Children with uncontrolled asthma are three to six times more likely to be hospitalized. It is necessary for parents and children to collaborate in order to help the child successfully manage their symptoms, yet few solutions exist to support this parent-child shared management. A novel mobile health application was designed and tested with 54 parent-child dyads in an eight-week pilot randomized controlled trial (RCT). The goal was to see if proper planning, communication and using an asthma monitoring watch would improve symptom monitoring in children with asthma. During this study, the watch detected the children’s asthma-related symptoms as they occurred. On a web application, both the parents and the children were asked to collaborate to enter reminders for medicine, summaries for any symptoms, etc. After 8 weeks, the parents and children completed semi-structured interviews where they reported any improvements in their asthma symptoms and general feedback regarding the app. We conducted qualitative analysis by writing codes based on the patterns observed in the user survey responses analyzed. Finally, we compiled our findings into a code book which serves as a guide for upcoming trials. The interview findings revealed that families found the intervention to be helpful for reducing asthma related flare-ups. However, the sync between the watch and the app, integration of the spirometer results with the app, and making the web application interface more kid-friendly are all recommended refinements to consider before the project is scaled for a larger clinical trial.

Skin is a densely innervated sensory organ, populated with various somatosensory receptors that help us perceive touch stimuli. Frequent injuries to the skin cause axon damage and lead to axon degeneration. Degenerating axons leave behind debris that must be cleared before reinnervation can occur and sensation is restored. We use adult zebrafish as a model to study injury and innervation because they have homologous cells and structures to humans and have transparent skin, allowing for high-resolution microscopy. Previous experiments in our lab revealed that skin-resident immune cells known as Langerhans cells (LCs) use highly motile protrusions to engulf axonal debris in the zebrafish epidermis. How are these dynamic protrusions regulated at a molecular level? Calcium signaling regulates phagocytosis and cell motility in other cell types, but the role of calcium signaling in LCs is unstudied. Through scale pluck assays and fluorescent microscopy, I have established a model for monitoring calcium signaling in LCs. I observed transient calcium flashes in LCs that varied in frequency, intensity, and subcellular location. I perturbed calcium flux using a calcium chelator and observed decreased flash intensity and protrusion length in LCs, suggesting that calcium signaling is required for protrusion dynamics. To investigate how calcium signaling affects engulfment, I imaged calcium flux in LCs during phagocytosis of apoptotic cells after laser injury. In contrast to the transient flux normally observed, LCs exhibited sustained high concentrations of cellular calcium during corpse engulfment. Because of the effects of perturbed calcium flux on intracellular calcium and protrusion motility, I hypothesize that perturbing calcium concentrations will inhibit LC phagocytosis. Identifying the molecular mechanisms underlying debris removal, such as calcium signaling in LCs, is relevant to understanding skin repair and disease states in which axon homeostasis is altered, including diabetic and chemotherapy-induced peripheral neuropathies.

Phytophthora is a group of destructive microscopic plant pathogens found in the Kingdom Protista. This water mold poses a substantial threat in the Pacific Northwest, as it infects rhododendron leaves. More Phytophthora are yet to be found here than are currently documented. Understanding the factors that contribute to the diversity of Phytophthora species found in an area is essential in predicting where the spread of this species is most likely. It is hypothesized greater levels of rainfall increase Phytophthora diversity. The sampling location, Chambers Creek in Pierce County, WA, is situated in an area of comparatively moderate rainfall; therefore, about three species are expected to be found. After baiting Chambers Creek with rhododendron leaves, PARP agar was inoculated, then Phytophthora was isolated. DNA was analyzed and bioinformatics was used to identify species present. By understanding the number of Phytophthora species present in local creeks, patterns in WA can be identified to help control this plant pathogen. Our samples identified three species of Phytophthora present in Chambers Creek. This matches our hypothesis that moderate levels of rainfall correspond to moderate species diversity. Based on the specific species found, we suggest to continue monitoring, but no urgent action is needed.

Compared to polymerase chain reaction with nasopharyngeal swab specimens, lateral flow assays (LFA) for detecting SARS-CoV-2 antigen in saliva are rapid, inexpensive, simple-to-use, and instrument-free, but have low sensitivity (ca. 11-40%) due to limited LFA sample capacities (ca. 50µL) and preparatory saliva sample dilution. To address this challenge, I developed an osmotic processor, a 3D-printed device, to concentrate target analytes through static and spontaneous osmosis for improving salivary-based COVID-19 LFA. The specimens loaded into the device were separated from an aqueous polymer solution using a dialysis membrane. The polymer solution with high mass polymer concentration resulted in an osmotic pressure difference that caused water transport from the saliva to the polymer solution, while the target analyte – SARS-CoV-2 nucleocapsid (N) protein – remained within the membrane. As the final solution contained the same N protein amount in a lower liquid volume, the overall sample was concentrated, thus improving the antigen detection limit ca. 60-fold, from 62.5 to 1 pg/mL. Combining the osmotic processor and saliva based LFA enables rapid, sensitive, simple, and inexpensive POC testing.

Phytophthora is a genus of oomycete water molds that harms plants. They are a group of fungi-like microbes responsible for several plant diseases, including blight, canker, dieback, and root rot. Even though approximately 30 species have been identified in Oregon and Alaska, the number of species currently present in Washington is yet to be determined. Our research focuses on the diversity of Phytophthora species. First, in an analysis of previously collected data and US Census data, the Phytophthora species diversity is higher in counties with a higher human population. Based on this analysis, we expected to find more than three species of Phytophthora present in Chambers Creek (our sample site) due to the high human population in Pierce County. Secondly, we sampled Chambers Creek for nine days using Rhododendron leaves, incubated the leaves for two days, then performed a PCR. The PCR product was sequenced using Sanger sequencing and a BLAST was used to identify the species present in our sample. Ultimately, we identified four species of the harmful plant pathogen Phytophthora present in Chambers Creek. From this, we conclude that there is a high diversity of Phytophthora present in this Pierce County water source. Phytophthora can be extremely harmful to trees and crops, and it has caused mass famines in the past. This research is vital because understanding patterns of its diversity allows us to monitor its presence locally; should a particularly harmful species be found, we can work to mitigate its effect on our ecosystems.

