at 6 months of age have been annotated. I anticipate that our findings will inform parents, researchers, and early childhood professionals about the presence of music in the auditory environment of young children, which will inform further work regarding how best to use music to support language acquisition.

The human fragile X messenger ribonucleoprotein (FMR1) gene was first characterized as having causative mutations responsible for the inherited intellectual disorder Fragile X syndrome. The protein encoded by the gene, fragile X mental retardation protein (FMRP), is a RNA binding protein which functions as a chaperone to mRNA during nuclear export to the cytoplasm. Drosophila melanogaster contains an ortholog of this gene, and has been a model organism for better understanding its role in behavior and in the development of neural anatomy. The D. melanogaster Fmr1 gene has multiple coding isoforms and previous research has demonstrated the existence of a non-canonical start codon present in a subset of those isoforms. Non canonical start codons are understood to be less efficient at translation initiation than standard start codons, but are thought to provide an alternative mechanism for isoform diversification and protein function. Our project aims to further understand the evolution of the non canonical start site through sequence analysis of the Fmr1 gene in different species of the Drosophila genus. We first annotated the gene in various Drosophila species using D. melanogaster as a reference species. We used RNA sequence data from the target species and localized homology searching as determinants for a non-canonical start site in species. Our preliminary results suggest evidence of a non-canonical start site for Fmr1 genes in a subset of closely related Drosophila species but a lack of evidence in species more distantly related, indicating its recent evolution as a gene expression mechanism. Moreover these results work to further increase the understanding of the evolution and utilization of non-canonical start sites in gene expression.

Pathogenic bacteria often promote their growth by introducing proteins into host cells they are infecting. Some of these introduced proteins hijack host cell ubiquitin signaling pathways. Ubiquitin (Ub) is a small protein that is involved in many signaling pathways within eukaryotic cells. In ubiquitin-mediated degradation, the degradation of a target protein is promoted by the attachment of chains of ubiquitin onto the target protein. The attachment of poly-ubiquitin chains is facilitated by a class of proteins called E3 ubiquitin ligases. Salmonella Typhimurium secretes into host cells an IpaH/SspH class E3 ubiquitin ligase called SspH1. My research investigates the mechanism for how SspH1 modifies its target protein, Protein Kinase N1 (PKN1), with poly-ubiquitin chains. I am investigating whether ubiquitin is transferred sequentially onto PKN1 or if a long poly-ubiquitin chain is first formed on SspH1 and then transferred en-bloc onto PKN1. Using biochemical methods, I will compare whether the rate of poly-ubiquitin chain synthesis is faster starting with unmodified PKN1 or mono-Ubiquitinated PKN1, an intermediate in the sequential addition pathway. I hypothesize that the formation of poly-ubiquitin chains onto mono-Ub-PKN1 is slower than the formation of poly-ubiquitin chains onto free PKN1. This would suggest that the mechanism does not occur through the sequential addition of ubiquitin. Learning more about the mechanism of SspH1 will allow us to both better understand the IpaH/SspH class of proteins and better understand how ubiquitin can be transferred onto protein substrates.

Adoptive T cell therapy presents a promising possibility of a safe and effective therapeutic for a wide range of cancers through utilizing CD8+ T cell-mediated killing of targets expressing tumor-associated antigens (TAA). However, developing these therapies require knowledge of a T cell receptor’s (TCR) antigenic specificity. My project aims to find the antigen specificity of CD8+ T cells from a patient with an exceptional response to hematopoietic cell transplantation (HCT). We hypothesize that leukemia-specific T cells may have helped prevent early relapse and can be identified in samples from this patient. Bone marrow T cells from the patient post-HCT were previously identified and their TCRs were sequenced. By using genetic engineering to knock out (KO) endogenous TCRs on a healthy donor’s T cells and then transducing the patient’s TCRs into the T cell via lentivirus, TCR-transduced healthy donor T cells can be prepared for functional testing. These TCR KO, TCR transduced lines are expected to provide more consistent responses in functional assays due to the presence of only one TCR on the cell surface. . The Long Killing Assay, a flow cytometry-based cytotoxicity assay requiring the targets to be in co-culture with T cells long enough to ensure T cell-mediated killing, will be utilized for functional testing. This will determine whether target cells presenting a variety of antigens are killed by the TCR-expressing T cells. After the assay, the target cells will be further studied and deconvoluted to determine the resulting TCR antigen specificity. This project will be important in refining our lab’s method of determining antigen specificity of reconstructed TCRs and potentially provide insight on the antigens responsible for antileukemic immune responses. 

High quality surveillance of antimicrobial resistance genes (ARGs) is critical for addressing the threat antimicrobial resistance poses to global health. However, the existing surveillance systems are centered around individual-level sampling in clinical settings. Hence, they are limited in that they do not reflect dynamics in community settings and require culturing for detection. The purpose of the study is to develop a surveillance method in wastewater that provides community level detection of ARGs. We are targeting β-lactam ARGs. DNA extracted from previously collected influent samples from Seattle’s wastewater treatment plant, were seeded with known gene alleles. We then applied Unique Molecular Identifier (UMI) PCR to amplify the alleles, used Nanopore sequencing, and developed bioinformatic pipelines for genomic data analysis. The pipeline translates the nanopore sequencing output (fast5) to genomic sequences (fasta), aligns them with an ARG database to determine the allele types, and graphically represent our alignments and produce interpretable figures from the data. We successfully completed the first allele sequencing and identification of two similar CTX-M alleles (genes for β-lactam resistance) that we inserted into samples. I am evaluating and validating this method with replicates of another β-lactamase ARG - KPC. The expected result at this stage is to successfully identify multiple β-lactamases alleles and test the enrichment of two different gene targets in one reaction. This more efficient and less expensive surveillance method in wastewater will facilitate ARG detection at the community level, providing public health agencies a tool that guides effective and regional-specific monitoring and intervention program design.

After a myocardial infarction, the heart develops non-contractile scar tissue that can impair blood circulation. Human pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (iPSC-CM). The Murry lab has shown that iPSC-CMs can be injected into infarct sites to remuscularize the heart and restore function. A major obstacle observed is ventricular arrhythmias that are due to features of immature iPSC-CMs through misaligned pacemaker activity between iPSC-CMs and the host heart cells. We seek to mature CMs to avoid this. Mature CMs have longer action potentials, larger rod shaped cells, T tubule presence, greater sarcomere organization, and greater contractile force. I hypothesize that overexpression of cardiac transcription factors (TFs) will promote maturation of iPSC-CMs. To test the cloning of our TF plasmids, I used PCR to amplify our gene insert, and confirmed by DNA gel electrophoresis that cloning was successful. To check the efficacy of the plasmid’s target protein expression, I transfected HEK-293FT cells with the lentiviral plasmid and showed upregulation of target TF protein expression by Western blot. After generating lentivirus, I infected iPSC-CMs and harvested these for gene expression analysis using quantitative PCR. I was able to see that expression of our barcode gene ranged 40-200 times higher in comparison to our housekeeping gene, demonstrating that lentivirus successfully overexpressed our target genes. I am currently testing a cardiac gene panel to see the effects of overexpression on cardiac associated genes, and whether TFs will promote maturation. I will do other assays to test the effects of overexpression on cell phenotype like measure action potential properties. If TF overexpression yields more mature cells, we hope to inject these cells into sites of damaged heart tissue without seeing ventricular arrhythmias. This could have great clinical potential as a form of regenerative medicine without posing major risk due to complications.

Cancer cells display dysregulated metabolism to meet the increased metabolic demands of rapid cell proliferation. While cancer cells enact metabolic changes to glucose metabolism, known as the Warburg effect, it is also important to consider the metabolic pathways involved in biomass synthesis which support cellular divisions. For example, the amino acid aspartate is a central facet of proliferating cell metabolism because it is a precursor to both purine and pyrimidine nucleotide synthesis, and essential for asparagine and arginine biosynthesis. Based on research from the Sullivan Lab and others, aspartate biosynthesis is essential for tumor cells to proliferate. However, the cellular mechanisms by which aspartate levels impact the proliferation rate of tumor cells remain unknown. Based on data collected in Sullivan lab, my mentor and I hypothesize that SDH inhibition blocks the production of a metabolic intermediate between succinate and aspartate that has an unknown function in sensing aspartate limitation and therefore dictating cell proliferation. Therefore, my project seeks to investigate how aspartate levels as well as several aspartate precursor metabolites in the TCA cycle govern the proliferation rate of tumor cells. In the first portion of the project, I measure proliferation rates of WT and GOT1/2 (converts oxaloacetate to aspartate) double knock-out (DKO) cells treated with or without the SDH inhibitor Atpenin A5 (AA5) in the presence and absence of aspartate. In the second portion of the project, I examine if modulating fumarate, malate, and OAA levels in the TCA cycle impact the proliferation rate of GOT1/2 DKO cells. I anticipate seeing decreased proliferation rate along with decreased levels of fumarate, malate, and OAA in DKO cells treated with AA5. This research project will contribute to the lab and cancer treatment by providing new insight into aspartate metabolism.

While vaccines help prevent infection from SARS-CoV-2, therapeutic drugs remain necessary to treat people who are infected. In my research, I develop peptide-based therapeutics, which are safe and readily bioavailable due to their low immunogenic response and low production cost. I help design peptide inhibitors against the Mpro enzyme, the main protease present in SARS-CoV-2. Mpro normally cleaves the virus’ pp1a and pp1ab viral polyproteins, which are important for viral replication and transcription. By creating a competitive inhibitor that binds to Mpro, I can prevent it from activating these proteins and thereby prevent viral proliferation. We began with computational design of peptide inhibitors using the stub-extension approach on the Rosetta Macromolecular Modeling suite, a peptide design computational platform. An effective drug both finds the active site of Mpro and binds to it more strongly than the natural substrate. First, our group started with selecting the most critical residues (stubs) that form well-characterized interactions in the active site. We then used Rosetta to add amino acids and cyclize the peptides. After generating cyclic backbones, we designed amino acid side chains with high shape and chemical complementarity to the active site. After filtering, we proceeded with the 50 best 7- to 11-mer peptide candidates. Afterwards, I used solid phase peptide synthesis to chemically synthesize these peptide designs, followed by cyclization and purification with High-Performance Liquid Chromatography. I helped to test their effectiveness in mass spectrometry-based inhibition assays. Overall, we have identified four promising peptides, and our most promising peptide is gzm1_1, which has an IC50 value (the concentration of peptide required to inhibit 50 percent of Mpro) of 0.076 µM. Through refining the structure of these inhibitors even more, I can improve their inhibitory effectiveness, enabling them to serve as the basis of an effective medication for people infected with SARS-CoV-2.

Unlike mammals, western clawed frog (Xenopus tropicalis) tadpoles are able to completely regenerate their spinal cord after tail amputation. This complete spinal cord regeneration is due to the ability of their neural progenitor cells (NPCs) to differentiate into neurons successfully. Our research focuses on two transcription factors—Meis1 and Pbx3– that are upregulated by regenerating neurons and are necessary for successful regeneration. We aim to elucidate how these two proteins are working together to guide successful spinal cord regeneration in X. tropicalis tadpoles. I am specifically investigating Meis1 and Pbx3 splice variant expression during neural regeneration. Previous work in mice found that different known splice variants of Pbx3 have different expression patterns. While X. tropicalis has two predicted splice variants each of Meis1 and Pbx3, nothing is known about their individual expression or function. I sought to fill in this gap by looking at Meis1 and Pbx3 splice variant expression in different tissues and over regenerative time. Based on previous research in mice, I hypothesize that both splice variants of Meis1 and Pbx3 have different gene expression patterns in different cell types over regenerative time. I aimed to investigate this hypothesis by doing two experiments. My first experiment was to study the expression of each splice variant over regenerative time by performing qPCRs in order to look at the presence of splice variant mRNA in uninjured, 24, and 72 hours post-amputation. For my second experiment, I made in situ hybridization probes specific for each splice variant to identify their tissue-specific expression patterns. Based on previous literature, we expect to see differential amounts of expression from each splie variants as spatial expression centralized on the spinal cord. Understanding the transcriptional network that is behind the regenerative mechanism of X. tropicalis can help us develop therapeutic tools to address spinal cord injury in humans. 

The purpose of this study is to establish biomarker assays for physiological stress linked to arsenic exposure and microbiome perturbation in freshwater snails. The ASARCO smelter in Tacoma, Washington polluted soil and water around Puget Sound with arsenic for nearly 100 years. Previous studies have shown that environmental pollutants can alter the gut microbiome and modulate host-microbe interactions in mice. However, little work has been done to understand how chronic exposure to environmental pollutants can impact the microbiota or physiology of primary consumers in aquatic ecosystems. To address this gap in knowledge, we are studying Chinese Mystery Snails (CMS) collected from three lakes in the Puget Sound region: Lake Killarney (20 ppm As), Steel Lake (2 ppm As), and Pine Lake (trace As). We hypothesized that snails exposed to higher concentrations of As are subject to greater levels of physiological stress than those with lower levels of exposure, and that this physiological stress is impacted by the microbiota of snails living in each environment. We measured relative levels of HSP70 expression in snail gut tissues to determine usefulness as a biomarker in our study. We also used inductively coupled plasma mass spectroscopy to determine the amount of arsenic in the tissues of CMS harvested from each lake. Next, we are isolating DNA from snail guts, water, sediment, and plants from each lake. The purified DNA will be subjected to 16S rRNA amplicon sequencing, allowing us to determine the relative abundances of bacterial taxa in each environmental compartment and the overlap between the snail gut vs local environment. Our future research will focus on validating additional stress induced biomarkers, while assessing microbiome alteration linked to the biomarker(s). This work will lay the foundation to future studies focused on understanding the links between arsenic exposure, chronic physiological stress, and microbiome composition.

Utopian thinking, a practice in which a person imagines what their ideal society would be like, could prime that individual to be more receptive to ideas of societal change. The presence of an internally generated standard with which to compare the real world may reduce the amount that a person rationalizes or subscribes to the current system, captured in the construct of system justification. Lower system justification scores may result in an individual being more open to accepting a need for change, and possible plans for carrying this change out. A 2018 study by Fernando and colleagues found that engaging in a utopian thinking exercise reduced system justification scores when compared to a control condition. However, it remains to be seen whether this effect will replicate, and whether it extends to attitudes regarding specific systems, such as the United States justice system. The current study asks survey participants to complete a five-minute writing exercise in which they are either asked to describe their idea of a utopia, or describe a normal day in their life. Participants will then complete a system justification scale, and will be asked to rate their support for two justice systems, the current system and a proposed restorative justice system. I hypothesize that utopian thinking will reduce system justification scores and support for the current justice system, and increase support for the alternative restorative justice system. If utopian thinking results in more openness to proposed new ideas, it could be used as a tool for political messaging and social change.

The Puget Sound region of Washington State has the largest urban ferry system in the world. The most traffic occurs within the central corridor routes, connecting various communities on the Kitsap Peninsula to the metropolitan area of Seattle. These routes are frequented by commuters as they provide a direct connection to the labor market. Traditional land-based modes of commuting, such as by personal vehicle or bus, are shown to negatively impact the commuter. Factors of these traditional commuting environments, such as perceived lack of control and unreliability of the route, act as stressors that can harm the commuter’s well-being in both the short and long term. Many aspects of the ferry environment, however, might avoid these typical stressors. I administered a survey to workers who commute from the Kitsap Peninsula to the Seattle area by a passenger ferry route, and collected data on stress, the perceived reliability of the route, and the positive and negative factors of the commuting experience. Preliminary results suggest that while ferry commuters experience stress due to the unpredictable nature of their route, other factors of the ferry environment cause the ferry commuter to feel overall less stressed than the typical commuter. I aim to supplement previous anecdotal evidence that ferry commutes do not harm worker well-being to the same extent as traditional modes of commuting. Given the unique experience and lessened detriments of ferry commuting, this study can advise researchers to consider ferry commuters as their own distinct class separate from other categories of commuting, and consider how their unique experience can influence transportation policy recommendations and challenge the traditionally negative perception of the modern commute.

Diffusion-weighted imaging (DWI) shows great potential for improving breast cancer detection and diagnosis. Primary findings from the ECOG-ACRIN A6702 multi-site, multi-vendor clinical trial indicate that DWI apparent diffusion coefficient (ADC) values may help reduce false positives and unnecessary biopsies. Gradient nonlinearity (GNL) correction was previously found to improve the accuracy of ADC mapping within and across MRI vendor systems. In this study, we evaluated the impact of GNL correction on breast lesion ADC measures in the A6702 dataset. The dataset comprised 81 suspicious breast lesions (28/81 malignant) in 67 women. Standardized DWI scans were acquired across 9 different MRI scanners. ADC maps were created from DWI scans, and ADC values were measured for each lesion. Direction-averaged GNL correction maps were constructed based on scanner-specific gradient specifications. ADC map correction was then performed through pixel-wise scaling by the GNL correction maps using custom software developed in MATLAB. Lesion ADCs before and after GNL correction were compared using a two-tailed z-test. ADC diagnostic performance (benign vs. malignant) was evaluated using area under the receiver-operating-characteristic-curve (AUC), and optimal ADC cutoffs were chosen to maximize specificity while maintaining 100% sensitivity. GNL-corrected lesion ADCs were significantly lower than uncorrected ADCs (1.12±0.29 vs 1.17±0.30x10-3mm2/s, p<0.001). GNL error in lesion ADCs varied across gradient systems (mean ∆ADCvendorA=0.14±0.08, ∆ADCvendorB=0.03±0.02, ∆ADCvendorC =0.004±0.01, p<0.001). GNL correction produced a slightly lower optimal ADC cutoff (1.33 vs. 1.35x10-3mm2/sec). However, no overall difference in diagnostic performance was detected: AUCuncorrected=0.78 (95% CI 0.68-0.88), AUCcorrected=0.79 (95% CI:0.69-0.89), p=0.22, and 18% potential biopsy reduction for both. This study showed GNL substantially affects lesion ADC measures, with significant variability across different vendor platforms. These findings suggest that GNL correction should be implemented to ensure uniformity and consistency in diagnostic breast lesion ADC measures across MRI platforms, especially for multi-center clinical studies.

Heart disease is the leading cause of death in the United States. Echocardiography is used clinically to highlight cardiac structures, wall motion, and contraction abnormalities to diagnose heart failure. Heart failure is classified into diastolic (relaxation) or systolic (contraction) heart failure using left ventricular ejection fraction (LVEF), a measurement of the amount of blood pumped during each contraction. Global Longitudinal Strain (GLS) analysis, which measures the stiffness and deformation of the heart wall during contraction, has recently emerged as a more sensitive metric of systolic function that predicts cardiovascular mortality in patients. Elamipretide (ELAM;SS-31) is a mitochondrial-targeted intervention that improves aging heart mitochondrial and diastolic function. In this study, we used an aging mouse model to compare sex-specific outcomes in systolic function by LVEF and GLS. We hypothesized that GLS would be a better predictor of systolic dysfunction in mice than LVEF, and that ELAM would repair aging systolic dysfunction. We compared young (4-6 mo) and old (25-26 mo) male and female mice using 2D echocardiography to obtain left ventricular parasternal short and long axis images. A cohort of aged male mice was imaged before and after 8-week ELAM treatment. These images were analyzed using Vevo LAB software using conventional echocardiography to measure LVEF and speckle-tracking echocardiography (STE) for GLS and LVEFStrain. Statistical analysis was performed using GraphPad Prism Software. Limited change in LVEF was observed by conventional echocardiography. Using STE, GLS and LVEFStrain declined with age. Treatment with ELAM restored GLS in aging mice to young levels. Here we show that the more sensitive STE analysis reveals that aging mice exhibit both systolic and diastolic dysfunction. The research supports our hypothesis that ELAM treatment improves systolic function in aging. Future treatments to target systolic dysfunction can be assessed in an aging mouse model using STE.

Cells communicate with each other and their environment via signaling pathways. When a cell receives a signal, it is processed by several proteins. Oftentimes, the same proteins are used by multiple pathways that process distinct signals and produce distinct cellular outputs. I am investigating how the cell is able to correctly process signals using Wnt signaling as a model pathway. Wnt signaling helps regulate cell growth and differentiation and is therefore tightly associated with many diseases. Both Wnt and insulin signaling processing utilize the enzyme glycogen synthase kinase 3β (GSK3β). Activation of insulin signaling results in the phosphorylation of GKS3β. However, if phosphorylated GSK3β from insulin signaling interacts with Wnt proteins, Wnt signaling can be inappropriately activated in the absence of a Wnt signal. The mechanism by which cells prevent improper activation of Wnt signaling is unknown. I am researching the role scaffold proteins play in signaling pathway insulation. Traditionally, scaffold proteins facilitate reactions by binding enzymes and their substrates, bringing them in close proximity to each other. It has also been shown that scaffold proteins can assist in promoting chemical reactions through mechanisms other than binding. Because the scaffold protein Axin binds GSK3β as well as PP2A, an enzyme that dephosphorylates GSK3β, I propose that Axin promotes insulation of the Wnt pathway. Using in vivo human cell culture assays, I have determined that Axin promotes dephosphorylation of GSK3β by PP2A through a mechanism more complex than bringing the two proteins in close proximity with each other. I will use in vitro kinetic assays to determine the underlying kinetic mechanism of this effect. Determining the kinetics of scaffold-mediated insulation will produce a model that can be applied to other signaling pathways and is important in understanding how to specifically target Wnt signaling for disease treatment without affecting other pathways.
 

Science and biology are intextricably linked to policymaking and thus interconnected to justice and injustice in our society. Yet, STEM education fails to emphasize this perspective. Our purpose in this research was to find ways undergraduate biology students can apply their scientific knowledge in the pursuit of justice, such that they can go beyond reading the text, thinking critically and answering questions applying science to the policymaking process. Accordingly, we asked, to what level of humanization do undergraduate biology textbooks frame scientific influence on policy as a tool for achieving justice? To investigate this, we, along with a larger team, analyzed six popular introductory biology textbooks searching for excerpts that contextualized science in society through ideas such as government policy or the judicial system. If an excerpt was found to contain these topics, it was coded based on topic and degree of humanization (in increasing order of humanization: none/scarce, detail, nuance, equity/inequity, or justice). For this project, we defined humanization as discussing or positioning science within the context of society or justice and/or injustice. We found that textbooks' contextualization of science within society is underdeveloped and does not foster critical thinking about the applications of science in policy and justice. Additionally, where this is addressed, there is limited depth in the text and breadth of examples. While our data suggests that these textbooks rarely contain humanizing concepts related to policy, our intent is not to suggest edits to the textbooks. Rather, we have identified trends in humanizing biology content that instructors can add to their curricula to contextualize the role of science in policymaking and justice in their classrooms. Additionally, these changes can encourage students to acknowledge the applications of their biology knowledge to policy in the pursuit of justice in society outside the classroom. 

Blackbody radiation is the name given to the phenomenon that makes warm objects radiate—it explains why a hot stove glows red, or why the sun is so bright. This radiation is well-understood in physics, but some theories of new particles suggest that at low temperatures there will be small deviations to the equations that normally predict the amount of power radiated by these bodies. I am conducting an experiment designed to search for these deviations. This experiment will involve measuring the radiated microwave power from a resonant cavity and a resistor as they are cooled to liquid nitrogen temperatures; standard physics predicts these power values scale linearly with temperature, which is the expected result. However, some expanded standard model theories predict a deficit of power at low temperatures due mixing of photons with new light particles. This research will help refine our understanding of blackbody radiation at low temperatures, including our understanding of the results from the UW Axion Dark Matter eXperiment (ADMX).

Hexokinases (HK) are enzymes that catalyze the first step of glycolysis – the phosphorylation of glucose to glucose-6-phosphate. Hexokinase 3 (HK-3), one of four mammalian HK isoforms, is detected in hematopoietic cells and tissues, especially in cells of myeloid lineage. Previous studies have shown that after myeloid cells undergo CRISPR/Cas9-mediated HK-3 gene disruption and differentiation to neutrophil-like cells, loss of HK-3 leads to no effect on glycolysis, but an increased cell death phenotype during differentiation to neutrophil-like cells. This study aimed to examine if HK-3 gene knockout causes a similar cell death phenotype in monocyte/macrophages cells, which are of myeloid origin. To test directly, THP-1 cells, which are monocyte-like cells that can be differentiated to macrophage-like cells, underwent CRISPR/Cas9-mediated HK-3 gene knockout via electroporation. This is a method that uses electric pulses to make pores within the cell and nuclear membrane. PCRs and DNA sequencing comparing wild-type THP-1 cells and HK-3-null THP-1 cells were done to confirm successful knockout. The HK-3-null THP-1 cells then underwent 48-hour, 250 nM PMA-induced differentiation to macrophage-like cells and were then stained with Trypan Blue to compare viability of wild-type macrophage-like cells and HK-3-null macrophage-like cells. No apparent difference was found in cell viability between wild-type macrophage-like cells and HK-3-null macrophage-like cells during differentiation and over six days post-differentiation. Cell function was not assessed. This suggests that further work should be to: (1) confirm these results by repeating the HK-3-null in THP-1 cell line process and remeasuring cell viability after differentiation, (2) perform the HK-3-null process in other cell lines to differentiate into other cells of myeloid origin to determine if cell death phenotype appears, and (3) determine if loss of HK-3 alters viability under culture conditions mimicking low oxygen conditions, such as found in hypoxic tissue areas during bacterial infection. Understanding cell viability differences from loss of HK-3 could give insight into the potential regulatory function of HK-3 during myeloid hematopoiesis. 

The modern education system has the opportunity to raise students with a well-rounded, interdisciplinary knowledge base, but that does not begin until textbooks (the backbone of many science curricula) include such topics. We wondered to what extent Multiple Ways of Knowing (MWoK), particularly in the topics of religion, culture, traditional medicine, and philosophy, are represented in introductory biology textbooks. The term MWoK describes these topics and recognizes that there is more than one way to learn and understand, crediting different cultures for their scientific contributions. This study is a spin-off from a greater project in which our team scored and evaluated six introductory biology textbooks on how much and how well they included a variety of social justice topics. Using the data gathered from the aforementioned study, we categorized the instances of MWoK by frequently occuring themes - religion, culture, traditional medicine, and other. We found that across over 9670 pages, these textbooks lack adequate discussion of MWoK, with only 32 mentions. Within these few instances, only one showed themes of justice or injustice, and one demonstrated themes of equity and inequity. Textbooks alone are insufficient for implementing the diversity of science in American classrooms. We envision this research as a starting point for instructors to integrate these topics into their classes. Incorporating more involved conversations about MWoK within the context of science can act as a way to foster a more inclusive learning environment wherein all students are able to engage more meaningfully with the material. Including MWoK in biology textbooks, and thus, biology curricula, would bring the STEM classroom one step closer towards increased diversity. 

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. 

Intracranial Epidermoid Cysts (IECs) are benign tumors located in the parasellar region and cerebellopontine angle of the brain. The incidence of IECs is ~1% of all intracranial tumors. These slow-growing, asymptomatic tumors are significantly enlarged when discovered which leads to trigeminal neuralgia, visual and hearing loss, tinnitus, severe face pain, and seizures. IECs are suspected to be a result of disruptions during neural tube closure during embryogenesis. Given their rarity, IECs are poorly investigated, and the mechanism of IEC formation remains unknown. Consequently, there is no drug therapy available up to date and surgery is still the only option. However, because the cyst lining adheres to important brain structures, a gross resection can result in deafness, facial weakness and chemical meningitis. These observations highlight the need to develop additional therapeutic options for these patients. We performed paired-end Whole Exome Sequencing (WES) on DNA isolated from sporadic IECs resected from 6 patients. From these, 4 were investigated with matching peripheral blood and 2 were sequenced without. We processed raw unmapped reads in tumor-normal and tumor-only mode as appropriate, following the GATK best practices for variant calling. Of the 5230 somatic variants found, 233 variants were found in protein coding regions (183 missense, 46 frameshift, 4 stop-gained/lost) suggesting damaging effects on the phenotype. Two mutations were identified in oncogenes (FCGR2B, PABPC1) and 2 mutations were identified in tumor suppressor genes (RSPO2, KMT2C), indicating possible tumor driver candidates. Gene enrichment analysis using the EnrichR interface revealed patterns of downstream microRNAs (miRNAs) regulation and signaling pathways implicated in the development of IECs. The validation of these findings and the study of their role in the IEC pathogenesis are currently under investigation in our laboratory. The identified driver mutations may serve as drug targets for the development of therapies to treat IECs.

Human adaptive immune systems consist of B- and T-cells, which respond to specific infections through the binding of cell-receptor proteins to surface proteins on pathogens. I aim to understand what receptors target post-acute sequelae of COVID-19 (PASC), commonly known as long COVID. To determine these receptors, I have analyzed subsamples of the T-cell repertoires (the collection of T-cell receptors in an individual’s immune system) of over 200 individuals — some healthy, and others with acute COVID or PASC. T-cell receptors (TCRs) are created through a combinatorial stochastic process called VDJ recombination, so I used a Bayesian network to learn a baseline model for how receptors are generated in individuals. Additionally, I have used the statistical model soNNia to characterize the functional selection (selection based on the reactivity of receptors to pathogens) on TCR repertoires. By applying information theory tools and utilizing the learned statistics of the generation and selection processes of these models, I have been able to quantify the variations in immune repertoires over time and between individuals. This analysis has enabled me to understand the correlations between these differences and the metadata of individuals and identify the immune-determinants that can predict the development of PASC. Using the soNNia models trained on immune repertoire data and comparing it to single-cell data, I intend to discern which receptors are most likely to be responsive to acute COVID or PASC. The results of this project would uncover immune factors that are predictive of the onset of PASC and how PASC progressively modifies immune repertoires. These insights could be used to inform treatments and preventative approaches to PASC.

The epidermis is a multi-layered tissue at the body surface made of cells called keratinocytes that protect humans from infection and dehydration. Keratinocytes undergo a unique program of differentiation while moving upwards in the epidermal tissue. During the final stage of maturation, each keratinocyte must eliminate its organelles and nuclei to allow flattening of the cells to form the uppermost layers that provide a water-tight seal for the body. To understand this crucial biological process, I aimed to train an online artificial intelligence (AI)-based image analysis algorithm called Biodock AI to detect key morphological features of differentiating keratinocytes and of engineered epidermal tissue. I annotated specific cellular and tissue structures in a set of microscopy images to train a new supervised AI model to recognize these features. In brightfield histological images of engineered human skin, I used an area selection tool to identify the boundaries of the epidermis to assess tissue thickness. To train our pipeline to recognize a common pathological feature, I next labeled nuclei that were improperly retained in the cornified layers of various drug-treated tissues. After the algorithm was trained by Biodock AI, our pipeline successfully replicated our tissue outlines and identified retained nuclei. Zooming to the subcellular level, I next labeled organelle features within fluorescence microscopy images of keratinocytes. I selected endoplasmic reticulum (ER) fragments that had broken off the tubular network during differentiation. Our AI-trained image analysis pipeline successfully identified ER fragments with high concordance with those annotated by a lab member. In summary, I successfully trained and implemented AI-based image analysis pipelines to detect tissue and sub-cellular features that characterize the process of epidermal differentiation. Our results demonstrate the potential of AI algorithms to accelerate imaging-based research to understand cellular differentiation and tissue pathology while mitigating bias and error from human investigators.

The ForwArd Search ExpeRiment (FASER), located 480 m downstream from the ATLAS beam interaction point, is designed to study light weakly-interacting long-lived particles (LLPs) and neutrino interactions. One of the main components of FASER, used to detect the decay products of LLPs, is the four tracking stations, each with three layers and 24 semiconductor tracker (SCT) modules. Particles leave electronic hit signals on SCT modules, and particle tracks reconstructed from hits are used for analysis. To exploit the excellent intrinsic resolution of the silicon microstrip detectors, high-accuracy alignment is required. A local χ2 alignment algorithm is designed and tested on both Monte-Carlo and collision data to perform software alignment on the tracking stations. By aligning the FASER tracking stations, the performance of track reconstruction from detector hits is substantially improved, indicating reconstructed tracks are more likely created from their corresponding hits. Furthermore, the alignment allows researchers to match particle track information with other detectors. For the showcase for my research, the software alignment shows promising results when compared to survey data.

Teacher burnout is a historical issue plaguing schools, but instead of school leadership focusing on decreasing teacher burdens, many teachers experience increased rates of burnout due to their focus being shifted to the added responsibilities to implement evidence-based practices (EBPs). Existing literature has shown that principals’ leadership type (i.e., transactional vs. transformational) and the implementation climate present (i.e., perceptions that EBP use is rewarded, expected, and supported) can impact teacher burnout. My research analyzes teacher burnout rates as they relate to leadership type and implementation climate when implementing Tier 1 (i.e., universal social, emotional, and behavioral programs and practices) services. As part of a larger ongoing study of a leadership-focused implementation strategy (i.e., strategies designed to improve adoption, fidelity, and impact of EBPs), principals, teachers, and school staff from 10 elementary schools within the same district completed a survey battery at the beginning of the school year. Data collected by the research team included things such as leadership type, implementation leadership, teacher burnout, and implementation climate. Preliminary analyses are ongoing to identify trends amongst school-building implementation leadership, leadership types, implementation climate, and teacher burnout when implementing Tier 1 EBPs, which can reveal how key implementation aspects relate to one another.

Duchenne Muscular Dystrophy (DMD) is a severe muscle wasting disease caused by deficiency of the protein dystrophy and affects approximately 1/3500 boys. Patients have shortened life expectancy due to cardio-respiratory problems caused by the disease alongside impaired ambulatory function. Previous studies have described the “waddling” gait in patients with muscular dystrophy but not in the animal model of DMD. Here we present the characterization of exercise ability and quantified gait metrics in a novel DMDmdx model that better represents DMD in humans. We used the Noldus Catwalk XT motion capture system to identify different gait parameters between DMDmdx and wild-type rats at 14-15 weeks of age. Compared to wild-type rats, DMDmdx has a reduced stride length and swing time in both front paws and hind paws. Time to max contact in DMDmdx rats is 5% faster than wild-type, but max intensity at time of max paw contact is 15% lower in DMDmdx. The “waddling” gait is indicated by 13% higher uses of 3 and 4 paws supported by DMDmdx during a run compared to wild-type. Subsequently, this led to a higher abnormal step pattern as similarly observed in patients with muscular dystrophy due to hip muscle weakness, thus resulting in the “waddling” gait. Gait pattern of the novel DMDmdx rat model reflects the impaired ambulatory function commonly seen in patients with DMD, thus making this a potentially useful outcome for understanding disease progression, therapies, and development of exercise guidelines.