As diseases like COVID-19 become endemic, it becomes more apparent that access to low-cost, user-conducted tests with high sensitivity and rapid results are necessary to help reduce the spread of disease and mitigate burden on healthcare and laboratory infrastructure. While paper-based colorimetric tests attempt to fill this gap, they have reduced sensitivity when compared to “gold-standard” tests such as RT-qPCR, which typically exhibit results with fluorescent reporters. The goal of this project is to detect fluorophore activity using a smartphone camera and flash, with zero modifications (or as minimal as possible). As many people have smartphones and the ability to take a picture, but fewer possess lab skills or access to a lab, we aim to develop a smartphone-based system with the highest sensitivity and lowest barrier for entry, that is capable of detecting a fluorescent output. We are experimenting with both biological and technological levers, including combinations of time-delay using FRET and long-lasting fluorophores. On the software side, we are looking into whether we can leverage a smartphone’s built-in bayer filter to better delineate between emission wavelengths of fluorophores, and using timed flashing and recording methods to detect the biological time delay. So far, we have collected preliminary data on colorimetric readout reactions and shown that the difference between reactant and negative control is apparent at fairly low concentrations (250 uM). We expect that with a simple filter setup, we will be able to excite and detect the fluorescent output from a fluorophore. Further research will aim to simply things setup further, to reduce the number of external modifications required for use. When coupled with a diagnostic test, these ideas could potentially bring any test that can be coupled to a fluorescent readout from the lab to the user, increasing accessibility and lowering the costs of such tests.

 Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that often results in deficits in communication, social skills, and emotion regulation. Additional concerns include disruptions to the sleep wake cycle that results from circadian rhythm dysfunction. 40% of individuals with ASD also have clinically significant anxiety, which tends to exacerbate pre-existing behavioral issues and social deficits. Previous studies suggest an association between increased sleep dysfunction and increased issues with anxiety in typically developing (TD) adults, and have insinuated a possible bidirectional relationship between the two. This study aims to look at the relationship between sleep quality and anxiety in adults with and without ASD. 89 adults (ASD=39) from a longitudinal five-site NIH-funded study on sex differences in autism were included. Participants completed the Munich Chronotype Questionnaire (MCTQ), a measure of the amount of sleep, based on sleep-wake times, and the Screen for Adult/Child Anxiety Related Disorders for a measure of generalized anxiety. Analysis will include (1) independent sample t-tests to examine group differences in anxiety and sleep duration and (2) correlations between sleep duration (MCTQ) and generalized anxiety subscore from the SCAARED measure for the group with ASD and the typically developing group. I hypothesize that individuals with ASD compared to TD will demonstrate higher anxiety scores and worse sleep quality. I also hypothesize that there will be a correlation between higher anxiety scores and shorter sleep duration. Additionally we will explore sex differences in anxiety and sleep. If sleep quality is related to anxiety, this might support the increased use of sleep behavioral interventions to improve mental health in individuals with autism spectrum disorders.

The functional connectivity of the brain evolves throughout the life of every individual. These changes, often referred to as neuroplasticity, can be impacted by a wide range of variables from diseases to eating a favorite dessert. How can these changes be modulated to treat neurological diseases and disorders such as post traumatic stress and major depressive disorder? My colleagues and I are intrigued with the prospects of neuromodulation as a therapeutic for abnormal brain connectivity and network dynamics, leading me to the question “At what rate do these connections accumulate and decay with optogenetic modulation; Optogenetics, a technique that uses light to activate or inhibit genetically targeted neurons, offers high cell-specificity and temporal resolution that allows us to zoom into the network dynamics and find more finely tuned results that can help in the development of neuromodulation therapies. I plan to use both single site and paired-pulse optogenetic inhibition to gain a clearer understanding of how functional connectivity behaves during and after repeated modulation periods followed by extended recordings of spontaneous activity with no modulation. By analyzing the pairwise coherence of the local field potentials collected using electrocorticographic recordings in non-human primates, I anticipate seeing targeted changes in functional connectivity when comparing before and after each inhibition session. By analyzing the rate of change in connectivity I plan to understand the timeline of neuroplasticity following optogenetic modulation, thus informing the development of future neuromodulation therapies. This research could have a profound impact on the future therapeutic paradigms for neurological and neuropsychiatric disorders that can accelerate recovery for individuals with these conditions.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder that is characterized by difficulties in social communication skills, and repetitive restricted behaviors. ASD has been associated with alexithymia, or difficulties processing one’s and/or own emotions. Prior research suggests, individuals with alexithymia are at increased risk of developing disordered eating patterns, particularly if they are male (Shank, 2019; Larsen, 2006). Disordered eating tends to be associated with other psychopathology and possible to be comorbid with ASD. Individuals with ASD are also at increased risk of developing disordered eating (Huke, 2013), however it is unknown if the relationship between alexithymia and disordered eating is also found in autistic individuals. Thus, the aim of this study is to investigate sex differences in alexithymia and disordered eating patterns in individuals with and without ASD. 108 adults (ASD n=58, Male n=35) were included; all participants had an IQ ≥70. Alexithymia was measured using the Toronto Alexithymia Scale which is a 20-item instrument describing feelings, identifying feelings, and externally oriented thinking. Scores ≥61 indicate alexithymia . Eating behavior was measured using the Adult Behavior Eating Questionnaire which is a 35-item instrument involving 8 subscales including hunger, food responsiveness, emotional overeating, enjoyment of food, satiety responsiveness, emotional undereating, food fussiness, and slowness in eating. We expect to see a correlation between alexithymia and eating behavior in the autistic group, similar to what has been previously found in non-autistic groups. We also hypothesize that this correlation will be present for autistic males but not autistic females. This study will contribute to better understanding if alexithymia and eating behavior patterns are found in individuals with ASD, and whether or not sex mediates this relationship. If this relationship were true, this may affect how interventions for ASD are administered and label alexithymia as a risk factor for disordered eating.

To navigate their surroundings, migratory cells respond constantly to many signals in their environment, including both chemical and electric cues. These signals are produced locally by other cells, pathogens, and in the context of electrical signals, by disruption to the normal ionic balance across cell boundaries. Disruption to this ionic balance will create a local electric field to which immune cells will respond to guide their movement and prevent infection. How cells sense or respond to this electrical cue is not known. To better understand this phenomenon, we are using HL-60 cells that are a migratory neutrophil-like human leukemia cell line, which we have found migrates to the cathodal pole of an applied DC electric field. We have identified a number of gene candidates related to glycosylation, the modification of proteins with the addition of sugar molecules, that reduce the directionality of HL-60 cells in an electric field. Using CRISPR interference to create cell lines with reduced expression (knockdown) for eleven of the gene candidates, we are studying how the loss of these genes alter migration. We used video microscopy to track their migration in 3D at different intensity levels of current to see how the loss of these genes affected cell movement when cells are exposed to an electric field. All of these knockdown lines showed marked change in the cell's response, with less persistence towards the cathode at higher currents than control HL-60 cells. Of these eleven, knockdown of UXS1, a gene that encodes for UDP-xylose that is used in the attachment of long sugar chains (glycosaminoglycans) to certain proteins on the cell surface, showed the greatest effect. Our results suggest that UXS1 is critical for neutrophils' ability to sense or respond to DC electric fields. 

With respiratory diseases, namely COVID, becoming exceedingly present, new diagnostic techniques are important in delivering quick and accurate results to patients. With the broad aim of creating a COVID breath test diagnostic, the project focuses on building a classifier able to detect various volatile organic compounds (VOCs). I applied a panel of VOCs to the antennae of the moth Manduca sexta and recorded the change in voltage across the antenna over time, also known as an electroantennogram (EAG). After recording the voltage response it is essential to extract important features associated with the response to avoid overfitting in the classifier. Each odor will have a unique dose-response curve. By identifying the pattern of intensity-related response for each odor, I have extracted important information that can be useful in classifying EAGs. Going forward, this project will be scaled to include COVID-related VOCs and multichannel experiments to measure electrical response in multiple areas of the antenna simultaneously. Multi-channel recordings will provide an increased number of important features for the classifier to use in learning the unique voltage signature related to each odor in our panel.
Preliminary data has shown that the administration of floral odors to the antenna elicits unique voltage signatures when recorded in single-channel EAGs. I predict that if we record from two sites in the antenna, we will observe a set of unique dose-response curves relative to the differential expression of olfactory sensors in the base and the tip of the antenna. These sets of dose-response curves may provide our classifier with better parameters to categorize and detect the presence of odors associated with COVID, as two dose-response curves must be consistent with an assigned odor’s electrical signature instead of only one. This may improve our chances of creating a classifier able to reliably differentiate between COVID and non-COVID odors.

Conjugative plasmids are extrachromosomal genetic elements commonly found in bacteria and are capable of being shuttled between different bacterial cells via a process called conjugation. The Luria-Delbruck Method (LDM) is a stochastic based modeling framework used to estimate the conjugation rate of a particular plasmid between bacterial strains. In my experiment, I am probing the theoretical experimental assumptions made by the LDM; in particular, I am testing that when there is variation in the precision of the selection assay, the application of a theoretical correction can result in accurate quantification of the conjugation rate. In the early phases of the experiment, I performed experimental assays in liquid medium to determine antibiotic concentrations in which donors (strains hosting a conjugative plasmid) and recipients (strains able to receive plasmids from donors via conjugation) die and transconjugants (recipient cells that have received plasmid from donor) grow. To test the theoretical correction, I chose two antibiotic concentrations that differ in the amounts of extinction occurring in the transconjugant population. I executed the LDM conjugation assay with these two conditions which produced equivalent conjugation rate estimates, as would be predicted if the correction factor is effective at mitigating the bias produced from variable amounts of transconjugant extinction in the selective conditions. My experiments demonstrate that the LDM continues to be robust in the face of violations to experimental assumptions which affirms the viability of applying the method to a wider range of bacterial populations with variable selective conditions and thereby broadens our ability to understand the dynamic movement of conjugative plasmids.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by social communicative impairments and sensory sensitivities. Additionally, the physical and social changes that occur with puberty may be a turbulent time for adolescents. Earlier pubertal timing has been correlated with higher internalizing mental health symptoms for neurotypical girls with earlier onset of menarche in females being tied with higher rates of depression that persists into adulthood. This study investigates the relation between pubertal timing and internalizing mental health problems for autistic and non-autistic female adolescents in a longitudinal study. 23 female ASD participants (ages 8 to 17) and 42 female neurotypical participants (ages 8 to 17) from a NIH-funded project investigating sex and gender differences in individuals with autism are included. Participant data was collected at a second timepoint, 3 to 8 years later. Data on pubertal development was collected using the Pubertal Development Scale, a parent or self-report measure of physical development. Depression and anxiety were assessed using the Child Behavior Check List, a parent-report behavioral checklist of mental health symptoms at the first time point, and a self-report version of the CBCL at the second time point. First, we examine pubertal timing variation by calculating residuals of a pubertal maturation by time regression plot. Second, we will investigate the relationship between puberty timing and depression and anxiety using a correlation test. To further analyze this relationship between pubertal timing predicting future depression and anxiety, we will run a multiple regression test. I predict that the relationship between pubertal timing and depression and anxiety will be greater for autistic girls than for neurotypical girls. This study’s data can add a neurodiverse perspective on how pubertal timing impacts mental health in females and could provide evidence for the need of interventions and additional support to adolescent females with ASD.