The first direct observations of gravitational waves (GWs) by the LIGO and Virgo interferometers in 2016 confirmed the predictions of general relativity for the dynamics of black hole mergers, and has since advanced the field of GW astronomy. These interferometers focus on detecting GWs in the Hz-kHz frequency range. There are no known astrophysical objects that emit beyond the 10 kHz range, motivating an interest in detectors for frequencies above this range which could lead to the discovery of new physics beyond the Standard Model. Theoretical potential sources of these high frequency GWs include mergers of sub-solar mass objects such as primordial black holes (PBH) or boson clouds from PBH superradiance. Resonant microwave cavity detectors such as the detector used in ADMX are shown to be sensitive to GWs in the GHz range, and generate a coherent electromagnetic (EM) signal through the GW-EM coupling if the GW frequency matches the cavity resonance frequency. To detect these signals, I use a matched filtering technique on power measurements collected from the cavity during ADMX’s Run 1B data acquisition period in 2018. I generated templates for a range of frequencies based on an expected signal a PBH merger would emit and convolved it with the data to find similar signals. This analysis may confirm or further constrain the existence of PBH sources within the GHz frequency range. PBHs could comprise a significant fraction of cold dark matter, and its detection allows us to probe the density fluctuations and phase transitions of the early universe with implications in galaxy formation and supermassive black hole formation.

The drug rapamycin has been shown to extend lifespan in model organisms ranging from mice to fruit flies, but little is known about how genetic variation affects the response to rapamycin. It is possible that some individuals might not respond to the drug, or may even respond negatively. To study the impact of genetic variation on rapamycin sensitivity, the Promislow lab has been using the fruit fly, Drosophila melanogaster, to test the ability of rapamycin to slow early development in different strains. They found a range of responses, from strains that were completely resistant to those that were highly sensitive. To better understand why resistant and sensitive strains differ, the Promislow lab analyzed their metabolome profiles. The metabolome consists of diverse small molecules, metabolites, that are fundamental to sustaining life in cells, and the relative abundance of metabolites is referred to as a metabolome profile. By comparing metabolome profiles, the Promislow lab found that the metabolome of sensitive larvae treated with rapamycin was similar to those of larvae starved of nutrients. One possible explanation for this observation is that sensitive larvae on rapamycin-treated food eat less food than resistant larvae. To test my hypothesis, I am measuring how much food they ingest, using dyed food, and scoring how much dye each larva ingests over time. If sensitive larvae eat less rapamycin-treated food than resistant larvae, then we can investigate why rapamycin affects feeding. If no significant difference is found between scores for sensitive and resistant larvae, then we can investigate why rapamycin affects early development in sensitive strains when their food consumption is not limited. By determining if sensitivity to rapamycin is accompanied by differences in feeding, then we could potentially manipulate feeding to sensitize flies, and possibly humans, to this beneficial drug.

Weight loss (WL) is recommended for people with obesity to mitigate cardiometabolic risk, but its effect becomes limited when reaching a WL-plateau (WL-PL), when the rate of WL becomes minimal despite efforts to continue losing weight. The biological basis for the WL-PL is not fully understood. The goal of this study is 1) to test two diet-regimens in the development of a WL-PL in mice with diet-induced obesity (DIO), and 2) to identify subsequent changes in mitochondrial function. We hypothesized that despite similarities in caloric intake, higher fat content will make high-fat diet (HFD-CR20) mice protect their adiposity and reach a WL-PL sooner than low fat diet (LFD-CR20) mice. We also hypothesized that mitochondrial function will be reduced in mice that have reached a WL-PL. To test hypothesis 1, individually housed DIO mice were divided into two groups, and were provided with 80% of their ad libitum caloric intake with either a high-fat, or low-fat diet daily for ~2.5 weeks. Body weights were recorded daily. A WL-PL was identified by weight stability (<0.5% change BW/day) following weight loss. HFD-CR20 mice reached a WL-PL phase after 10 days of caloric restriction. LFD-CR20 mice did not achieve a plateau within the study time frame. LFD-CR20 mice lost more weight than HFD-CR20 (-10.8% ± 2.2 vs. -5.2% ± 1.8, p<0.05 respectively), which was attributed to a greater loss in adiposity, measured by an EchoMRI, (-23.3 g ± 6.0 vs. -3.3 g ± 3.0, p<0.05). To test hypothesis 2, mitochondrial function was assessed by high resolution respirometry at the study endpoint. We will further analyze this data to identify differences in mitochondrial function attributed to the WL-PL. This work will improve our understanding on the biological mechanisms behind resistance to weight loss to help advance obesity treatments in humans.

Supported through the University of Washington Campus Sustainability Fund, this project is developing and conducting a 3-year (2020 -2023) monitoring plan to assess the establishment and growth of the camas meadow at the Burke Museum’s yard. Designed and installed as part of the newly constructed Burke Museum the camas meadow is an approximately 10,000 sq. ft. area of the site that contains a planting mix of Northwest native meadow species including several species of camas (Camassia spp.) a culturally important plant for regional tribal communities. I conducted field monitoring via quadrat sampling, a traditional method of assessing biodiversity. Quadrat sampling utilizes an evenly divided square frame placed on the ground. I then record the specific vegetation found within the frame. Twenty-three different plots across the camas meadow were assessed monthly using this method, with special attention paid to the locations and prevalence of Camassia spp. and Lupinus rivularis. I predict that regular maintenance via weeding, mowing, and exclosure will have positively correlated with the biodiversity scores and camas establishment found in the quadrat sampling. The purpose of this monitoring is to determine the effects that design and management have on the establishment and biodiversity of the camas meadow in order to develop a strategic, long-term management plan that is culturally engaged, built on collaboration and conversation and interpreted through perspectives that are grounded in local traditional knowledge as much as scientific approaches to botany and environmental understanding. While more locally working closely with UW Grounds and the Burke through the Camas Meadow caretaker to mitigate increased costs for maintenance while ensuring the ecological viability and sustainability of the project, our intention with this work is to assist other organizations and communities seeking to establish this habitat type in Washington State and the broader Northwest region. 

Even though Ivermectin was only shown to work against the COVID virus in vitro, and even though the FDA warned against its use in humans, many media outlets reported that research showed it was effective against COVID. The fact that they often did so without any caveats may have led readers to make unwarranted inferences and act on them, believing that their actions were supported by science. In order to mitigate against such outcomes, a 2017 paper that has been cited over 600 times so far urges authors to include Constraints on Generality (COG) statements. But do COG statements in fact work as intended? In the present experiment, participants were randomly assigned to read popular press articles with COG statements or those without, and answered a series of questions about the articles. We hypothesized that the inclusion of COG can (1) reduce the likelihood of prematurely making overly-confident and overly-generalized inferences after reading reports of research findings; (2) reduce the likelihood of acting on such inferences (e.g., ingesting untested substances); and (3) improve their understanding of science as a long-term, cumulative effort to reduce, but never eliminate, uncertainty. The data collected so far provide partial support for these hypotheses. We hope our findings will help the quality of popular press reporting of scientific findings, avoiding sensationalistic and over-confident content.

The CandyCollect is a lollipop-inspired device that utilizes microfluidic channels to capture oral bacteria in saliva. The original intent of this device was to successfully collect oral Streptococcus pyogenes (S. pyogenes) from saliva samples and screen for strep throat. Using in vitro experiments, we were able to capture and elute S. pyogenes using the CandyCollect. To explore the functionality of the device, we performed in vitro experiments to capture and elute two different strains of commensal bacteria, Staphylococcus aureus (S. aureus) and Streptococcus mutans (S. mutans). Collection of bacteria using the CandyCollect would allow us to sample from the general population rather than strictly individuals infected with S. pyogenes, allowing the CandyCollect to be used in various applications. The method of bacteria elution we performed was compatible with polymerase chain reaction analysis. We conducted an IRB (institutional review board) approved study to compare the CandyCollect to two other standard saliva collection methods (ESwab™ and SpeciMAX Stabilized Saliva Collection Kit™). Our results showed that the CandyCollect was able to capture both of these commensal bacteria strains when present. We then performed a human subjects study to compare the detection of the CandyCollect to the other two methods. We focused on detection of S. mutans and S. aureus due to their high prevalence in healthy adults. Our results showed that for participants in which a given bacterium (S. mutans or S. aureus) was detected in one or both of the commercially available methods, CandyCollect devices had a 100% concordance with those results. Surveys were sent out to participants to assess the comfort of the sampling methods. The CandyCollect was the preferred method of sampling. Based on the results of this study, we are hoping to one day incorporate the CandyCollect into clinics for strep throat diagnosis to replace more invasive current methods.

Gift-giving is a central component for many people in terms of expressing affection through increasing perceived closeness as well as strengthening relationships. Giving gifts can also facilitate requesting help or asking someone for a favor. According to Aknin and colleagues (2018), these ‘motivated gifts’ decrease recipients’ anticipated satisfaction and willingness to perform the favor which could negatively impact relationships. The current study proposed that this decrease in prosocial tendencies could be explained by the self-determination theory of basic psychological need satisfaction (BPNS), that is, that humans require three psychological needs to flourish and live optimally (autonomy, competence, relatedness; Ryan & Deci, 2000). Fulfilling these needs is said to facilitate the integration of autonomous motivation, which was hypothesized to be the mediating role between motivated gifts and prosocial behavior. Participants (N = 426) were recruited for an online survey in which we randomly assigned them to read a hypothetical scenario where they imagine being asked a favor by a friend either with or without a motivated gift. The current study was unable to replicate the findings from Aknin and colleagues’ (2018) study and found no significant differences in participants’ BPNS, but did find that motivated gifts caused a decrease in autonomous motivation. Additionally, motivated gifts were not found to cause a change in whether a participant would be willing to participate in an unrelated study. Altogether, the findings suggest that motivated gifts do not necessarily have a negative impact on prosocial behavior which, considering the primary reason gifts are given, is good news for relationships and prosocial tendencies.

An individual’s capacity to remain healthy in times of stress is exceedingly difficult, and there is sufficient support that stress contributes to poorer eating habits such as consumption of high-sugar and high-calorie foods. The impact of stress, and anxiety in particular, can be mitigated by engaging in healthy adaptive cognitive processes such as metacognition. A limited amount of research has investigated the impact(s) of adaptive metacognition on participants’ food choices specifically. However, some previous studies show that guiding metacognitive processes may improve food choice. The goal of the present study is to determine if metacognition can moderate stress and help participants navigate everyday health choices. Participants were recruited at the University of Washington Tacoma to complete the study online. Food choice was measured by a pretest-posttest design providing the participants with a forced-choice computerized simulation of various drinks i.e., sugar-sweetened beverages (SSB) and water. Metacognitive guidance was given to participants in the form of knowledge-based information about SSB and reflection on metacognitive monitoring and control. Participant stress was measured using the Perceived Stress Scale (PSS), eating habits with the Intuitive Eating Scale-2, and participants completed a demographic questionnaire. It is expected that metacognition guidance through knowledge, monitoring, and control may positively moderate the impact of stress on food choice. Specifically, students will opt for beverages with fewer sugar grams after the metacognitive guidance, and perceived stress will be correlated with the change in food choice before and after the guidance. Metacognitive guidance may prove to be another tool in helping individuals reduce their sugar intake and thus decrease likelihood of developing sugar-related health conditions such as diabetes and overnutrition.

Oral tolerance to antigens is a mechanism by which immune responses are inhibited to prevent chronic inflammation and tissue damage in response to common exposures, such as dietary antigens or commensal bacteria. Regulatory T cells (Tregs) are an important cell type in such regulation of immune responses, especially in the intestines. There is, however, heterogeneity within Tregs, including a subset that expresses the transcription factor RORgt. However, the mechanisms by which RORgt+ Tregs carry out their suppressive function are currently unknown. Intestinal Tregs are induced in the mesenteric lymph nodes (MLNs) by antigen-presenting dendritic cells (DCs) that migrate from the gut. Antigen transfer from DCs to Tregs is crucial for the development of oral tolerance and DCs are largely regarded as being upstream of Tregs. However, it has also been shown that Tregs play a role in DC migration via a CTLA-4-mediated mechanism. Because this DC-Treg relationship is not fully understood with regard to Treg heterogeneity, I have examined the changes in DC populations in a mouse model where the RORgt+ Treg population alone has been ablated. To do this, I developed a new panel of antibodies to use in flow cytometry in order to characterize the subpopulations of DCs in the intestines and related organs. I harvested and processed murine spleens, MLNs, Peyer's Patches, and small intestine lamina propria in order to compare the populations systemically and locally. I anticipate seeing fewer DCs in the mice lacking RORgt+ Tregs and more DCs in the small intestine and Peyer's patches. This work furthers our understanding of the intricacies of the intestinal immune system. This knowledge can be applied to vaccine research, as RORgt+ Tregs have been implicated as suppressors of immune response to oral vaccines. Intestinal immunity is also of interest in allergy and gut inflammation research.

Cystic Fibrosis (CF) is a recessive genetic disease that affects roughly 70,000-100,000 people worldwide. CF is caused by mutations in the CFTR gene, which encodes for a channel that regulates the flow of water and ions into and out of the cell. CFTR deficiency results in mucus buildup and other defects that lead to increased bacterial burden and inflammation. However, whether or not CFTR deficiency itself is sufficient to initiate or potentiate epithelial inflammatory responses is unknown. Inflammasomes are multiprotein complexes that upon activation initiates a lytic form of cell death called pyroptosis and the release of pro-inflammatory cytokines including IL-1B, a hallmark of CF. Previous studies have shown that inflammasomes are expressed in the epithelia of mice and humans. Although CF is largely a disease that affects airway epithelium, other tissues such as the skin and gut are also affected by CFTR dysfunction. Moreover, we and others recently found that inflammasomes play an important role in host defense of epithelial barriers, including the intestinal epithelium. Thus, we hypothesize that inflammation in the gut of CF patients may be caused or enhanced by inflammasome activation. To test this possibility I used both human and mouse intestinal organoids as models and ran a Forskolin-induced Swelling (FIS) Assay to assess CFTR function in presence and absence of a CFTR inhibitor (CFTRinh-172). Having established this model system, I am now evaluating the impact of CFTR function on inflammasome activation. We anticipate that our work may reveal a link between CFTR dysfunction and inflammasome activation which may provide further insight into how inflammasomes affect inflammation in the gut epithelia of CF patients.
 

Type 1 Diabetes (T1D) is one of the most prevalent chronic diseases among teens in the United States. Teens with T1D facing socioeconomic disadvantage are disproportionately negatively affected by the burden of T1D and are at-risk for poor mental and physical health outcomes. Limited access to diabetes-related technology may be one factor contributing to these disparities. The purpose is to describe diabetes technology (continuous glucose monitors and insulin pumps) use among teens with T1D according to neighborhood-level socioeconomic disadvantage in Washington. The research also aims to investigate links between neighborhood disadvantage and diabetes outcomes and test whether diabetes technology moderates this relationship. We abstracted demographic, clinical, and psychosocial data from medical records of teens aged 13-18 seen for T1D clinical care at Seattle Children’s Hospital in 2019. We determined state-relative decile scores (1-10) for neighborhood disadvantage using the area deprivation index (ADI) tool, which considers income, education, employment, and housing quality factors. Diabetes management was measured using A1c, a 3-month average of blood sugar levels. Diabetes distress was measured using Problem Areas in Diabetes-Teen, a self-report measure of the emotional burden of living with diabetes. I will perform a descriptive analysis calculating the percentage of participants using diabetes technology for each ADI score and for low and high disadvantaged groups using cutoffs from previous literature to reveal technology use patterns. I will conduct linear regressions exploring relationships between neighborhood disadvantage and outcomes (A1c and diabetes distress) and enter diabetes technology as a moderator. We predict that higher neighborhood disadvantage will be associated with poorer diabetes management and higher distress, but this will vary as a function of technology use. Altogether, this research highlights use of a neighborhood-level tool to identify patterns for an at-risk group of teens and may help to identify intervention targets for public policy addressing health disparities.

As of 2021, there were approximately 38.4 million people living with HIV who require routine viral load testing. Viral load testing returns a quantitative measure of viral concentrations and is indicative of antiretroviral therapy efficacy and adherence compliance, with lower viral loads correlated to better health outcomes. Quantitative polymerase chain reaction (qPCR) is the gold standard for measuring viral load, but is not accessible to many clinics and patients around the world due to its long assay times and requirements of specialized equipment and highly trained personnel. As a result, qPCR is limited to centralized testing facilities far from the point-of-care (POC), leading to delayed results or loss of follow-up. Our group has addressed this by developing an HIV viral load test using recombinase polymerase amplification (RPA), which has a 20 minute sample-to-answer time and is more appropriate for POC settings. Our test involves the formation of discrete fluorescent nucleation sites which can be counted to estimate the viral load. However, our test fails to accurately quantify higher HIV viral loads (>3,000cps/rxn). We have difficulties distinguishing between individual sites at these higher copy numbers due to sites merging. In this project, I address the limited dynamic range of this test by performing RPA between two glass slides and investigating the effects of different slide thicknesses and concentrations of polyethylene glycol (PEG), a crowding agent used in RPA reactions. I perform nucleation site analysis using computer vision techniques to measure nucleation site radius and intensity and study how these factors affect site diffusion and amplification. By analyzing nucleation site behavior, we demonstrate potential for an HIV viral load test with a higher dynamic range and gain a better understanding for RPA nucleation site formation, ultimately helping to improve access to testing and treatment for people living with HIV.