The SirT6 gene is protective against systemic aging by enhancing DNA repair, decreasing inflammation and epigenetic deacetylation, and maintaining efficient lipid and glucose metabolism. Studies have shown that SirT6 function gradually decreases with increasing age with a loss in protective mechanisms. The role of SirT6 in brain aging has not been well established, and would be of interest to determine possible therapeutic targeting for conditions of age-related neurological dysfunction including cognitive impairment and dementia associated with Alzheimer’s disease. A mouse line was obtained from the NIH National Mouse Repository with genetic inactivation of one allele of the SirT6 gene (SirT6-/+) resulting in a partial loss of function. Older aged mice were tested in a spatial navigation learning task for exploratory behavior and memory. SirT6-/+ mice were unable to find escape holes as quickly as unaltered littermates (SirT6+/+) with a P-value of 0.003 significance. This observation suggests that partial absence of SirT6 function is associated with cognitive impairment. Following cognitive testing, mice were humanely euthanized, and brains collected and formalin fixed for immunohistochemistry staining to identify specific biomarkers of aging pathways including gamma H2AX for DNA damage response, HDAC-2 for epigenetic alterations, MCP-1 for inflammation, nitrotyrosine for oxidative stress, and p21 for senescence. Stains will be digitally imaged and heat maps generated for quantitative analysis of specific areas of brains from SirT6-/+ and SirT6+/+ mice. Results are expected to provide new knowledge on the role of aging pathways in the downregulation of SirT6 and age-related decline of learning and memory, and insight into possible conditions for development of more severe neurodegenerative diseases associated with dementia such as Alzheimer’s disease.

Using simple behavioral analysis (SimBA) and Deep Lab Cut (DLC), we can create predictive behavior classifiers using pose estimation (PE) data obtained through DLC. PE is a computerized technique to track and predict the location of mice by training the video dataset with labeled frames using specific regions of interest (ROIs). With this, we can create machine-learning (ML) predictive classifiers of complex social behavior in SimBA. Social behaviors and interactions are difficult to manually track due to their rapid successions. To overcome this, I plan to use ML classification using our SimBA pipeline for behavioral classification allowing us to exceed human performance and increase throughput and consistency. I plan to create accurate classifiers for social behaviors that I will use to analyze the behavioral motifs of mice undergoing an operant social stress procedure. First, we train male and female C57BL/6J mice to self-administer (SA) their same sex cage mate. Experimental mice are then subjected to either physical stress for males or witness stress for females. Following social stress, non-reinforced SA is used to assess social reward seeking. Next, social interaction (SI) tests are performed to document time spent approaching the familiar same-sex conspecific cage mates and the aggressive CD-1 mice. All behavior was recorded, and transferred to DLC, followed by frame extraction. Using these frames, we trained the operant behavioral dataset to track the orientation of the mice. Next, we evaluate the dataset for a low error margin as observed by a continuous plateau of iteration loss. Although not complete, I expect to create behavioral classifiers for mice during social decision making in a social reward context following social stress inclusive of sex differences. Providing descriptive statistics of both movement and probability of successive behaviors as they occur in real-time.

Biological age generally occurs earlier or later than chronological age and can be used as a biomarker for aging intervention studies. The rate of wound healing is known to decrease with increasing age, such that a simple skin wound might demonstrate overall aging of all organs and tissues of the body. This concept was tested in a mouse model of wound healing consisting of a 2 mm through and through ear biopsy. Cohorts of male and female C57BL/6 mice at 5, 13, and 21 months of age were biopsied, and the area of closure measured over 3 weeks. Ear biopsy cores were used to obtain DNA methylation signatures to a database of age-related signatures to compare biological age. The 5-month-old mice showed a higher percentage of wound healing than the 21-month-old mice 2.5 weeks following the biopsy with a p-value of 0.04. DNA methylation results used to calculate expected wound healing percentage correlated with measured healing percentage with an R2 value of 0.82. Additional validation of biological age will use a computational paradigm to assess severity of age-related lesions on an inter and intra-organ basis. Preliminary observations from this study indicate that a simple skin biopsy procedure can be used to predict biological age, with important translational implications for the treatment and prevention of aging and age-related diseases.

Alzheimer’s disease (AD) is a complex neurodegenerative condition that commonly affects aged populations; therefore, increasing resilience to aging may increase resilience to age-related diseases including AD. To test this concept, a preclinical mouse study was designed to investigate the effects of anti-aging drugs rapamycin, acarbose, and phenylbutyrate in combination (cocktail) previously shown to delay aging phenotypes in mice. Cohorts of C57BL/6 mice, 22 months of age and of both sexes, were fed standard chow containing the drug cocktail or standard control chow for two months. Mice were then given an intravenous injection of a neuronal-specific adeno-associated virus (AAV) vector consisting of Aβ and phosphorylated (p) tau, the major pathogenic components of AD, or AAV sham, and followed for another three months. Mice were then tested for cognitive function using a spatial navigation learning task. Overall, AD mice treated with the drug cocktail showed faster learning times, specifically in trial 2 for females (p<0.05) and trial 3 for males (p<0.05), compared to AD mice not receiving the drug cocktail. Following humane euthanasia, brain tissues will be assessed for Aβ and ptau neuropathology and pathways of aging using a combination of special stains and digital imaging to determine the cellular and molecular effects of the drug cocktail. The preliminary observation that the drug cocktail can prevent cognitive impairment associated with the early stages of AD suggests that targeting resilience to aging has potential translational implications for the early diagnosis and treatment of AD in human patients. Future efforts will work to establish molecular evidence of these observations and validate results in additional models of AD.

Ovarian cancer is the deadliest gynecological malignancy with an estimated 13,270 women in the United States to die from it this year. Because of its nonspecific symptoms, it is often diagnosed at advanced stages, resulting in lower survival rates. The majority of current in vitro models for ovarian cancer use established cancer cell lines that lack clinical and translational relevance due to their inability to capture the high inter- and intra-tumor heterogeneity. To study tumor heterogeneity and cancer initiation, induced pluripotent stem cells (iPSC) have emerged as a new powerful tool that are both amenable to gene editing and differentiation into different cell types. One group has previously reported the generation of fallopian tube organoids from human iPSC. However, the generated organoids lack the characteristic tube-like structure as well as components of the microenvironment that removes the possibility to address structural factors and the functional role of the extracellular matrix. Bioengineered devices can be used for geometric cues and controlled release of biomolecules that can guide spatiotemporal cell and tissue organization. This project focuses on establishing a model by seeding iPSC-derived fallopian tube epithelium into lumenized microfluidic devices for further study on cancer initiation and drug screening. To confirm the generation of relevant cell types, I will benchmark the iPSCs at different time points throughout the differentiation protocol with qPCR and immunostainings. We expect results to show specialization of iPSCs towards the fallopian tube epithelium lineage and their further maturation (cilia beating and mucus secretion) once incorporated in the designed device. Once a model is established, further work can be done on elucidating factors such as substrate curvature, genetic mutations, and infections on tumorigenesis.

Substance use disorder (SUD) can be defined as the misuse of pharmaceuticals, illegal drugs, and alcohol, and suggested to be due to the co-opting of existing pathways in the brain controlling natural reward. The endogenous opioid dynorphin (dyn), signaling via the kappa opioid receptor (KOR), has shown great promise in being targeted as an effective treatment strategy for SUDs. However, the role for dyn-KOR signaling in natural reward seeking and specifically, the location of its action in the brain are unknown; information that is critical in informing intervention times for treatment. The Dorsomedial Striatum (DMS) is an area of the brain where dynorphin is ample, and it’s crucial for reward-seeking behavior. However, what dyn is doing to influence these behaviors is unknown. To study natural reward-seeking, we set up an operant behavior task where the mice learn to nosepoke (seeking) into an “active” port for sucrose (reward), whereas nosepoking into an “inactive” port yields nothing. While wild-type mice learn this behavior, we observe that mice lacking DMS dyn are slower and do not seek as many rewards upon learning. This suggests that DMS dyn is necessary for reward-seeking behavior. To determine if the converse is true, we artificially boosted DMS dyn release using in vivo optogenetics. We show that when we stimulated dyn release during reward delivery, mice enhance their seeking behavior. Hence, we hypothesized that dyn may help shape the value of a reward, thereby impacting seeking. We designed a reward devaluation task, where animals are given free access to the reward prior to a session of reward-seeking, to decrease/devalue their seeking. We observe that wild-type animals decrease their responding, but animals lacking DMS dyn don’t devalue their seeking. Altogether, we reveal that dyn transmission in the DMS during reward shapes seeking, thereby enabling value-guided flexibility of reward-seeking behavior.

Autism Spectrum Disorders (ASD) refer to neurodevelopmental difficulties in communication and social interaction. It is thought that 94% of autistic adults have used camouflaging behaviors at some point in their lives, meaning that they have developed certain behaviors to blend in the social world and to “hide” their autistic differences. Camouflaging behaviors include: masking - hiding the autistic features; compensation - practicing certain behaviors to compensate for certain social shortcomings; and assimilation - trying to fit in so they are not singled out (Hull et al., 2018). We are interested in the relationship between camouflaging behaviors and social communication in individuals with and without autism. Data from 85 participants (42 ASD, 48 females) ranging from 15 to 23 years old from the NIH funded study on sex and differences in autism were included in the analysis. Autism diagnosis was confirmed via standardized tests and all participants had an IQ of 70 or higher. Participants completed the Camouflaging Autistic Traits Questionnaire (CAT-Q), a 25-item questionnaire that tests the degree of using camouflaging strategies, and Vineland Adaptive Behavior Scales, a parental interview that informs the diagnosis of intellectual and developmental disabilities. We predict significantly higher camouflaging behaviors and lower socialization skills in the autistic group compared to the non-autistic group. We predict a positive correlation between CAT-Q scores and Vineland socialization scores in the autistic group, since by resembling their peers will make their parents report better social skills. We also predict that the correlation between masking and social skills will be higher in females than males in both groups, as females are found to have higher social motivations (Cook, Ogden, & Winstone, 2018). Camouflaging may prevent others from recognizing the symptoms of autism and fail to get diagnosis. Therefore, it is important to detect camouflaging behaviors so autistic children get timely treatments.

Organic mixed ionic electronic conductors (OMIECs) are a class of semiconducting compounds that have recently sparked major interest due to their unique ability to conduct both ions and electrons when electrochemically doped. This unique property of OMIECs make them great contenders for applications in biosensing and neuromorphic computing, since they are soft organic materials that can change their state of conductivity. The rate at which these polymers can undergo redox reactions is known as the polymer’s doping kinetics, which is an important parameter for understanding these materials. In this work, I measured the doping kinetics of the commercially available polymer, poly(3-hexylthiophene-2,5-diyl) (P3HT), using UV-Vis spectroelectrochemistry to measure the electrochemical oxidation rate in solutions of different anionic species and solution concentrations, as well as varying the film thickness. I predicted that anionic species of larger sizes, at higher concentrations, results in faster doping kinetics of P3HT. I also expect to see faster doping in thinner films of P3HT, when compared to thicker films. My results show that the choice and concentration of the electrolyte plays a large part on the kinetics of electrochemical doping. I showed that using electrolytes that have larger anions were able to generate a faster doping kinetics. Increasing the electrolyte concentrations also increased the kinetics of doping the polymer. I also found that the thickness of the polymer film, when decreased, resulted in a faster doping kinetics. Using P3HT as a model system, I have examined the effects of anion, electrolyte concentration, and polymer film thickness, which are important parameters to understanding the factors that go into making these materials good conductors for a range of applications.