College students often struggle to get a sufficient amount of sleep (7-8 hours) due to balancing their academic, social, familial and financial lives. This study investigates whether education of sleep and its physiological and neurological effects on the body, encourages college students to improve their sleep quality and lowers their levels of stress and anxiety. We surveyed students via Qualtrics asking their levels of stress and loneliness, along with their sleep quality before and two weeks after attending a wellness workshop detailing the importance of sleep and how to better manage sleep. Using the perceived stress scale, perceived anxiety scale and sleep quality scale, we ran the results in an SPSS software, and we found a statistically significant decrease in perceived stress scores before (M = 28.77, SD = 7.41) and after (M = 26.50, SD = 7.91) the workshop, t(56) = 29.33, p < .001 and anxiety scores before (M = 38.07, SD = 11.42) and after (M = 35.89, SD = 8.09) the workshop, t(55) = 24.94, p < .001, along with a significant increase in students’ sleep quality scores before (M = 17.05, SD = 2.552) and after (M = 17.68, SD = 3.222) the workshop t(55) = 50.44, p < .001. Although there was a decrease in the sample sizes from each survey (n=57 in pre-survey, n=40 in postsurvey), the decreases in stress and anxiety, along with the increase in sleep quality, still suggest teaching students methods to manage their sleep through interventions such as the workshop, could decrease their levels of anxiety and stress. Using this knowledge, we seek to find ways to implement better education of physiological wellness in institutions to lower students’ levels of stress and anxiety in school in hopes that they can become better students and better citizens of their communities.

CRISPR Activation (CRISPRa) is a powerful discovery-based tool to evaluate gain-of-function en masse. Applied successfully to emerging cell therapies, this technology offers tremendous promise to inform next-generation therapy design. Despite this potential, translation of CRISPRa to primary cells, including T lymphocytes, has been impeded by poor transgene expression. Based on prior reports of dCas9 genotoxicity, we suspected that CRISPRa could be exerting a toxigenic effect on CAR T cells, and thereby selecting against clones with high expression. To test this hypothesis, three separate constructs were designed with inhibited transcription and/or translation of the CRISPRa transgene. Following delivery of the constructs to donor T cells, analysis by flow cytometry revealed similar levels of cell yields and no net increase in dCas9 marker positivity across all CAR T cell subsets. Further epigenetic experiments and drug studies with anti-silencing compounds revealed that transcription of the CRISPRa transgene was severely inhibited. Collectively, these findings suggest that the CRISPRa transgene does not exert a toxigenic effect on CAR T cells; rather, low CRISPRa expression is caused by transgene silencing. Targeted efforts to mitigate silencing of the CRISPRa transgene are thus warranted to achieve adequate implementation to therapeutic cell subsets.

Particle-laden turbulent flows are important in both natural and industrial contexts. The particles in many of these processes, such as the formation of ice crystals in clouds or the paper-making process, are anisotropic, with directionally-dependent drag coefficients. Generally, anisotropic particles are free to rotate as they are advected by the carrier fluid. However, external forcing from gravitational and magnetic fields, the larger scale flow, and active behavior can restrict the particles’ orientation, fixing their anisotropic resistance with respect to the reference frame. The dynamics and statistics of symmetric particles in isotropic turbulence are well-studied, but the effect of anisotropic forcing on the transport and behavior of asymmetric particles is less well-understood. We studied these dynamics by conducting Lagrangian particle tracking in simulated isotropic turbulence from the Johns Hopkins Turbulence Database. Computer simulations of 54,000 randomly-placed particle tracers were run in Python, with anisotropy introduced by directly scaling the velocity of the tracer-particle at each simulation time step. We examine how increasing a particle’s resistance to motion in one direction in isotropic turbulence impacts the transport and dispersion statistics in all three directions. We find that increasing tracer anisotropy decreases diffusivity in the direction of velocity scaling as expected, but the diffusivity in the unscaled directions increases such that the total diffusivity remains roughly constant. Studying the dynamics of these simulated anisotropic particles in turbulence will provide a better understanding of how turbulence mixes both particles and the fluid itself, which can then be applied to particle-turbulence interactions in both environmental and industrial contexts.

The periodontal ligament is a connective tissue that anchors teeth to the bony socket and is crucial in providing nutrition for the survival and functioning of the human body through the mastication of food by teeth. Because the periodontal ligament is anatomically sealed off from the oral cavity, no non-invasive techniques currently exist to investigate it in-vivo, making the development of sound in vitro models critical for periodontal research. With periodontal disease affecting over 743 million people worldwide, in-vitro research to develop regenerative therapies to replace diseased periodontal tissue is urgently needed. To achieve this, we have developed a novel 3D in-vitro model, which closely mimics the in-vivo periodontal ligament. The 3D tissues are fabricated by inverting an array of silicone posts into silicone molds containing a cell-collagen gel mixture in a 24-well plate. Once the tissues have been incubated and the collagen polymerizes, magnetic mechanical force stretches tissues on posts. The post deflection is used to calculate tissue stiffness and contractility. Preliminary data shows a reduction of contractile force in the tissue constructs after 24 hours of mechanical stretching. I expect to find similar outcomes through additional experimentation. Understanding the periodontal ligament’s response to mechanical force is crucial for its effective restoration and ensuring that it is mechanically sound. The novel in-vitro 3D model that we have developed provides a valuable opportunity to better comprehend the ligament's response to these forces. By performing further experiments with this model, we can gain a deeper understanding of the periodontal ligament, allowing for informed decisions when it comes to replacement and repair in patients. This model offers controlled and repeatable experiments, providing more accurate insights into the biology of the periodontal ligament and contributing to the advancement of periodontal disease treatment.

The world faces an unprecedented magnitude of ecological challenges: climate change and rapid biodiversity decline being chief among them. Sustainability, in a holistic sense, provides a means to help elevate these ecological challenges while creating long-term societal benefit. Certain nations are ‘better’ at destroying the Earth than others with The United States being among the worst perpetrators. In this project, I will explore why the US is so far from a sustainable society and how that ‘sustainability gap’ is fed by a kind of individualism unique to America. The main question: How does American culture’s emphasis on individualism affect the United States’ ability to create and foster a sustainable society? More specifically, I will dive into the phenomena among American society that emphasizes that “I” is more important than “we” and how that sociology plays out and relates to the United States’ struggling efforts towards sustainability. The main mode of this research will be the gathering of and analyzing information from books, articles, surveys, etc. from various authors and disciplines. The product formed from this research will be an synthesis essay that will demonstrate my research understanding of the information that I have gathered. This essay will act as an interdisciplinary connection between multiple subjects--from sociology and philosophy to environmental science and sustainability--with the intention of highlighting the complexity of culture’s unique influence on sustainable action. I expect to discover that American culture’s individualistic nature remains a hardened barrier to true sustainable, communal action influencing nearly every aspect of American life and society. Thus, I will also include recommendations for where America might have the capacity to change from the sociological and environmental perspectives. This kind of interdisciplinary research is crucial to understanding how culture and science conjunctively influence each other to incite or inhibit progress.

 Children are tasked with listening and learning in noisy environments where many people are talking at the same time every day. Conceptual models of listening under complex conditions posit that working memory plays a role in the ability to extract the target speech from the distracting noise. Hard-of-hearing (HoH) children, in particular, do worse listening in noisy environments. Thus, in this study we investigate the relationship between working memory and multitalker speech perception in HoH children. We hypothesized that we would observe a correlation between working memory and speech perception in both HoH and the typical hearing comparison group. Moreover, we expected that HoH children will have worse multitalker speech perception thresholds overall, suggesting difficulty perceiving speech under complex acoustic conditions. We tested 7-to-18-year-old HoH children and an age- and biological sex-matched typical hearing (TH) comparison group. Working memory was assessed in both visual and auditory domains; participants were asked to recall either a sequence of visually presented letters or auditorily presented numbers. Multitalker speech perception thresholds were obtained under 2 conditions: Collocated noise and Segregated noise. In the Collocated Noise condition, the target speaker spoke sentences from 0° azimuth with two additional distracting talkers, referred to as maskers, collocated from the same location. In the Segregated Noise condition, the distracting talkers were spatially segregated to ±90° azimuth, while the target talker remained at 0°. Preliminary analyses suggest that a relationship between working memory and multitalker speech perception is observed. These results will advance our understanding of why HoH children experience difficulty perceiving speech under noisy conditions and have the potential to lay the groundwork for novel assessment and intervention strategies to improve how TH and HoH children listen and learn in a noisy classroom. All authors participated in data acquisition, analysis, and preparation of this presentation.

 Hemophilia A is an inherited bleeding disorder caused by mutations in the human coagulation Factor VIII (FVIII) gene resulting in mild to severe loss of clotting ability when bleeding. While protein replacement therapy has greatly decreased the mortality and morbidity of HA, 30% of patients develop FVIII neutralizing antibodies (inhibitors) that bind to FVIII protein in the blood and initiate clearing of FVIII from circulation leaving patients vulnerable to life-threatening uncontrolled bleeding events. N-linked glycosylation is a post-translational modification known to modulate the functional and antigenic properties of proteins. Deletion of glycans at the N2118 site of FVIII has been shown to significantly reduce the immunogenicity due to the elimination of a potent glycopeptide epitope that can interact with and activate T-cells, however, more investigation is needed to characterize the humoral immune response in detail. Here we describe efforts to optimize a method for the production and isolation of cell-derived mannosylated peptides for use in ongoing humoral immune response characterization experiments. We chose to use cell-derived peptides rather than synthetic peptides so the glycoforms will closely resemble the plasma-derived human FVIII and cell-produced recombinant FVIII proteins used clinically. SDS-PAGE and LC-MS analysis confirmed our peptides contain the desired mannosylated glycoform and preliminary results demonstrate that the peptides were able to generate a robust immune response in HA mice as intended. Going forward, these peptides will be used to generate an immune response in mice so we can characterize the inhibitor binding affinity to our peptides, assess the specificity of splenic CD4+ T cell receptors and determine the T helper cell differentiation status via cytokine assays. The findings of these experiments will hopefully lead to the development of a more immune-tolerant FVIII protein product for protein replacement and gene therapy treatments for HA.

 Solid tumor cancers are capable of releasing chemicals that are detectable in body fluids. These cancers can cause severe acidosis within the patient, resulting in the change of structure and function of Complement serum proteins, membrane-bound regulators, and receptors that interact with various cells and mediators. A better understanding of the mechanistic interaction between the complement system and tumor-induced acidosis can provide a new direction in cancer immunotherapy research. Here, we investigate the effect of pH on binding between Heparin, Complement Factor H (CFH), and C3b. We found that in controlled in vitro assays conducted through biolayer interferometry, CFH and Heparin bind better to C3b in pH 6.0 but only in lower concentrations of CFH. As for pH 7.4, the binding between CFH, Heparin, and C3b is lower in comparison; however, the binding between only CFH and Heparin is higher at pH 7.4 than at pH 6.0. Our results demonstrate how Heparin, Complement Factor H, and C3b are likely to function in a tumor microenvironment where the pH is generally lower. We anticipate this research to be a starting point for investigating the role of the Complement System in tumor growth and researching the implications of its biological actions with respect to the development of anticancer therapy.

The blood-brain barrier (BBB) is a physiologic interface between the bloodstream and the brain. The BBB has tight junction (TJ) proteins that restrict blood and toxins from entering the brain and allow important nutrients and ions to pass through. In recent studies, we found a significant increase in the TJ protein, occludin, in microvessels (MV) isolated from the parietal lobe of female subjects of advanced age with Alzheimer’s dementia (D) relative to aged match females (F) without dementia (ND) (n=8 D and 8 ND, age range: 79-99, mean: 93 years for both groups). There were no significant differences in occludin between MV of male subjects with and without dementia, who were of a much younger average age (mean: 74 years old). To further evaluate this finding, we examined MV in cut sections of the superior parietal lobes of a separate group of F subjects with D and ND via immunohistochemistry (IHC) (n=12 D, age range: 82-93 and n= 5 ND, age range: 83-98). We also measured levels and distribution of occludin in isolated MV from the same original group of female subjects with D and ND utilizing immunofluorescence (IF). Preliminary analysis of IHC was not able to detect differences in occludin, but initial IF studies support higher levels of occludin in MV derived from F D subjects. Current studies are evaluating occludin localization in both groups of brain MV. In summary, we have data that occludin is increased in brain MV from female subjects of advanced age with D, relative to ND. Ongoing studies with IHC and IF will confirm differences in levels and determine localization. The increase in occludin in F D MV is an unexpected finding, and could reflect a compensatory mechanism within brain MV in the context of advanced age and Alzheimer’s dementia.

Many curricula today fail to connect STEM with the issues students and their communities face. This shortcoming can lead to increased dropout rates and equity gaps especially in early STEM courses. With the COVID-19 pandemic and the George Floyd uprising, fostering students’ ability to tie STEM into the issues of social justice have never been more important. In this study, we investigate whether a Course-Based Undergraduate Research Experience (CURE) centered around the public health implications of indoor CO₂ can give students a greater understanding of the relevance of STEM to social issues and help them see STEM as a tool to solve problems in their communities. Students were administered a brief pre-survey before beginning the CURE. They were then introduced to the low-cost CO₂ sensors and the public health implications of high indoor CO₂. Students formed groups, formulated their research questions, and collected data. Students then analyzed their data and presented their research to their peers. A post-survey was administered following the CURE. We are currently analyzing the results of the surveys and post-curriculum interviews. The preliminary results suggest that connecting CUREs to relevant social issues in early STEM courses is a powerful tool that not only teaches students to interpret the world around them but also to change it.

Sensory signal processes– specifically how visual systems perform– function under the limits imposed by physics. One such physical limit comes in the detection of light. Light is divided into discrete amounts of energy called photons. Because of this division, light is inherently variable and such variance sets limits on detection in the retina. Understanding how a population of cells in the retina work together to detect and encode the motion of moving objects during a given time interval is pertinent to unraveling the internal mechanisms of the retina. This property is fundamental across varying light levels to understand visual adaptation in low-light conditions. So far, the only thing studied in low light is if our system can detect the existence of a flash or dim light, but not deduction of movement and time information. Rod vision has traditionally been viewed as slow from measurements such as flicker fusion. Flicker fusion is the frequency at which flickering light is perceived as continuous– through studying varying light levels, we can directly study sensitivity to the timing of different frequencies (temporal resolution). These measurements suggest that rod cells have extremely poor temporal resolution. This in turn suggests that the ability to detect moving objects– which relies on timing information– would be poor at low light levels. Through extracellular electrophysiological experiments conducted using a 512-channel multi-electrode array, we recorded the electrical activity of neural firing from a population of at least 5000-1000 retinal ganglion cells in the primate retina to show that there is high temporal precision in star-light levels due to compensatory mechanisms in the retinal ganglion cells and adjunct circuits.

The placenta is a crucial fetal organ providing oxygen and nutrients to the developing infant. Researchers typically use placental cell models to study the placenta, which are derived from immortalized cells. The use of in-vitro placental cell models is important because human samples are difficult to obtain, and placental biology is highly species-specific. Despite this, our understanding of the characteristics of these cell lines and how they compare to placental tissue samples is limited. This project aims to determine which placental cell model most directly reflects the gene expression of the human placenta. RNA sequencing data from the placental cell models HTR-8/SVneo, JEG-3, BeWo as well as data from primary trophoblast cells was obtained using the Gene Expression Omnibus (GEO) database or through lab-generated datasets. Data for each cell line was combined into a single dataset of shared genes (n=6835) and individual datasets of genes unique to each cell model. For genes that were unique to each cell model, I performed KEGG pathway analysis and characterized placenta specific genes using the Human Protein Atlas (HPA). BeWo cells expressed the highest number of unique genes (n=355) and shared the highest number of genes with the primary trophoblast cells (n= 1,167). Pathway analysis showed that genes unique to primary trophoblast cells (n=2661) were overrepresented in 24 pathways, unique BeWo genes were overrepresented in 15 pathways, while unique HTR-8/SVneo genes (n=355) were overrepresented for a single pathway- the neuroactive ligand-receptor interaction. Placenta specific genes were expressed within the uniquely expressed genes for JEG-3 (n=1), BeWo (n=1), and primary trophoblast cells (n=17), but not HTR-8/SVneo. Ultimately, the results from this project will provide a tool to evaluate differences in placental cell models and aid the placental biology research community in understanding which cell line is most representative of human placental tissue samples.

After someone suffers from a heart attack, heart tissue can become scarred. Scarred heart tissue cannot regenerate, leading to many dangerous outcomes, including dysrhythmia, chronic heart failure, and even sudden cardiac death. Therefore, there is a need in the tissue engineering field to create a “band-aid” that could functionally replace damaged heart tissue. Towards this goal, I propose using open microfluidic patterning techniques to generate a suspended heart tissue that could reinforce and replace unviable heart tissue. To generate a suspended heart tissue, we utilize an open microfluidic patterning removable rail that interfaces between two polydimethylsiloxane (PDMS) posts such that an open channel is formed around the posts. Combining human vein endothelial cells (HUVECs) and enzyme-degradable polyethylene glycol (PEG) hydrogel, we pattern the cell-gel solution through the open channel. After the hydrogel has gelled, the rail is removed from the post, revealing a suspended tissue model that acts as the vasculature of the heart tissue. Then, a second patterning rail is placed around the first generated suspended tissue that has a larger open channel and we repeat the protocol, now using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) in fibrin (a physiologically relevant extracellular matrix of heart cells). Once the fully gelled tissue is made, the construct is treated with an enzyme that specifically degrades PEG, resulting in a hollow core within the hydrogel structure, releasing the HUVECs into the newly formed cavity through the tissue where they can localize around the walls, thus generating an endothelized vascular chamber. Overall, this platform models the inner walls and lumen of a blood vessel, thereby representing an endothelium. Eventually, to further this project, I intend to integrate this blood vessel within a thick, multilayered engineered heart tissue patch and test its efficiency in providing structural support and useful circulation.