Every winter, those residing within the eastern half of the U.S. are slammed by powerful storms that pound cities with snow – costing millions of dollars in damage, halting travel, and impacting businesses and schools. Yet, like a thumbprint, each of these winter storms are unique and they can possess a range of tracks, structures, and intensities. This research project aims to provide a greater understanding of the development of such storms and the causes of precipitation variability within them by focusing on a single storm that hit the Midwest on 17 February 2022 as a part of a research flight conducted during the Investigation of Microphysics and Precipitation for Atlantic Coast-Threatening Snowstorms (IMPACTS). I analyzed data from multiple sources to explore the large scale environmental conditions and the fine scale precipitation structure. The environmental conditions included a strong thermal contrast, or a frontal boundary, that provided the lift needed for precipitation as well as sub-freezing surface temperatures that allowed for precipitation to fall as snow. Analysis of the vertical cloud and precipitation structure from radar data collected during the flight revealed regions of higher reflectivity where snow was heavier than in other portions of the storm. Microphysical properties, such as particle sizes, shapes, and ice water content were different within the region of high reflectivity than outside it. Additionally, the strengthening front at the 700-hPa pressure level coincided with the localized region of enhanced reflectivity observed by the ground-based and airborne radars. The high-resolution radar and microphysics data collected by the IMPACTS airborne instruments are used to help identify precipitation-defining processes within these storms, and ultimately will increase the accuracy of snow prediction and remote sensing of snowfall from space-borne instruments.

Climate change has resulted in environmental changes that pose direct and indirect challenges to marine organisms. One such organism that has been widely studied is the common murre (Uria aalge), which has experienced decreased reproductive success and adult survival in response to climate change-induced ocean warming. Because common murres molt flight feathers relatively synchronously, they may be especially vulnerable to environmental stresses, such as the challenge this may pose on obtaining food. In this study, we explore the degree to which shifting environmental conditions impact adult survival of common murres at a physiologically sensitive point: flight feather molt. The data we used for this study include: monthly expert-verified observations of bird carcasses from the Coastal Observation and Seabird Survey Team (COASST) citizen science program collected from the outer coast of Northern Washington south down to Humboldt, California; significant wave height data from the National Data Buoy Center (as a proxy for storminess); spring transition date (from the Columbia Basin Research website) as an indication of annual production potential; and Bakun upwelling index (from the Pacific Fisheries Environmental Laboratory website) as a measure of upwelling strength and production potential. We annualized both the bird and environmental data over the years 2003-2021. For this project, my role was to conduct statistical analyses using the statistical program R. I employed generalized additive mixed models to determine the relationship between the proportion of adult carcasses in molt and the environmental variables. To select models, I used Akaike Information Criterion corrected for small sample size (AICc). Our research will give insights into how the combined effects of physiological and environmental stressors may impact upper trophic seabirds as climate change continues to intensify.

Autism spectrum disorder (ASD) is a developmental disorder characterized by deficits in social communication and interactions, and repetitive behaviors or interests. Children with ASD are more likely to show food selectivity than neurotypical (NT) children, and their restricted food consumption may be associated with nutritional deficiencies. Eating disorders are serious mental health conditions that can have long term health-related consequences. Understanding behavior surrounding eating habits is paramount to developing treatments that are effective for children with autism. In youth without autism, self-compassion impacts eating behaviors. Higher self-compassion is causally linked to lower eating pathology. This study aims to extend this work to examine whether self-compassion influences eating behaviors in adolescents with ASD. Participants included children with ASD (n = 37) and children with NT development (n = 52) between the ages of 13 - 17 years. Parents of participants completed the Child Eating Behavior Questionnaire, and participants completed the Self-Compassion Inventory for Youth survey. We compared emotional overeating and emotional under eating to the self-compassionate coping scale and self-punitive coping scale. Given the stereotyped and repetitive behaviors of adolescents with ASD, we expect a similar relationship between self-compassion and eating behavior seen in studies with neurotypical individuals, to also exist within an ASD group. Specifically, we hypothesize: (1) a positive correlation between emotional overeating and self-punitive coping, (2) a positive correlation between emotional under eating and self-punitive coping, (3) a negative correlation between emotional overeating and the self-compassionate coping scale, and (4) a negative correlation between emotional under eating and the self-compassionate coping scale. This study will offer more insight into the role of self-compassion and eating behavior of ASD adolescents. If the hypothesized correlations exist within the ASD and NT groups, we can look for similar risk factors of disordered eating behavior within ASD adolescents.

From playing sports to driving a car, our brain constantly receives a barrage of complex visual information. Previous work shows that recognizing two things (e.g., words, animals) presented simultaneously is harder than recognizing one, suggesting that the brain has a limited ability to attend to multiple visual objects at once. In this project, I examine whether American Sign Language (ASL) experience affects sign recognition. Specifically, I hypothesize that signers and non-signers may differ in their ability to attend to multiple signs at once. Participants look at the center of a monitor where a reference letter sign appears, followed by test letter signs on both the left and right. On each trial, either the left or right sign is relevant (single-task condition), or both signs are relevant (dual-task condition). Participants respond, with a button, whether the relevant test sign(s) match the reference. The dual-task deficit (the difference in performance between single- and dual-task conditions) measures how well people can attend to multiple simultaneously presented signs. I compare the dual-task deficit across hearing non-signers, hearing fluent signers, and deaf signers. Previous work has shown a large dual-task deficit for written English letters and a small deficit for objects. I hypothesize that signers’ will have similar dual-task deficit to those of written English letters, while non-signers’ dual-task deficit will be small, similar to objects. So far, preliminary results from two non-signers support this second prediction. This project will reveal how the different sensory experiences and language demands of deaf and hard-of-hearing populations alter how their brains process visual information. Understanding these differences can help improve accessibility for these marginalized groups in public spaces. For example, it can help improve methods for presenting sign information to Deaf students to improve learning outcomes.

Neuropsychiatric disorders pose a difficult challenge for healthcare providers. Treatments for such disorders vary in efficacy and come with detrimental costs for patients and their communities. Historically, preclinical animal models have failed to incorporate the nuances of volitional human social behavior. This project used chronic social defeat stress to induce depression-like behaviors in male and female mice, this was followed by self-administered social interactions within an operant chamber in which lever presses were reinforced by social contact. The goal is to develop preclinical animal models that can be assessed to identify mechanisms responsible for stress-induced social motivation. The mice will be injected with a nuclear localized tag (oNLS) and viral retrograde tracer rAAV2-retro-GFP. Male and female mice will train to self-administer social interaction with a sex and age-matched housing partner over the course of ten 12-trial sessions. Next, experimental male and female mice will be subjected to physical and witness defeats followed by operant social self-administration. Before and after the 10-day operant social stress sessions, we will test social reward seeking via non-reinforced self-administration of social reward followed by a progressive ratio test. Brain tissue will be collected and prepared for immunohistochemistry and iDISCO+ whole-brain clearing for cfos labelling. We predict results will show differential cfos activity in sexually dimorphic brain regions such as the hippocampus, prefrontal cortex, amygdala and the bed nucleus of stria terminalis. We determine that operant social stress can be used to discern differences in social motivation in male and female mice as a result of stress-induced factors. There is great potential in using whole-brain activity mapping to identify brain structures activated during social reward following social stress, as this can also serve as a technical resource for the field by identifying relevant non-canonical brain regions and circuits that govern such behaviors.

Aging is a complex indicator for the development of age-related diseases. Resilience to aging is defined as the ability to experience a stressor and return quickly to homeostasis. It is important to assess biological age quickly and accurately to fine-tune intervention strategies for non-resilient aging populations. The excision repair cross-complementation group 1 (ERCC1) is an endonuclease that plays a vital role in mediating the nucleotide excision repair of DNA, as well as being involved in the interstrand cross-link and double-strand break repair of DNA. Knockdowns of the gene significantly reduce the lifespan of the mouse due to an accumulation of damaged DNA. Previously it has been shown that wound healing is robustly correlated with DNA methylation clocks and age-related pathological features. In this study, we aim to understand whether a non-invasive wound healing assay is sensitive to accelerated aging phenotypes in an ERCC1 -/â–µ progeria model. In a preliminary study, mice 5 months old had a 2mm hole punched in each ear. The area was measured at 0 and 16 days time. The resulting healed area was compared against a standard curve where it was shown the accelerated aging mice were aged significantly older than the wild type (p<0.05). The lifespan of the mice also correlated with the wound healing (p<0.05). These findings indicate that a simple wound healing assay may be able to accurately assess biological age. Clinically, the ability to intervene and treat aging patients before they develop age-related pathology may help lengthen healthspan and postpone the onset of associated diseases. Future work will look to strengthen these findings with molecular evidence and pathological support.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by differences in social interaction and communication, as well as repetitive behaviors (APA 2013). Per Kanne and Mazurek (2011), 56% of 1380 children and adolescents diagnosed with ASD engaged in some form of aggression towards family, teachers, or peers. Aggressive behaviors can prevent youth with ASD from being able to engage in learning opportunities and community events. An important factor in decreasing rates and severity of aggression is through the identification of social and environmental factors that may impact the stability of aggressive behaviors. The aim of this study is to look at factors that may impact the stability of aggressive behaviors in a longitudinal sample of autistic and non-autistic youth. 44 participants (22 ASD) aged 8 to 18 years, from the NIH funded longitudinal study on sex differences in autism were included in the analysis. Aggression was measured using the Child Behavior Checklist (CBCL), a parental report measure on problem behaviors, which includes items related to self aggression (self injurious behaviors) and other-aggression (aggression towards peers and adults). Data was collected at baseline and 3 to 8 years later. We expect low IQ and younger age will be related to higher aggressive scores on CBCL at baseline. We predict that youth with greater social improvement between timepoints and use of psychotropic medication will have lower levels of aggression over time. Identification of factors that impact changes in aggression over time can aid in implementing interventions to aggression and improve quality of life.

Possession Sound, WA, serves as an important foraging area for a group of seasonally resident gray whales Eschrichtius Robustus. This group of predominantly Eastern North Pacific Gray Whales that visit Possession Sound (Sounders) arrive as early as January and continue migrating towards other feeding grounds near May. If the frequency of sounds within the hearing range of these gray whales (50 to 5000 Hz) exceeds comfortable levels, they may have difficulty foraging. To monitor these relationships, we looked at the frequency and amplitude of sound near Mt. Baker Terminal (MBT) in Possession Sound, WA, from January 2021 to May 2021 with a specific focus on sounds from 50 to 5000 Hz. To gather this data, we used a SoundTrap 300 HF hydrophone, which is currently mounted to a dock at MBT. From January 2021 to February 2021, the hydrophone recorded continuously at a 96-kilohertz sampling rate. From March 2021 to May 2021, the hydrophone was set to record the first fifteen minutes of every hour at the same sampling rate. Preliminary research shows that Possession Sound has a consistent prevalence of ambient and intermittent noise within the established frequency range and peak amplitudes as high as 164 dB. This level of noise may have the potential to negatively affect gray whale behavior and foraging success. Future research should be conducted to see how current and future vessel slow-down trials will impact the frequencies and amplitude of local noise. Future research should also include comparing the MBT sound profile to other sites in the Salish Sea to better understand if the amplitude increase is unique to the area or a cause for wider concern.

The interaction between incoming salt water from the ocean, which is driven by tides, and exiting freshwater from rivers drives circulation through estuarine environments. As a result of the interaction between the incoming water and the exiting water, nutrients and sediment are moved around the estuary. This study focuses on the interaction between tides and the Snohomish River as it enters the Possession Sound estuary, located in Everett, Washington. To acquire data, I deployed a boat-mounted acoustic Doppler current profiler (ADCP), which uses sound to measure water current direction and velocity. I collected these data at varying tide stages at three different sites between the Snohomish River’s southern output to nearby Mount Baker Terminal, located 3.6 miles southwest of the river output. I have collected 8 samples over the course of 7 months at both ebb and flood tide stages. Each transect survey lasted 3-6 minutes and collected data from the first 20 meters of the water column. To get accurate analysis of current velocity and direction I split the data into categories based on depth. Preliminary analysis shows a southward current at many sites during all tide stages and depths. This raises questions about the scope of the river influence and the potential for southward currents regardless of tidal stage. However, further analysis of current velocity and river discharge are needed. Due to the complexity of the currents in the area, understanding how the river and the tides are interacting can provide a greater understanding of how these currents are impacting the dispersal of sediment and nutrients throughout the estuary.