Developmental Language Disorder (DLD) is a hidden neurodevelopmental impairment that impacts an individual’s ability to learn, comprehend, and use language to express their thoughts. While this is a common disorder (about seven times more prevalent than Autism Spectrum Disorder), there is limited research in adults with DLD. The primary goal of this project is to investigate articulatory variability during speech production in adults with DLD. Previous research shows that children with DLD have more articulatory variability during speech production compared to age-matched peers. On the other hand, other studies indicate comparable variability between groups. However, stimuli, kinematic measures, and inclusion criteria vary by study. Here, I will use state-of-the-art vocal tract magnetic resonance imaging (vtMRI) to capture the movements of the entire vocal tract, rather than focusing on just lip movements. Adults with and without DLD will produce sentences in the scanner. Sentences will be linguistically more and less complex. If I find increased articulatory variability in the DLD group during simple and complex sentences, I would interpret that as evidence of a general speech motor control difference. If I find increased articulatory variability only during more complex sentences, I would indicate that increased language load reduces speech motor control in this population. These data can help us build a better model of what might be happening in the brain in adults with DLD, ideally eventually leading to improved diagnoses and treatments for this common disorder.

Glioblastoma Multiforme (GBM) is an aggressive and highly heterogeneous form of brain cancer. Due to the heterogeneity of gene expression in GBM within and across patients, patterns in expression of these genes may provide new insights into targeted drug treatments. In this study, I utilized the predictive computational model built by using the Mechanistic Inference of Node Edge Relationships (MINER) algorithm based on data from The Cancer Genome Atlas (TCGA) and clinical outcomes in order to identify enriched biological pathways that affect GBM patient survival. 3797 gene groups, or regulons, and associated transcription factors were analyzed across the 526 TCGA patients. Of these regulons, the top 10 transcription factors and regulons which were most associated with survival through upregulation or downregulation were identified based on Cox hazard ratios. Additionally, individual gene functions were grouped into higher-level pathways. From there, I created a network map of causal and mechanistic influences to identify known and novel driving mutations of GBM. The resulting network included established GBM-driving mutations such as mutations in TP53 and NF1 genes. Additionally, novel GBM-associated mutations were found, such as mutations in the ATRX, PDGFRA, and CUX2 genes. The results of this exploratory study could be used to identify biological pathways for targeting by candidate chemotherapeutic drugs, incorporating drug interactions into the network map. Furthermore, increased accuracy in GBM prognoses may be achieved by examining regulon activity of newly-diagnosed GBM patients.

The National Archives in Seattle provides access to permanent records created by Federal agencies and courts for Alaska, Idaho, Oregon and Washington in its 56,000 cubic foot warehouse. Researchers, historians, government officials, and the general public consult the facility for many reasons including genealogy, tribal records research, land use, geographic study, and citizen engagement in federal activities. Not only do the archival records hold information from the past, the ways and spaces in which documents have been held form a hidden history that reflects our culture. In 2020, NARA’s Seattle location was announced to be sold. Although community members gathered to successfully prevent the closure, the vulnerability of archival spaces was exposed in this process.Site/Archive/Cite uses mixed reality to intervene into the hidden history of the NARA building, establishing an augmented ghost archive that questions how materiality and meaning shifts when translating materials from a physical to digital space. The art installation examines the relationship between the public, the archival record, and its physical presence in our region. Revitalizing archival materials using new technology allows viewers to explore how we view and store records of the past. This mixed reality experience furthers public conversation regarding the role of digitization in archival access. My role includes researching fabrication techniques to create digital and physical components of the exhibit. I have been building software to allow images of archival materials to appear in the gallery space and be responsive to the viewer's presence within an AR experience. I support finding methods for installing the exhibit and planning components for the larger work including plexiglass with motion sensing lighting. A teaser of our larger exhibit was shown at the Seattle Art Book Fair in May, 2023, and acted as a test “trial” for the larger exhibit being developed.

Injuries to the spinal cord are among the most debilitating injuries to the human body. The task of repairing this dense network of nerve cells and fibers has been seen as insurmountable. However, new techniques emerging from the field of regenerative medicine have illustrated the possibility of encouraging the body to repair these injuries on its own. In the Wills Lab, we study the model organism Xenopus tropicalis, or the Western clawed frog, which has the incredible ability to regenerate its spinal cord and associated tissue following amputation. My project focuses on how Xenopus uses the classic developmental morphogen Sonic Hedgehog (Shh) to recapitulate the dorsal-ventral patterning of the spinal cord during regeneration. Previous research in a closely related organism showed that a switch to non-canonical mode of Shh signaling, not involving the Gli family of transcription factors, was important during regeneration. In order to investigate this further, I used chemical inhibitors of the canonical Shh signaling pathway to confirm this pathway’s limited importance in terms of gross regeneration. Next I used in-situ hybridization to visualize the expression of Shh and its associated genes along a regenerative time-course. It is anticipated that inhibition of the canonical pathway will decrease these expression levels and confirm the selectivity of our inhibitors. An understanding of the role of key biological signals like Shh will be integral in the development of regenerative therapies for spinal cord injury.

For Saccharomyces cerevisiae (S. cerevisiae) to survive, they must either synthesize nitrogenous bases or reside in a nitrogenous base-rich environment. If there is a mutated enzyme along the pathway that makes the nitrogenous base uracil, then the organism would be unable to synthesize uracil and die without uracil. When S. cerevisiae is grown in 5-Fluoroortic Acid (5-FOA) media, the URA3 enzyme will catalyze 5-FOA into a toxic intermediate that causes cell death when incorporated into RNA. In URA3 mutations, the intermediate is not produced, and S. cerevisiae can survive. In an experimental setup where we explored mutation patterns in aging S. cerevisiae cells, we used URA3 to select mutants that grow on 5-FOA media when uracil is provided. However, when analyzing the mutant sequences, there were fewer URA3 mutations than expected. We performed Sanger sequencing on the URA3 gene in individual mutants and were surprised to find that some did not have any mutations. We, therefore, sequenced the whole genome of those mutants and found that they had missense mutations in the URA6 gene. We then analyzed the URA6 locus from the original pool. Across the URA6 gene, the mutations appeared randomly spread with the possibility of some mutation hotspots, indicating that they could be loss of function mutations. In contrast with the URA3 mutants, we observed URA6 mutants were able to grow 5-FOA media when uracil was not provided. There may exist an alternative pathway for URA6 S. cerevisiae mutants to synthesize uracil and survive. To further study this observation, we plan on analyzing the function of the URA6 gene in 5-FOA media compared to the URA3 gene and the components of the URA6 pathway. Ultimately, the result of this study could clarify nitrogenous base production pathways in S. cerevisiae and the impacts of 5-FOA on S. cerevisiae.

mr-DWL is an epoxy-based, negative photoresist that can be used in both the DWL66+ direct write laser system and the Nanoscribe two photon polymerization system. mr-DWL shares several advantageous properties with SU-8 resist, such as high aspect ratio imaging, robust thermal and chemical resistance, and excellent biocompatibility, but has the added advantage of being more sensitive at higher wavelengths. The use of SU-8 at the Washington Nanofabrication Facility (WNF) is problematic, as prototyping with it is expensive and inefficient. Here, we explore mr-DWL as a possible alternative. The properties of SU-8 make it suitable for a variety of technologies including MEMS devices, microfluidics molds, cantilevers, waveguides, and inkjet nozzles. However, it is incompatible with the DWL66+ direct laser write system at the WNF. As such, researchers must prepare a new photolithography mask and perform contact alignment for each design iteration. Repeated mask preparation can render process development slow and expensive if a device’s design changes often. mr-DWL is compatible with maskless direct write lithography, so would substantially lower the time and cost for researchers developing microfabrication-based technologies. In this study, we explore the viability of mr-DWL as an alternative to SU-8 photoresist by spin coating wafers with mr-DWL 5 and 40 to various thicknesses and conducting an exposure series using the WNF’s Heidelberg direct write tool. These wafers can then be measured and characterized by WNF's metrology suite to narrow down the exposure parameters required for film uniformity and feature accuracy.

Design of de novo proteins that can conformationally switch between two defined structural states proves to be promising, as allostery allows control over many metabolic and signaling pathways in nature. My work focuses on computationally designing allosteric protein complexes whose oligomeric state can be controlled by molecular signals. Right now, I am working on designing and characterizing switchable protein ring complexes that can specifically switch between different cyclic symmetries in response to peptide binding. I am also designing homodimeric proteins whose affinity of assembly can be allosterically modulated by the addition of various peptides, both natural and synthetic. I produce these designs through molecular cloning, protein expression, protein purification, and size exclusion chromatography. I structurally characterize them by electron microscopy and functionally characterize their binding affinities and cooperativity through nanoBIT and fluorescence polarization. The ultimate goal of this work is to generate allosteric building blocks that can be used as components in switchable nanocages for targeted drug release, biosensors for peptides, protein logic gates and molecular motors.

Understanding how species of kelp perform under different environmental factors is critical for the management of kelp farming and its effects on carbon sequestration. In this study we evaluated how low salinity and water temperature affect two kelp species: bull kelp (Nereocystis luetkeana) and ribbon kelp (Alaria marginata). Pulse-amplitude modulated (PAM) fluorometry was used as a proxy for health, while wet weight and the hole-punch measurement method determined growth. At Friday Harbor Laboratories (San Juan Island, WA), we collected and exposed ribbon and bull kelp to different treatments under grow lights for a standard photoperiod (12 hours/day) in closed-circulatory sea tables for 72 hours. Our treatments were high temperature (20ºC/ 30PSU), low salinity (12ºC/ 25PSU), high temperature with low salinity (20ºC/ 25PSU), and control (12ºC/ 30PSU). The results of the stress test via PAM fluorometry demonstrated 0% survivability of bull kelp for both heat and the combination of heat and salinity at 24 and 48 hours (Fv/Fm = 0) while ribbon kelp survived. When comparing percent change of wet weights, ribbon kelp gave a p-value of <0.05, with significant differences between combined treatment with control and salinity treatments, whereas bull kelp gave a p-value >0.05, however temperature treatments of bull kelp did not survive (Kruskal-Wallis test and Dunn post-hoc). These results indicate that heat has a more significant effect on kelp than salinity. This data is increasingly relevant as the environmental effects of climate change increase global temperatures and could identify which kelp species are most vulnerable.

Capillary microfluidics devices automate point-of-care diagnostic assays because of their instrument-free operation, small size, and low material cost. However, capillary microfluidics currently require expensive fabrication instruments limiting rapid prototyping and deployment in low-resource settings. State-of-the-art capillary microfluidics are fabricated by using digital light project stereolithography (DLP-SLA) 3D-printers that offer high resolution (40 µm) but are expensive ($10,000 – $20,000). Liquid crystal display (LCD) SLA printers have recently emerged with comparable resolution and much lower cost ($300 – $1000). However, our initial experiments with LCD-SLA printers exhibited defects and post-processing issues. Our goal is to optimize the fabrication process of capillary microfluidics using an inexpensive LCD-SLA printer (Anycubic Photon Mono 6K, ~$400) and calibrate performance relative to a DLP-SLA printer (CADWorks Pr 4K, ~$15,000). By varying printing parameters including UV power, exposure time, layer height, retraction speed, and resin choice, we found optimal conditions that produced microfluidic channels with comparable dimensions on the Anycubic and CADWorks printers (i.e. coefficient of variation <20%). With printer settings of 40% UV Power, 1.8s exposure time, 20 µm layer height, and 0.1 mm/s retraction speed with CADWorks clear resin, the Anycubic 3D printer produced microchannels down to 100 μm, the smallest feature size we achieved with the CADWorks printer. Optimizing 3D-printing of capillary microfluidics using inexpensive LCD-SLA printers like the Anycubic has the potential to enable rapid prototyping of point-of-care diagnostics in low-resource settings. Next steps include printing more complex microfluidic components including domino valves and trigger valves.

Molecular and synthetic biology are rapidly growing fields, with wide applications in medical diagnostics and treatment. These areas of study require iterative experiments, which greatly increases cost, volume of reagents needed, and potential for human error. The field of digital microfluidics, which involves the manipulation of droplets on an electrode board, provides an automated process of carrying out iterative molecular biology experiments to mitigate these issues. We are developing the PurpleDrop, a digital microfluidic device that manipulates droplets via Python 3 scripts that define droplet movements in experimental protocols. The surface on which the droplets move is coated with Teflon, making it hydrophobic and allowing drops to move across without getting stuck. A major problem in the field is called ‘fouling’, which is when droplets that contain protein bind to the hydrophobic surface. The hydrophilic portions of the protein are exposed, making the surface effectively hydrophilic. This prevents droplets from moving across the fouled area and interrupts experiments. I am investigating the idea of a droplet that can remove protein from a fouled area (which we call the ‘Zamboni drop’), similar to a Zamboni machine that cleans and smooths an ice rink. This is a novel idea because preventative methods, rather than reversal methods, make up the majority of published anti-fouling strategies. I have explored several protein denaturants, or chemicals that bind to and break down the structure of proteins, to use as the Zamboni drop. I have demonstrated that guanidinium chloride, a common denaturant, can undo fouling on a Teflon-coated surface, so I am translating these results onto the PurpleDrop and automating the use of the Zamboni drop. Developing a fouling reversal process will allow for running longer and more complex experiments on the PurpleDrop, thereby expanding its capabilities in molecular biology research.

Osteoporosis is characterized by decreased bone mineral density (BMD) and increased bone fragility, putting patients at higher risk for fractures. Osteoporosis has a strong genetic component, as indicated by data suggesting that BMD is 50-85% heritable. The aim of genome-wide association studies (GWAS) is to identify genetic variants common in the population that influence disease-related traits. A prior GWAS identified 56 loci harboring variants associated with BMD, including the XKR9 locus. The XKR9 locus comprises several genes, and the causal gene at the locus is currently unknown. This project focuses on TRAM1, a gene in proximity to XKR9, and TRAM2, the homolog of TRAM1. TRAM1 plays an important role in stabilizing proteins during translocation into the endoplasmic reticulum. Though the function of TRAM2 is not well characterized, it is suggested to play a role in collagen translocation. Currently, no in vivo studies have been published investigating the TRAM genes. The goal of this project is to determine how TRAM genes impact bone morphology in zebrafish. We have isolated zebrafish with germline mutations for tram1, tram2, and a combination of the two. Micro-CT scans were generated at 90 days post fertilization, and ImageJ and FishCuT were used to quantify the impacts on vertebral bone morphology. tram1 mutants exhibited significant changes in tissue mineral density (TMD) while tram2 mutants showed no significant changes in bone morphology or mineralization. Additionally, fish with homozygous mutations in tram1 and heterozygous for tram2 showed no significant changes, however double mutants for both tram1 and tram2 were embryonically lethal. These findings identify overlapping and distinct roles for tram genes in vivo. They also provide evidence that variants at XKR9 could act through TRAM1 to influence BMD thereby introducing translocation as an important factor underlying genetic influence on osteoporosis risk.

North America is experiencing more frequent and intense wildland fires. Most public health research into exposure of wildland fire smoke is retrospective (backward-looking) examining emergency room visits, hospitalization, and emergency medication usage. In 2021 the Theberge Lab enrolled participants (n=64) in a research opportunity to map and study the inflammatory response of wildland smoke exposure. Participants self-draw liquid whole blood for baseline, smoke events, and 3- and 6-month post season follow-ups. A key advantage of this prospective study is the ability to compare participants’ exposure samples and surveys back to their own pre-exposure baselines. Blood samples are taken by the participants in the comfort of their home using technology developed in our lab called the homeRNA kit. The single use kit allows for a small whole blood sample (100-500 μL) to be withdrawn using the Tasso-SST™, stabilized with RNAlater™ , and mailed to our lab for processing and analysis. While providing samples a series of survey questions are answered. As study coordinator, I assembled sample kits, triggered when to send exposure kits by tracking multiple maps for smoke, shipped and received sample packages, and interfaced with participants. After all samples and surveys are completed, I examined the survey answers in relation to self-reported symptoms, self-reported well-being, general usability, and willingness to use the homeRNA kit during a disaster event. This research intends to provide real-time symptom data from exposure to smoke and to test the feasibility of the homeRNA kit. In the future, the homeRNA kit could be deployed for disaster events, research that requires frequent blood samples, and as a mail-in diagnostic tool between clinics and patients.

Despite the widespread success of antiretroviral therapy (ART) in the treatment of HIV, poor adherence to ART can lead to adverse patient outcomes including drug-resistance and treatment failure. Adherence measurement is therefore vital for efficacious HIV treatment. The REverSe TRanscrIptase Chain Termination (RESTRICT) assay measures antiretroviral drug levels based on the inhibition of DNA synthesis by HIV reverse transcriptase (RT) enzyme. RESTRICT currently incorporates a fluorescent output from PicoGreen™ dye, but this readout method requires resource-intensive equipment. We seek to transition RESTRICT to colorimetric readout enabling simpler optical equipment and increased suitability for point-of-care use. We tested leucocrystal violet (LCV) as a colorimetric alternative to fluorescent dye. Colorless LCV reacts with double-stranded DNA (dsDNA) to form crystal violet, producing clear visual output. We performed RESTRICT comparing LCV to PicoGreen™. Positive control reactions containing RT enzyme provide maximum signal while negative controls containing buffers only provide minimum signal. To provide viable results, LCV needed to distinguish between positive and negative controls. While PicoGreen™ fluorescence provided this distinction, LCV did not. To troubleshoot, we compared two DNA templates: one that was optimized for fluorescence RESTRICT, and another that was 10-fold longer, increasing the potential for differentiation between controls. Absorbance readings produced no significant difference between positive and negative controls values using both DNA templates (p-values 0.2236 and 0.3370 respectively). Thus, LCV is incompatible as a method of colorimetric detection through RESTRICT. Further research into alternative dyes is needed to optimize a colorimetric output for RESTRICT.

This study aimed to assess the prevalence of cphA-genes, which encode for a CphA β-metallo lactamase (MBL), in Aeromonas bacteria. Aeromonas isolates investigated were collected from wetland and non-wetland locations within the University of Washington Bothell, where crow fecal matter contamination and runoff from North Creek may contribute Aeromonas spp., among other bacteria. Some of the methods I used involved isolation of Aeromonas by plating fecal samples and water samples (on filters) on Ampicillin-infused blood agar, Meropenem (MERO)-infused LB agar and Cefsulodin-Irgasan-Novobiocin antibiotics (CIN) agar. The 41 collected isolates (26 isolates from fecal samples and 15 isolates from wetland water samples) that tested positive on Meropenem were further tested by Modified Hodge test and Modified Carbapenem Inactivation Method (MCIM) test. Results indicated that two (5%) LB+MERO isolates were cphA-positive, which I tested by PCR with primers directed to the cphA gene. These samples were positive both by the MHT and CphA-PCR. Both the cphA+ isolates originated from stream water samples, and they were collected on two different days, indicating the possibility of an alternative source for the CphA β-lactamase-encoding gene other than crow fecal runoff into North Creek. As MBL-producing species pose a potential health risk to the environment and residential areas in proximity to the river, further investigations must be performed. Future studies will involve testing more fecal and wetland water samples to definitively establish the source of cphA containing Aeromonas.

Despite decades of research very little is known about how mitochondria control stress responses. Therefore, new and innovative models are needed to understand the mechanisms of mitochondrial stress response. We developed a new mouse model of skeletal muscle mitochondrial stress to mimic the aging process in young animals to determine if mitochondrial oxidative stress replicates age-related skeletal muscle and mitochondrial dysfunction. We generated a mouse model to induce skeletal muscle mitochondrial redox stress to mimic skeletal muscle aging by knocking down superoxide dismutase 2 (SOD2). My project was to determine whether this model works in vivo. I fed animals a Doxycycline (DOX) chow diet (0.625g/kg) to induce SOD2 knockdown (KD). After 3-week DOX feeding, tissues were collected and processed for western blotting (WB). WB's were run for SOD2 in gastrocnemius, quadriceps, liver, kidney, heart and brain tissue. Normally, SOD2 is expressed in all tissues that contain mitochondria, so comparing SOD2 expression levels across tissues in KD animals validates tissue specificity. HNE adducts, a marker of oxidative stress, were measured by WB to confirm increases in oxidative stress associated with SOD2 KD. Three-week DOX feeding showed significant decreases in SOD2 protein in gastrocnemius (p<0.001) and quadriceps muscles (p<0.0001) compared to unfed littermate controls of the same genotype. There were no differences in SOD2 protein in heart, brain, liver or kidneys between DOX and control groups. HNE protein adducts were also significantly increased in skeletal muscle of DOX compared to controls (p<0.05). SOD2 is knocked down in skeletal muscle in response to DOX feeding. The increase in HNE adducts confirms that the knockdown of SOD2 causes an increase in oxidative stress. This model can now be used to explore the physiological mechanisms of inducing mitochondrial redox stress in young animals to recapitulate the effects of aging in a controlled manner. 

Risk variants of apolipoprotein L1 gene (APOL1) increase the risk of chronic kidney diseases in populations of African ancestry. We seek to study this disease, which is unique to humans, in human kidney organoids derived from induced pluripotent stem (iPS) cells. However, APOL1 is not expressed in organoids at baseline. While interferon (IFN)-gamma is a potent inducer of APOL1 expression, our prior experiments suggested that IFN-gamma itself disrupts organoid structures, limiting the degree to which the specific effects of APOL1 can be assessed. To improve the kidney organoid system as a better platform to model APOL1-associated nephropathy without IFN-gamma stimulation, I am establishing an APOL1 inducible expression system in kidney organoids. I hypothesize that cell lines with the risk variants will demonstrate an accelerated rate of degradation compared to the non-risk variant, modeling risk variant-dependent cytotoxicity. The inducible expression system will be established by generating iPS cell lines encoding Tet-On system sequences, enabling both tunable and temporal control of APOL1 expression using doxycycline. Plasmids were constructed by PCR amplifying and inserting the targeted sequences into a homology-dependent repair template with a doxycycline-inducible promoter. Ligation had to be done multiple times due to the high prevalence of self-ligation of the backbone vector and backward insert orientation. However, we found that adding alkaline phosphatase to dephosphorylate 5’ ends of the backbone vector significantly improved the integration rate and led to the successful construction of plasmids. Next, CRISPR-Cas9 gene editing will be utilized to introduce the APOL1 gene variants into the AAVS1 safe harbor locus in iPS cell lines, which can be differentiated into kidney organoids. This project aids in isolating the phenotype of APOL1 on human kidney organoids with various cell types, which will be a valuable tool in developing an in-vitro pathophysiological assay such as use in therapeutic drug discovery.

Delirium is an acute disturbance in cognition. It’s a common and serious problem in hospitalized elderly patients, associated with increased morbidity & mortality, prolonged hospital stay, and decreased functional independence. The modified Hospital Elderly Life Program (H.E.L.P) pilot was implemented in Harborview Medical Center (HMC) acute care units in 2022. Through simple activities, sensory enhancements, and cognitive orientations, volunteers helped in preventing and reducing delirium symptoms in hospitalized patients 65 years and older. The purpose of this project is to explore barriers and facilitators to successful volunteer visitation. Trained volunteers attempted visitations using a list of patients from the geriatric medicine team. Reorientation, reading, and reminiscence were conducted. Volunteers completed an electronic survey after each visitation, including questions like length of visitation, and perceived barriers. I conducted quantitative and qualitative analysis of the surveys at the end of the pilot. From June to December, 2022, 375 surveys were collected. 50% of visitations lasted 15-60 minutes, 41% lasted less than 15. Volunteers identified 3 barrier categories: patient’s conditions, like pain/agitation; conflicts with hospital activities such as surgery prep; communication challenges related to patient’s illness, such as speech impediments, and lack of resources, such as no interpreter available for non-English speakers. Perceived top facilitators are the patient's positive attitude, consistent visits, & allowing the patient to lead conversations. One surprising finding is the most prominent patient condition related to identified barriers--daytime sleeping--shouldn’t be a barrier at all. Daytime sleeping disrupts the normal sleep-wake cycle and could contribute to increased delirium. Looking into the future of the H.E.L.P Program at HMC, we should consider more nurse involvement to help with coordination of volunteer visitations. We should increase resources so volunteers are better suited for a variety of situations with patients, to increase confidence and reduce barriers during visits.

Photoreceptor neurons in the retina require increased energy to function in the dark, with their mitochondria undergoing turnover throughout the day and night. Mitochondria change number and gene expression profiles throughout this cycle. Here, I investigated whether levels of mitochondrial RNA (mtRNA) and mitochondrial DNA (mtDNA) fluctuate throughout the day-night cycle in cone cells. To assay this, I used RNAscope probes to conduct in situ hybridization on sections of zebrafish retina collected at different timepoints. This was followed by immunohistochemistry to label mitochondria, confocal imaging, and analysis to quantify levels of mtDNA and mtRNA. I saw a preliminary increase in signal from a probe detecting mtDNA and mtRNA collected at 11PM compared to 9AM. This correlates with an increase in mitochondrial number. These findings support the idea that an increase in mitochondrial number and gene expression at night supplements the increased energy needs of photoreceptors in darkness.

Cyanobacteria are ancient single-celled photosynthetic organisms, prevalent throughout Earth's oceans. Over billions of years, cyanobacteria have evolved genes that enable them to survive across a diversity of adverse, ever-changing environmental conditions. However, researchers are faced with the problem of not understanding the role of many of these genes. This research project entails tracking down the function of some high variance genes in marine Synechococcus, an important model organism and a genus of cyanobacteria. We will test gene function by generating knockout strains in which a gene of interest is inactivated, and testing the growth of these mutant strains in various conditions. In particular, our project focuses on the importance of the flavodoxin gene, which codes for an electron transport protein that is involved in photosynthesis and is expressed in response to iron scarcity. This gene inactivation is done with a plasmid, which is a genetic structure in bacteria that can replicate itself independent of bacterial chromosomal replication, that’s enabled to knock out the flavodoxin I gene when inserted into Synechococcus cells. We insert the plasmid into our Synechococcus cells and once the DNA is taken up by the cell, we then use CRISPR technology to remove the gene. Successfully creating the flavodoxin knockout of Synechococcus establishes the procedures necessary for generating knockouts of other genes that could be expressed in similar patterns as flavodoxin. Ultimately, this research furthers our understanding of how Synechococcus’ genes allow it to adapt to various environments and contributes to ongoing research on how organisms might withstand the pressures of Earth’s ever-changing climate.

We have developed a workflow to integrate the CandyCollect with an at-home rapid antigen testing kit for strep throat (Areta Strep A Swab Test). The CandyCollect is an oral sampling device that utilizes open microfluidic channels to capture pathogens in the user’s saliva. The device is a polystyrene stick that has open microfluidic channels milled into the head and is then oxygen plasma treated to ensure that pathogens adhere to the surface. In addition to this, the device has a strawberry flavored isomalt hard candy that covers the head of the stick to act as a timer to ensure adequate sampling time. The device was originally developed as a noninvasive alternative to throat swabbing for strep throat testing in children. Previous experimentation both in vitro and through multiple human subject studies have shown that the device is effective at capturing pathogens for downstream in-lab analysis. This new iteration of the project focuses on incorporating the CandyCollect into at-home testing. The Areta Strep A Swab Test requires a direct throat swab, which may be uncomfortable for users, especially children. We have done in vitro experimentation with Streptococcus pyogenes to explore if integration with the rapid test was possible. After successfully substituting the kit provided swab with our CandyCollect device while still maintaining the quality of test results, we aimed to improve the workflow to increase the limit of detection. We did this by both increasing the elution time and decreasing the volume of the elution solution. This work expands the potential of the CandyCollect into new diagnostic applications, and further demonstrates the CandyCollect’s functionality as an alternative option to traditional oral sampling methods.

Group B Streptococci (GBS) are gram-positive bacteria that asymptomatically colonizes the vaginal tract of approximately 18% of women worldwide. However, during pregnancy GBS in the lower genital tract can ascend into the uterus and infect the placenta and baby resulting in preterm birth, stillbirth, and neonatal infection. We have used a nonhuman primate (NHP; pigtail macaque, Macaca nemestrina) model to determine differences between GBS strains that confer different levels of invasiveness. The objective of the study was to determine if there were differences in gene expression among animals infected with a “progressive” versus a “localized/resolved” infection. We hypothesized that a greater inflammatory response would be associated with a “progressive” GBS infection compared to the “localized/resolved”. Twenty one NHP received either a choriodecidual inoculation of: 1) 1-3 X 10^8 colony forming units (CFU) of hypervirulent GBSΔcovR (n=15) or, 2) saline (n=6). Cesarean section was performed at preterm labor or 1-3 days after GBS infection or 7 days after saline inoculation. Placental chorioamniotic membranes were sampled near the inoculation site. GBS infections were categorized as “progressive infections”, “localized/resolved infections”, or “resolved” infections at the time of preterm labor or 3 days after GBS inoculation. Next, we prepared mRNA libraries from placental chorioamniotic membranes near the GBS inoculation site, which were sequenced using the NextSeq 550 platform. Data were normalized and then analyzed by Single Gene Analysis, Gene Set Analysis, and Ingenuity Pathway Analysis. The analysis is currently ongoing and will be ready to summarize during the “Revision Window”. Prevention of GBS infection in pregnancy is complex and is likely influenced by multiple factors, including pathogenicity, host factors, and the vaginal microbiome. Understanding mechanisms influencing the invasiveness of GBS infections during pregnancy will facilitate the development of novel therapeutics and vaccines.

In the wake of the Nazi Holocaust, Yizkor, or "memorial" books were written to commemorate and preserve the legacy of many European Jewish communities that were destroyed by Fascist forces. Many of these texts appeared following the Pogroms of the late 19th and early 20th century, but proliferated following the Nazi Holocaust. These texts were written in Hebrew, English, Yiddish, as well as many languages spoken by Holocaust survivors and their descendants. Much of my family originates from a Slovakian town by the name of Sečovce. My Hebrew studies here at the University of Washington allowed me to translate this integral piece of Jewish history and my family's history. Much of my family history is told through this book, including the infamous story of the murder of my great-great-grandparents in chapter eleven. It is with solemn dedication that I have undertaken this project. It is no small task translating the memory of a community so close to many hearts, including my own. This project allowed me to conduct genealogical research, recovery of maps and other archival materials, and exploration of Eastern European custom and cultural values along with translation in an effort to better understand and preserve memory of the past. It is a great privilege to know so much about my family history. Many Jews around the world have lost their ancestral history due to its intentional destruction at the hands of the Nazi regime. And many more histories sadly have no one left to tell them.

Nature uses only four nucleobases to store genetic information in DNA. However, additional synthetic bases which use Watson-Crick pairing have been developed and are known as non-standard bases (NSBs). NSBs P, Z, B and S incorporated alongside standard bases A, G, C and T compose DNA strands using a new genetic alphabet. Nanopores offer the potential capability for direct single-molecule sequencing of DNA containing non-standard bases (NSBs). Using a voltage gradient, DNA strands were directed through a nanopore, the biological membrane protein MspA, while we measured the ion current through the pore over time. In studying the effect of NSBs on the ion current through the pore, we observe current measurements corresponding to the Z base have a different noise profile compared to other bases. We hypothesize this noise may be associated with pH-dependent protonation of the base. To test this hypothesis, we conducted experiments with identical sequences in buffers of pH 8 and pH 7, as Z is known to have a pKa of 7.8. I analyzed the noise from the ion current signals to look for signs of protonation. I found increased current noise values associated with the Z NSB in pH 7 compared to pH 8, while the canonical A base had no change in noise values from pH 7 and pH 8, supporting the hypothesis that the increased current noise is due to protonation of the Z base. In addition to indicating potential sensing abilities of nanopores for probing protonation kinetics of DNA, this research contributes to a better understanding of the fundamental mechanisms that control the currents in nanopore sequencing of DNA.

Adoptive cell therapy with T-cell receptor-engineered T cells (TCR-T cells) has shown promising results in a subset of Acute Myeloid Leukemia (AML) patients. To advance this therapy to a broader patient population, we are developing engineering strategies to enhance the persistence and tumor-targeting ability of TCR-T cells. The interaction between the upregulated Fas ligand in the AML microenvironment and the surface-expressed Fas receptor on T cells initiates negative, apoptotic signaling, leading to T-cell apoptosis. Our laboratory has developed a T cell engineering strategy, Transform Switch Receptors (TSR), which combines the inhibitory Fas ectodomain with a costimulatory signaling endodomain to effectively convert negative signals into positive signals within T cells. We aimed to evaluate the efficacy of different TSRs for the ability to enhance T cell antileukemia activity and exhaustion resistance, by utilizing in vitro killing assays and in vivo survival studies in murine models. The study implements a genetic engineering technique to incorporate the TSRs into T-cells via viral transduction. The efficiency of transduction is quantitatively evaluated using flow cytometry. The modified T cells are then assessed for the ability to eliminate target leukemia cells in vitro, through serial killing assays. TSR-T cells exhibited improved antileukemia efficacy compared to TCR only unmodified T cells in eliminating leukemia cells. The effectiveness of TSR-T cells is substantiated through in vivo assessments in an AML mouse model. TSR-T cells exhibited enhanced capability to eliminate leukemia cells compared to unmodified T cells, resulting in improved T cell activity and survival. Further research may be undertaken to improve the design of TSRs to be effective against solid tumors with hostile microenvironments.

Inherited retinal diseases (IRDs) are a diverse family of disorders which cause vision loss and retinal degeneration. With only 1-2% of the genome being protein-encoding, genetic variation within the expansive noncoding genome is critical to the development of disease phenotypes in the retina. Macular Telangiectasia Type II (MacTel) is an IRD resulting in disruption of central vision and greatly impacting vision-related quality of life. MacTel has an estimated prevalence of 1 in 1000 individuals, affecting approximately two million people globally. Though MacTel etiology largely remains unknown, accumulation of improperly degraded lipids within the retina is a leading hypothesis in its pathogenesis. Additionally, genome-wide association studies have implicated numerous loci in the development of MacTel, including the novel gene locus ceramide synthase 4 (CERS4). As CERS4 plays a critical role in the synthesis of lipid precursors and is highly expressed in the retina, it stands as a promising candidate for influencing MacTel development. We hypothesize that cis-regulatory element (CRE) mutations are central to the genetic frameworks underlying MacTel. We aim to characterize the sufficiency of putative enhancer regions to drive gene expression. We have identified potential CERS4 enhancer regions through a machine learning approach using adult human retina ATAC sequencing datasets. Sufficiency of candidate enhancer regions will be evaluated by insertion to a barcoded reporter library and electroporation into mouse retinas. Following proof of sufficiency, we will perform saturation mutagenesis on identified enhancers to investigate the impact of all possible single nucleotide variants (SNVs) within these regions. The results of our investigation will aid in identifying SNVs of interest within the CERS4 locus, potentially implicating specific mutations towards the development of MacTel. Greater understanding of CRE mutations will improve early clinical diagnosis and inform future therapies for patients with MacTel.

The axion is a hypothetical elementary particle beyond the Standard Model which serves as a candidate for dark matter, a mysterious form of matter that accounts for about 85 percent of total matter in the universe and is difficult to detect by conventional means due to its noninteracting nature with electromagnetic radiation. The ADMX (Axion Dark Matter eXperiment) is an axion haloscope which uses a strong magnetic field to convert axions inside a microwave resonant cavity into microwave photons that could be registered by the detector when the photon signal frequency is the same as the cavity’s resonant frequency. However, within a dataset, the axion signal can be hard to estimate and pinpoint due to the presence of RFI(radio frequency interference).In our research, we developed a new two-parameter statistical model that accounts for RFI appearance and estimates both the axion signal and RFI from a dataset. We show that contrary to the old one-parameter statistical model, which is currently utilized for processing datasets and does not account for RFI appearance, the new two-parameter model yields more accurate and consistent axion signal results regardless of RFI magnitude while the old one-parameter model fails to provide reasonable results at large RFI frequencies. The new two-parameter model would benefit ADMX by providing greater overall accuracy and precision for the search of axion dark matter signals and faster scan speeds.

CD123 is a cell surface protein expressed on neoplastic cells and underlying cancer stem cells in a variety of hematological malignancies, specifically acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin lymphoma, and blastic plasmacytoid dendritic neoplasm, making it a rational drug target for antigen-specific immunotherapeutic approaches. In some cases, however, cell surface CD123 antigen level may not be high enough to elicit a response with an immunotherapeutic, and so we set out to identify mechanisms of regulation of CD123 expression through genome-wide CRISPR screens, and through testing of CD123 expression level changes following treatment of human AML cell lines with clinically validated AML chemotherapeutic drugs. Using commercial anti-CD123 antibodies, I established baseline CD123 expression level on a variety of AML cell lines. I then treated selected cell lines for various times with chemotherapeutic agents, including azacytidine, histone deacetylase (HDAC) inhibitors, the BCL2 inhibitor venetoclax, all-trans retinoic acid (ATRA), doxorubicin, or cytarabine, and measured for altered CD123 level. Utilizing a FACS-based sorting approach on cells that were lentivirally transduced with a genome-wide CRISPR sgRNA Brunello library covering over 19,000 genes, we identified genes that were associated with either increased or decreased expression of CD123. A subset of these CRISPR screen hits were subsequently validated in a small number of AML cell lines using CRISPR/Cas9 sgRNA against specific targets implicated in CD123 expression changes.

High prevalence of antimicrobial resistant (AMR) pathogens is undoubtedly an emergent global health crisis. AMR is exacerbated by factors such as the overuse and misuse of antimicrobial drugs and a changing climate. Overusing antimicrobial drugs causes selective pressure that leads to favorable mutations of bacteria. Through mutations, bacteria acquire mechanisms that interfere with the function and effectiveness of antimicrobial drugs. AMR is a threat to global health because, with ineffective last line of defense antimicrobial drugs, we will be unable to treat the most severe infections. Our research focuses on developing pipelines that detect low abundance antibiotic resistance genes (ARGs) on a local scale. This work contributes to the broader context of AMR on a global scale because applications of AMR surveillance across the globe can inform us about the nature of AMR. In my work, I am examining the implications of seasonality in AMR alleles in Seattle. During the dry season in Seattle from July to September, we may find a higher diversity of AMR genes and in particular, unique alleles of AMR genes. The amount of rainfall influences the concentration of bacteria carrying AMR genes. I hypothesize that higher rainfall typically occurring from October to March will lead to a lower diversity of AMR genes. I am assisting in developing a workflow that uses a two-step, unique molecular identifier (UMI) PCR to enrich AMR genes in wastewater. After amplification, the PCR product undergoes long-read Nanopore sequencing and through bioinformatic analysis, I can identify what AMR alleles are present. By gathering data regarding environmental conditions such as rainfall, and wastewater flow rate, in addition to using our current workflow of amplification PCR and Nanopore sequencing, I can identify what AMR alleles are present in relation to season.

LINE-1 (L1) is the only autonomous transposable element active in human genomes. Due to L1’s ability to insert, delete, and rearrange genetic material; it is associated with more than a hundred diseases like cancers and autoimmune disorders. Our ancestors and present-day humans have developed several defense mechanisms to prevent the spread of L1 and maintain the integrity of our genomes. However, L1 continues to persist and expand within the population despite fitness costs. We think L1s are evading host regulation yet the exact mechanisms are unknown. Restriction factors are proteins that directly counteract L1 replication. Some of them target L1 via protein-protein interaction and we hypothesize that acquisition of mutations in L1 can result in disruption of the interaction and escape. Restriction factor TEX19 (testis-expressed 19) is of interest because: it is expressed in early embryo (same as L1), it is rapidly evolving in mammals, and it restricts L1 in vitro by labeling it's proteins for proteasomal degradation. How and where exactly L1 binds to TEX19 is still unknown. My research aims to determine the specific sequence of amino acids within human L1-ORF1p that is necessary and sufficient for direct interaction with TEX19. I will conduct co-immunoprecipitation and pull-down assays on fragments of L1-ORF1p and TEX19 co-expressed in bacterial/human cells. I expect to identify the region of L1-ORF1p that is responsible for binding to TEX19. Identification of this region would provide significant insight into how TEX19 and L1-ORF1p interact. This data is essential for understanding how L1 has evolved in the past and how it may evolve to evade restriction factors like TEX19 in the future.

Adeno-associated viruses (AAVs) are used for the delivery of genes capable of specific gene expression. There are different types of these AAVs, and their variation stems from their ability to target certain cells. To make a virus target these cells and deliver the desired transgene, a recombinase-dependent gene expression is packaged into the virus. This creates a viral vector, a packaging system that allows for precise, guided expression. These viruses can then be used in experiments to observe any differences in behavioral and genetic effects. Determining the titers from these viruses are important for the comparison of effects between experiments. There are different methods to quantify AAVs, one of which is commonly known as qPCR. This method, however, is tricky to quantify accurately and requires expensive reagents and equipment. By using ethidium bromide-stained agarose gels, we created a rough titer quantification technique easily accessible to other labratories, reducing cost and time required to obtain a titer. In this project, I used serial-two fold dilutions of a standard virus control and viruses of unknown titers. To determine the titer of the viruses, the virus samples were loaded onto a gel and the intensity of the bands were analyzed. A linear regression model based on the virus control was created and the equation was used for the calculation of the titers. The viruses titered from the same company as the control were off by 25% of the established titer and the viruses titered from a second company were off by 80% of the established titer. From this, we believe these results are within company-to-company reproducibility and this technique is accessible to other laboratories.

Leishmaniasis, caused by various species of Leishmania, is a global public health burden, with the World Health Organization reporting over a million cases in the last 5 years, leading to over 20,000 deaths annually. Leishmania and closely related organisms have the unique ability to glycosylate thymidine (Ts) residues to form a base termed Base J. Previous work by the lab has shown that de novo insertion of J is initiated by a protein called JBP2 that is targeted to specific regions of the genome by J2-TDP, a Tudor domain protein. Tudor domains bind to methylated arginine or lysine residues that are most commonly found in histones. To determine if targeting JBP2 to a genome location was sufficient to initiate formation of J, we wanted to target JBP2 to a region of the genome that does not normally contain J. To achieve this, we took advantage of the ability of the tetracycline repressor (TetR) to bind to the operator (TetO) by fusing JBP2 and J2TDP separately to TetR and expressing the proteins in Leishmania harboring a TetO cassette in a locus that does not contain J. I constructed a cassette with the necessary components which contain in order- a drug selectable marker, TetO sites, and a GFP, as a reporter gene to monitor any possible effects J has on the cells when inserted. Using CRISPR/Cas9, I inserted this construct into Leishmania that expressed the TetR fusion proteins. I then grew these strains in the presence or absence of Tet and then determined if J was present at this location. The results of these findings will give us a deeper understanding of the molecular mechanism(s) responsible for this specificity as well as this will offer potential opportunities for development of novel therapeutic agents against Leishmania.

At the site of vascular injury, platelets aggregate, activate, and exert contractile force to form a strong, stable platelet-rich plug. The balance of the blood glycoprotein von Willebrand Factor (VWF) and its protease ADAMTS13 are vital for regulating hemostasis. Under conditions of high shear flow, VWF initially arrests platelets at the wound site, while ADAMTS13 regulates VWF’s activity by cleaving VWF into smaller components, which ensures a thrombus does not occlude a vessel. Clinically, an imbalance of VWF and ADAMTS13 is correlated with an increased risk for thrombosis and/or bleeding. Α high VWF concentration relative to ADAMTS13 activity increases platelet adhesion and promotes large thrombi to form. While it is known how the concentration of these proteins contributes to the size of a platelet-plug, it is unknown how they impact stability and contractile force. If platelet-plug stability and contractile force are unable to overcome larger drag forces due to the size of the plug, thrombi are likely to embolize and travel downstream, which puts a patient at high risk for a stroke or heart attack. My approach is to use a novel microfluidic device that measures the force of platelet-plugs formed under shear flow, in parallel with a protocol I developed to fluorescently label a small percentage of platelets and track their movement. This is research in progress, but I hypothesize that a high VWF:ADAMTS13 ratio will have lower platelet-plug stability and contractile force. The study will explore the mechanical mechanisms by which a high VWF:ADAMTS13 ratio is indicative of thrombosis, leading to future diagnostic and therapeutic techniques.

Chimeric antigen receptor (CAR) T cells are used to treat blood cancers; however, neurotoxicity is a common complication that can be life threatening. The neurotoxicity patients may experience includes language and cognitive disorders, seizures, and cerebral edema and hemorrhage. In our previous research on a mouse model of CAR T cell toxicity, we found that leukocytes plugged 11.9% of brain capillaries in CAR T cell treated mice along with an increase of Intracellular Adhesion Molecule (ICAM-1) on brain capillary endothelial cells. We hypothesized the effects of capillary plugging may contribute to neurotoxicity, hence to better understand the mechanism, I am exploring the effects of different cytokines we observe in patients with neurotoxicity on ICAM-1 expression on human brain microvascular endothelial cells (HBMECs). I treated cultured HBMECs in 10, 100, and 1000 picograms/mL of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, CXCL10, GM-CSF, TNF-α, and IFN-γ for 24 hours. Then, I used flow cytometry to measure the amount of ICAM-1 and VCAM-1 expressed by HBMECs. Vehicle controls are included, and isotype control, single stains, and live dead stains are used for flow cytometry. I am comparing the change in the median brightness of ICAM-1 and VCAM-1 expression on HBMECs in response to each cytokine. Interestingly, we found IL-1β significantly increases ICAM-1 expression even with doses slightly above normal blood levels (2.5pg/mL). There were significant increases with TNF-α and IFN-γ only at doses reflecting highly elevated levels in the blood (1000 pg/mL), whereas there was no response to high doses of IL-2, IL-6, IL-8, and IL-10, CXCL10, and GM-CSF. Further studies blocking cytokines that greatly induced ICAM-1 expression in a mouse model and test if that will reduce neurotoxicity without affecting effectiveness of CAR T treatments would help us understand the underlying mechanisms of what causes neurotoxicity.

Neutrinos are fundamental particles involved in many important universal processes; however, because they only interact via the weak force and gravity, reliably detecting neutrinos directly is notoriously difficult. A new strategy is to study neutrinos through interactions with enhanced cross-section, like coherent elastic neutrino-nucleus scattering (CEvNS), in which the neutrino interacts with the nucleus as a whole (coherent) while conserving kinetic energy (elastic). However, due to the low energy of nuclear recoil in CEvNS, not all nuclei can produce detectable recoil if the recoil energy is on the order of the noise fluctuations in other background radiative processes, as recoil energies become indiscernible. Sodium iodide (NaI) is a candidate for detectable recoil, and I am characterizing the background spectrum of NaI crystals to see if NaI has low enough rates of background processes to be used in CEvNS studies. I analyzed previously collected NaI background spectra to calibrate the event energies and to perform a waveform analysis to distinguish physics pulses from electronics noise. The resulting spectra are used to determine the background rates in the crystals. These routines were converted into scripts to automate the same analysis for future data. Measuring the energy of CEvNS nuclear recoil can help characterize neutrino-quark interactions, which the coherent nature of CEvNS amplifies, providing unprecedented sensitivity to searches for non-standard interactions between neutrinos and matter. Improved characterization of CEvNS also allows for novel checks of predictions made by the Standard Model of particle physics, and has broader applications in understanding supernovae (which produce large quantities of neutrinos) and searches of dark matter candidates that may interact with neutrinos.

The COVID-19 pandemic demonstrated the importance of screening large numbers of individuals for respiratory pathogens. However, existing sample collection methods, including throat and nasal swabs are unpleasant and invasive, especially for younger patients. The CandyCollect device was designed to alleviate these barriers to efficient sample collection by utilizing specially engineered and surface-treated polystyrene lollipop sticks to capture pathogens from saliva. Prior studies by our team demonstrated the CandyCollect’s effectiveness in collecting commensal bacteria in samples kept stable through ambient temperature shipping, which were then eluted from the CandyCollect and detected and quantified with standard quantitative Polymerase Chain Reaction (qPCR) assays. Here we aim to augment this functionality by testing CandyCollect’s effectiveness with viruses in addition to bacteria. In our Institutional Review Board (IRB) approved clinical study STUDY00013842, we recruited 25 participants from the general public nationwide who were presenting symptoms of any respiratory disease, and sent each a testing kit containing CandyCollect devices in addition to a selection of the current gold standard sampling devices: an oral swab, nasal swab, and a spitting tube. Participants self-collected samples using provided devices and returned kits to our lab for qPCR analysis. Analysis revealed the detection of pathogens Streptococcus pneumoniae, respiratory syncytial virus (RSV A), and rhinovirus (the virus causing the common cold). Of the first five participants with positive signals on at least one of the conventional methods studied, all had concurrent positive CandyCollect signals, suggesting that the rate of successful sample retention of the CandyCollect for these common infection vectors is comparable to that of the currently employed sampling methods. Additional samples continue to be tested with qPCR, and further analysis of these results will be conducted. With this initial effectiveness of pathogen detection, we plan to further streamline the CandyCollect device for younger users, and improve efficiency in clinical settings.

Evaluating the risk of developing breast cancer is an important aspect of cancer care as it can allow for more tailored screening strategies and preventative therapies. Clinicians use multiple measures to determine a patient’s risk of developing breast cancer, including breast density on mammography and genetic mutations. Background parenchymal enhancement (BPE) on magnetic resonance imaging (MRI) has shown promise to improve stratification of breast cancer risk in women at high-risk of cancer development. BPE is the increase in signal intensity of normal breast tissue on dynamic contrast-enhanced (DCE) MRI after the administration of contrast agent. Despite BPE having an association with an increased risk of breast cancer development, the biological basis of this increased enhancement is unknown. The aim of this study is to investigate what biologically drives BPE by connecting quantitative MRI measurements with pathological markers from normal breast tissue. Our study cohort includes women that received prophylactic mastectomies and DCE-MRI scans acquired ≤1 year before surgery. From mastectomy specimens, pathological measures of COX-2, VEGF, and Ki-67 are used to measure inflammation, vascular recruitment, and proliferation, respectively. To quantify BPE, I used in-house software to correct pre-contrast images using N4 bias field correction and segment the whole breast. I then applied the breast mask to the pre-contrast MRI and used fuzzy c-means clustering to automatically segment fibroglandular tissue (FGT) from surrounding fat, generating an FGT mask. This mask was then applied to the DCE-MRI series, which includes pre- and post-contrast images, to calculate BPE, which is the mean percent enhancement across FGT. As part of ongoing work, I will obtain more specific measurements in quadrants of the breast from which the pathology specimen was derived. I will then correlate BPE measurements to the pathology measures to determine if any associations exist between BPE and inflammation, vascular recruitment, and proliferation.

Obesity is a condition characterized by excessive fat accumulation, resulting in increased risk for chronic diseases like cardiovascular disease and diabetes. Weight loss can effectively reduce the burden of cardiometabolic risk factors, but weight loss maintenance is difficult to achieve. Mitochondria are key organelles within cells that are responsible for the breakdown of substrates to produce energy. Mitochondrial dysfunction is implicated in obesity, but little is known about the role of mitochondrial dysfunction in weight loss maintenance. Additionally, females are often underrepresented in obesity research, partly attributed to female mice being more resistant to develop obesity compared to males. The aim of this study is to compare mitochondrial function in liver, adipose tissue, and skeletal muscle, following weight loss in female mice with diet-induced obesity (DIO). We hypothesized that obesity would result in a reduction of mitochondrial function across tissues, and weight loss to further reduce it. We provided CB6F1 female mice with a high fat diet, where 87% of them developed DIO. DIO mice were separated into two groups: one underwent 20% caloric restriction for 4 weeks (HFD-CR), and the other group remained on ad libitum high fat diet for the same intervention (HFD-AL). A healthy weight control group was maintained on a regular chow diet. Their weight and food intake were recorded daily. Body composition was assessed twice, before and after the 4-week intervention period. We had an unexpected finding, where mice lost 5-10% of their body weight prior to the intervention period. HFD-AL mice regained lost weight at study endpoint, while the weight of HFD-CR mice remained weight reduced until study endpoint. CR mice had lower adipose tissue mass compared to HFD- AL mice. Future analyses will include comparisons of mitochondrial content and function in different tissues. Findings will provide more insight into the effects of weight maintenance and regain on mitochondrial function.

Obesity is associated with mitochondrial dysfunction. A study previously conducted in our laboratory produced preliminary data suggesting a reduction in T-cell mitochondrial function in subjects with obesity relative to healthy weight controls. The purpose of this present study is to determine whether changes in T-cell mitochondrial function (MITO) reflect MITO changes occurring in liver and skeletal muscle, which are known for having greater influence in glucose homeostasis and energy expenditure. We hypothesize that mice with diet-induced obesity (DIO) will exhibit reduced MITO in T-cells that will correlate to a decline in MITO in liver and skeletal muscle. Adult male C57Bl/6J mice were divided into two groups–the control group was fed a standard chow diet whereas the experimental group was fed a high-fat diet for fifteen weeks. Body weight and food intake were measured every week. Body composition was performed at the endpoint. MITO was measured via high-resolution respirometry in permeabilized liver tissue, skeletal muscle fibers, and splenic T-cells. DIO mice had higher body mass than standard CHOW [52.8g±1.8 vs. 35.1g±2.6] that was explained by an increase in fat mass [19.9g±0.9 vs. 6.1g±1.8] and lean mass [31.5g±1.7 vs. 26.1g±1.1]. We found that differences between DIO and CHOW in mitochondrial leak respiration, maximal oxidative capacity, maximal electron transport chain, and ADP sensitivity were not the same across all tissues. We will proceed to determine which aspects of MITO are correlated between different tissues and assess if variations in respiration are associated with differences in mitochondrial content. An improved understanding of how DIO affects different types of cells regarding oxidative capacity might provide key insights into the development of therapeutics and other preventative approaches to improve immunity and cardiovascular fitness in obesity.

This research project aims to investigate how urban ecological design interventions can contribute to the restoration of the vitality of the Belltown neighborhood in Seattle in a post-Covid world. Specifically, the project will utilize an interdisciplinary framework that bridges the fields of landscape architecture and social science through the lens of urban ecology theory. The research methods that will be utilized in this project include investigation, interpretation, and design projection. Investigation will involve the analysis of the current state of the neighborhood, focusing on revealing the hidden urban ecological systems. Interpretation will involve a critical review of the findings from the investigation and the development of design interventions to address the challenges identified. The hypothesis of this project is that small, incremental design interventions that reveal hidden urban ecological systems will foster creative engagement of users with places and natural processes at play, encouraging the care and stewardship for healthier communities and ecosystems in the long term. To support this hypothesis, the project will conduct a thorough review of existing literature on urban ecology and design interventions, as well as collect data through observations and interviews with community members. The expected results of this project are specific and measurable, including the successful implementation and evaluation of an interactive story map to reveal urban ecological systems at play in the neighborhood, and finding potential locations for proposed design interventions. Ultimately, the project aims to contribute to the broader goal of designing future urban grids using the urban ecology framework to reconcile people with their streets in a post-Covid world.

Phonemic proficiency refers to the ability to manipulate the phonemes, or unit of sounds, in a word. Speech perception deficits has been linked to lower phonemic proficiency, as children have difficulty isolating, manipulating, and blending sounds that they cannot perceive. Autistic children commonly report difficulty perceiving speech in noisy real-world environments where many people are talking at the same time, such as a classroom or playground. Thus, in this study, we investigate the relationship between phonemic proficiency and multitalker speech perception in autistic children. We hypothesized that we would observe a stronger correlation between speech perception and phonemic proficiency in autistic children compared to the neurotypical comparison group. Moreover, we expected that autistic children will have worse multitalker speech perception thresholds overall, suggesting difficulty perceiving speech under complex conditions. We tested 19 7-to-10-year-old autistic children and a comparison group of 19 age- and biological sex-matched neurotypical children. The Phonemic Proficiency subtest of the Weschler Individual Achievement Test – Fourth Edition was administered to assess phonemic awareness. Multitalker speech perception thresholds were obtained under 2 conditions: Co-located Noise and Segregated Noise. In the Co-located Noise condition, the target speaker spoke sentences from 0° azimuth with two additional distracting talkers, referred to as maskers, co-located from the same location. In the Segregated Noise condition, the distracting talkers were spatially segregated to ±90° azimuth, while the target talker remained at 0°. Preliminary analyses suggest that a relationship between phonemic proficiency and multitalker speech perception is observed. These results will advance our understanding of the difficulty autistic children have perceiving speech and have the potential to lay the groundwork for novel assessment and intervention strategies to improve how children with and without autism listen and learn in a noisy classroom. All authors participated in data acquisition, analysis, and preparation of this presentation.

Facile deposition of thick, even photoresist layers remains a challenge in microfabrication facilities. This difficulty stems from the spin-coating process, which often fails to yield uniform films at thicknesses on the order of hundreds of microns. The shortcomings of this deposition method pose a particular challenge to the development of microfluidic devices, which often rely on thick resist layers in their designs. Engineers at the Washington Nanofabrication Facility (WNF) are considering SUEX and ADEX dry film photoresists as an alternative to SU-8, a common epoxy photoresist that is frequently employed in the fabrication of microfluidics. Like most photoresists, SU-8 is supplied in the form of a liquid solution, which must be deposited onto substrates by spin-coating. By contrast, SUEX and ADEX are supplied as films that may be applied to substrates by hot-roll lamination, yielding resist layers with exceptional uniformity at thicknesses up to a millimeter. The lamination process offers several key advantages to spin-coating: less expensive equipment, reduced exposure to solvents, minimal mess and waste, and a shallow learning curve. As an undergraduate research assistant (URA) at WNF, I am incorporating SUEX and ADEX films into double-layered microfluidic device fabrication workflows.  I will determine whether these laminate photoresist films improve the overall process in terms of simplicity, speed, and cost compared to existing methods reliant on SU-8.  This work consists primarily of lamination, photolithography, and various metrology methods such as microscopy and stylus profilometry. If I am successful, the laminate-based process could be utilized by WNF users and staff to ramp up the production and testing of a wide range of technologies.

Monitoring wildflowers in different ecosystems is important for understanding how each species responds to their surroundings. These responses to ecological differences are essential in predicting how they will respond to climate change. This study demonstrates the accessibility of ecological research and bioinformatics in lowlands and mountain contexts. We chose to compare the floral phenology along the trails of Green River College and Mount Si. Consistent monitoring is necessary to understand the changing world. Two fundamental questions arise from such comparative analyses between the different ecological contexts. How do species differ in their response to environmental conditions and which species are unique to each context? We used ArcGis Survey123 to record the flora present each week along GRC and Mt Si trails. This web platform allowed us to efficiently take photos of the species, log abundance, phenological characters (buds, leaves, blooms, or fruit/seeds), and GPS data. This was achieved with a cellular phone while students learned about botany through an immersive education. We then uploaded the information to Qlik, a web tool which creates visualizations of qualitative and quantitative data that are easy to interpret. The differences in elevation of the GRC trails (20m to 120m) and Mt. Si trails (140m to 1,200m) show major changes in the bloom phenology of species present along the elevation gradient. In addition, we compare geospatial data across the growing season. Our research contributes a detailed understanding of individual flora cycles and abundance. The development of an inclusive, educational, citizen/science component for biology classes allows species to be tracked efficiently for plant conservation. Additionally, data collections may be used to build a checklist of flora present in the area to further aid in the conservation and understanding of ecology.

This project examines national identity formation and conceptualization within popular stories in late medieval and early modern England. To do so, I consider a series of chivalric romances and ballads circulating in 14th through 16th century England that center Sir Gawain and/or his interactions with the often mysterious Green Knight. Previous scholarship on Sir Gawain has noted how he often appears in stories about incorporating new territories and people into the imagined England of King Arthur. Building from that scholarship, I find that his interactions with the Green Knight follow that pattern; furthermore, the transformation of their interactions across different stories demonstrates the ways differing ideas of national belonging and exclusion circulated within this medium simultaneously. Depending on the story, the Green Knight shifts from a mysterious figure outside the bounds of English control and understanding to a friendly subject of Arthur’s court who facilitates the conquest and assimilation of other territories in Arthur’s name. Each story presents its idea of England and English history as definitive and stable, but taken together they create a picture of a more complex and dynamic national mythos and identity in the process of being formulated and reformulated. The end result of my project includes both an academic paper targeted at the scholarly conversation on this corpora of texts and a public-facing website designed to introduce others to these texts and to encourage further exploration and thought on the topics of medieval literature, historical inquiry, and the concept of the nation by those currently unfamiliar with them. This combination of public-facing and academic work encourages broader conversation on historical processes of nation-building and imperialism and the stakes of those processes for their modern iterations.

Stress associated with financial instability can manifest as poor decision-making or self-indulgence when making choices that impact health. However, practicing self-compassion can buffer against the influences of stress. Whether self-compassion can reverse previously made unhealthy food choices is unexplored; thus, we are investigating the buffering effects of self-compassion on food choices during times of financial stress to determine if participants will improve their food choices. Additionally, are investigating the underlying role that self-compassion has on compensatory health behaviors. We hypothesize that after we have induced financial stress, participants will have greater preferences for foods with greater health risks (e.i. heavily processed) but after an intervention of a self-compassion exercise, participants will alter their food choices to reflect healthier eating habits, specifically, foods with lesser health risks. Finally, we hypothesize that after the self-compassion exercise, those with higher compensatory health behaviors will prefer foods with greater health risks. Participants will be recruited from an undergraduate psychology pool at the University of Washington Tacoma campus to complete the study online. First, stress will be induced through a writing prompt, recalling a recent state of financial stress. Participants will then be asked to complete a food choice task by using vouchers to choose foods from a list of 20 items. After completing a self-compassion exercise, participants will be then asked if they would like to revise or maintain their decisions from the previous food choice exercise. Finally, participants will complete the compensatory health belief scale and a demographics questionnaire.The hypothesis will be tested using a paired samples t-test and correlation, which indicates a desired sample of 34 (based on a power analysis using p = 0.05, and a power of 0.80). Data collection has begun and sample size will be achieved by March 2023. 

Colorblind ideology is the belief that ignoring or downplaying race leads to racial equality. People who express this ideology are often seen as racially prejudiced, because they are seen as ignoring racism instead of solving it. When Bob says “I don’t think people’s race matters,” for example, others may assume that Bob is ignoring contemporary racism. My research asks: what psychological factors cause this perception, versus the perception that people who endorse colorblind ideology are actively egalitarian? I asked participants to read vignettes and rate their perceptions of the characters therein. Overall, White American characters who express colorblind ideology were indeed more likely to be seen as racially prejudiced than those who made control statements unrelated to race. However, this was not always the case. International students, political conservatives, and those who did not agree that White Americans are generally racially prejudiced were less likely to see colorblind ideology as indicative of racial prejudice. What might account for this? I conducted semi-structured interviews and an online survey and found that some participants (15-20%) interpreted my survey questions in unexpected ways. For example, while I intended for the question “How much do you agree that in general, White Americans are racially prejudiced?” to refer to White Americans’ racism against others, some participants instead thought about White Americans suffering racism themselves. This is an example of just how different people’s interpretations of racial statements can be. I hope my research helps us better understand how people can perceive others’ intentions regarding race, in turn informing how we talk about race. If Bob really is committed to eradicating racism, the first step is to clarify that he is aware of racism and that he wants a society in which race, not racism, does not matter.

In storytelling, mental state terms (MSTs) are words that attribute thoughts, feelings, emotions, and perspectives when describing the internal state of the characters. Although MSTs have been shown to be indicators of literacy and Theory of Mind development among monolingual children, less is known about how bilingual children who were exposed to two languages simultaneously develop MSTs. In this study, we aim to examine the usage and development of MST among Mandarin-English bilingual children by coding the narrative elicited using two wordless-picture books, Frog Goes to Dinner and Frog Where are You? by Mercer Mayer. The data was collected from sixty students enrolled in 1st grade (N = 20), 3rd grade (N = 21), and 5th & 6th grade (N = 19) in a Mandarin-English Dual Immersion program. The research team coded MSTs into three categories (thoughts, desires, and feelings) and examined lexical diversity by analyzing the different meanings of the word "think" in both languages across all three groups. We expect to see more MST usage in older children and also an association between Mandarin and English MST categories. We may also see more lexical diversity in Mandarin compared to English due to cultural and linguistic differences in narrative production. Understanding the trajectory of MST development can further our understanding of how bilingual children acquire languages and the potential cross-linguistic differences between Mandarin and English. This study may also help practitioners in the field of speech-language pathology understand the importance of comprehensive bilingual assessments which measure skills in both languages.

Macular telangiectasia type II is a late-onset retinal degeneration disease which causes loss of central vision and a disruption in cell class proportions in the retina. Genome-wide association studies have identified a point mutation in the 5q14.3 enhancer as associated with MacTel. This enhancer has been shown to regulate the activity of microRNA 9-2. To elucidate the function of this particular enhancer on retinal health and cell class composition, both enhancer knockout and miR9-2 mice models were generated. In adult enhancer knockout mice, there were no significant changes in cell class composition compared with wild-type mice. In the miR9-2 knockout mouse model, it was found that at the 5 week time point, there was a significant increase in müller glial cells. Müller glial cell loss has been observed in Mactel patients, and these cells have been shown to play a crucial role in maintaining proper vascular networks. Future experiments to determine the effects of enhancer and miR9-2 loss on vasculature in the retina would help further identify the role of the 5q14.3 enhancer and its targets on retinal health.

Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) is a protein that modulates cytoskeletal actin dynamics, vascular trafficking and is involved in various signal transduction pathways. Upon phosphorylation by protein kinase C or binding of calcium-calmodulin, MARCKS then translocates from the membrane to cytosol. Previous studies from our lab revealed a supporting role of MARCKS as an HIV-1 Gag proximal protein for HIV-1 production. The exact cellular mechanisms remain unknown. My project was to verify if there is a phenotypic variance between wildtype (WT) and MARCKS-null THP-1 monocytic leukemic cell lines after they are differentiated into macrophages. One of our research scientists performed a phenotypic screening using THP-1 cells and preliminarily found MARCKS-null THP-1 cells differentiated to macrophages using 250nM phorbol 12-myristate 13-acetate (PMA) were unable to de-differentiate back to monocyte as compared to WT THP-1 cells. To further explore THP-1 MARCKS-null phenotype, I generated a MARCKS-null cell line using CRISPR/Cas9 gene editing with delivery via MARCKS guide RNA/CAS9 ribonucleoprotein (RNP) complex by electroporation. The introduced RNP complex caused a double-stranded cut on the MARCKS gene. I generated a THP-1 null cell population which was verified by western blotting for the presence or absence of MARCKS. To evaluate THP-1 null cells, both WT and THP-1 null cells were treated with 250nM PMA for 48 hours. The MARCKS-null cells differentiation resulted in fewer, more dispersed cells and less flattened phenotype as compared to WT THP-1 cells. Thus, my results verified the existence of a phenotype difference, indicating that MARCKS plays a significant role in cellular events during the de-differentiation process. Ultimately, our goal is to further identify the role of MARCKS on THP-1 differentiation. Future experiments will focus on verifying the viability of differentiated cells and examining if the differentiated cells underwent inflammatory cell death or apoptosis. 

One of the most cutting-edge instruments to probe the origins of our universe is the Murchison Widefield Array (MWA), a low-frequency telescope array located in Western Australia. Arrays like the MWA are best equipped to detect light from the Epoch of Reionization (EoR), which started about 400 million years after the big bang. Throughout this epoch many of the universe’s first stars and galaxies began to form, reionizing the surrounding neutral hydrogen gas. Understanding this period of cosmic history is essential because it reveals the properties of the first stars and galaxies and also impacts modern-day astronomy, for example by providing constraints on dark matter and information about present-day star formation. New ultra-wide angle images are being processed, hopefully giving us snapshots of the early universe. For this project, I searched and corrected for near-horizon contamination from astrophysical and anthropogenic radio emissions not caught by our flagging algorithm SSINS. I read processed Healpix data cubes into a jupyter notebook and Fourier transformed the data in two directions (the XX and YY polarizations) so that I am able to pick out radio frequency interference. After doing this for multiple images, I analyzed whether the consistent appearance of interference is significant enough to reevaluate the flagging software we use. By finding and correcting contamination that wasn’t flagged by SSINS, I was able to support the effort to create the world’s deepest reionization power spectrum measurement, the most powerful tool we have to probe the Epoch of Reionization. I hope to find and correct enough images of radio frequency interference so that we can improve the SSINS pipeline.

E2F transcription factor 1 (E2F1) is a significant factor that drives proliferation in castration-resistant prostate cancer (CRPC). Understanding how E2F1 is regulated will give insight into potential targets in E2F1 high CRPC tumors. A previous scientific paper on lung cancer proposed a hypothetical model that Meiotic Nuclear Divisions 1 (MND1) can promote the activity of E2F1, and our lab is interested in if it applies to highly proliferative prostate cancer. RNAseq of two independent CRPC metastasis datasets determined the expression of E2F1, MND1, and KLF6 in androgen receptor positive (AR+) and negative (AR-) CRPC metastasis specimens, as well as the correlation between transcription factors and proliferation. Survival rates of patients with MND1high and MND1low tumors were determined. Additionally, MND1 knockdown using siRNA, verification by qRT-PCR, and viability assays were performed to analyze the impact of silencing MND1 on prostate tumor cell proliferation in vitro. Multiple protein extracts from prostate cancer lines with different antibodies to MND1 were used to assess the expression of MND1, and correlate the level of MND1 transcript knockdown at the protein level in siMND1 treated cells. Our immunohistochemical analysis showed that CRPC is highly proliferative compared to primary prostate cancer. CRPC metastases with high proliferation scores also exhibit relatively high E2F1 and MND1 transcript. Additionally, MND1 expression was associated with shorter survival times in the SU2C cohort. However, silencing MND1 in prostate cancer cells had a limited impact on tumor cell proliferation in vitro. We conclude MND1high patients’ shorter lifespans and their high proliferation score are associated with the high expression of E2F1 and MND1 in CRPC. But while MND1 is associated with proliferation in CRPC, knocking down MND1 expression in prostate cancer cells in vitro had limited impact on cell proliferation suggesting MND1 can promote, but is not vital to E2F1 driving proliferation in CRPC.