Local bathymetry in estuaries strongly affects water current direction and speed. Water current and speed, or the water flow, determine the circulation of nutrients and oxygen within the estuary. To assess the relationship between water currents and slopes in an estuary, I utilized a boat-based acoustic doppler current profiler (RD Instruments Workhorse 600 kHz ADCP) to measure water speed and direction with depth. Based on preliminary analysis, water direction is consistent at surface level but becomes inconsistent beyond that. Speed is mostly between 0 cm/s and 50 cm/s, but becomes more varied beyond 30 meters. However, that may be due to inaccuracies in measurement, as data collection becomes less consistent beyond that depth based on our current ADCP configuration. The average direction of data at each depth doesn’t change significantly with depth. In summary, speed is mostly consistent with depth near a slope, whereas the direction is not. So, the bathymetry may have a greater effect on water direction than speed. For future work, investigating these patterns with higher resolution datasets will shed more light on any potential patterns.

The Everett Marina is a heavily developed area that has significantly altered water flow from the Snohomish River compared to its natural state. Anthropogenic alteration of this environment led to buildups of sediment, which has required dredging at increasingly shorter intervals leading up to the present. In order to explain sediment accumulation in the marina, this study examines the movement of currents and the abundance of sediment using an Acoustic Doppler Current Profiler (ADCP); data were recorded from June 2020 to August 2021 and compared to recent data I recorded from February and March of 2023. I hypothesized that current direction would be variable and speed would be sluggish due to the shallow depth. In addition, I hypothesized that seasonal changes such as snowmelt in spring and rain in winter would impact the velocity of the currents. Because sediment movement is largely impacted by currents, I hypothesized that sediment abundance would reflect seasonal changes in water flow. Analysis of water current data between June 2020 and August 2021 demonstrates a significant correlation between the velocity of North/South currents and the magnitude of tide stages, but river discharge levels appear to have less of an effect on current speed in the marina. The susceptibility of this area to tides more than river discharge suggests that it is easier for sediments to accumulate in one area as opposed to being swept away. Future analysis will compare this past data to the present and investigate potential correlations with sediment abundance.

The association of eukaryotic DNA with histone proteins serves not only to package entire genomes into the nucleus of a cell, but these histone-DNA functional units, called nucleosomes, are hubs for biochemical signaling that regulates gene expression. In the Chatterjee lab, we are fascinated by the transcriptional biology of the small ubiquitin-like modifier protein 3 (SUMO-3), a posttranslational modification that occurs on histones and has been correlated with reduced gene expression. Previous members have demonstrated that SUMO-3 stimulates the activity of transcriptionally repressive enzymes by binding with a scaffolding protein called CoREST1. Hence, my focus has been to understand the functional details of the SUMO-CoREST interaction, particularly how cancer-associated mutations in the SUMO-interacting motif (SIM) of CoREST1 affect its ability to bind SUMO-3. To answer this question, I started by using solid-phase peptide synthesis to prepare truncated CoREST SIM peptides bearing the mutations of interest. I then utilized these peptides, along with SUMO-3 enriched in nitrogen-15, for two-dimensional nuclear magnetic resonance spectroscopy. By comparing the chemical shifts of [¹⁵N]-SUMO-3 with and without the presence of each peptide, I could assess the effects of mutations on the proportion of bound and unbound species in solution. Of special interest was an acidic residue in the hydrophobic core of the CoREST SIM, which distinguishes it from canonical SIMs found in other proteins. Excitingly, my results indicate that substitution of this amino acid with lysine, a mutation found in gallbladder cancer, ablates binding. I observed a similar effect for other mutations in the hydrophobic core of the CoREST SIM. Using these results to guide studies with full-length CoREST in biochemical assays, my research will identify the effects of these mutations on downstream biochemical pathways that may be misregulated in human cancers.

Deep learning methods for protein sequence and structure generation have shown remarkable success in many design scenarios when combined with structure prediction networks such as AlphaFold2. Despite this advance, many design challenges such as de novo binder design still haven’t been fully solved. Diffusion-based models have demonstrated considerable success in image and language generation yet their application in protein design has not yet been fully explored. Recently, the development of a protein diffusion model called RoseTTAFold Diffusion (RFdiffusion) has shown significant success in protein design and enabled us to explore the challenging problem of designing protein binders. Here I demonstrate utilization of RFdiffusion towards generation of de novo binders to disordered major histocompatibility complex (MHC) peptides. Specifically, we took an MHC peptide from KrasG12D and used RFdiffusion to generate a diverse range of structures that can bind this peptide. To optimize the sequence of these structures we used ProteinMPNN. We used AlphaFold2 to predict the structures of these optimized binders in complex with the peptide and saw promising interaction metrics. Further, structure prediction of the designs in complex with Kras wild type (WT) peptide resulted in lower AlphaFold2 confidence metrics of the interaction occurring. This is a promising preliminary result that RFdiffusion can generate fully de novo MHC-mimics, which can differentiate between neoantigens and WT peptide. Many cancers are caused by a single point mutation such as KrasG12D, thus, designing protein binders with point mutant specificity is exciting as it allows for targeting of disease causing proteins over healthy WT proteins. 

Trauma-induced coagulopathy is a severe complication of trauma that alters the normal mechanism of blood clotting through a number of complex factors. If clots are hypercoagulable, there is risk for dangerous vascular blockages. Conversely, if the clotting is hypocoagulable, it can lead to fatal hemorrhaging. Prior research indicates that actin has a major impact on platelet activity and blood clot formation. Actin is a highly abundant cytoskeletal protein that forms long, insoluble filaments. When released into the blood during cellular death, these filaments have complex effects on blood clot formation. Actin filaments can be integrated into the scaffolding of the clot, increasing strength. My experiment aims to investigate the roles of actin and on human blood clotting. Healthy donor whole blood in 3.2% sodium citrate was spiked with either a saline control or recombinant human skeletal muscle-derived actin (final concentration 200 nM) and allowed to incubate for 5 min. Samples were then activated with either 10 mM adenosine diphosphate (ADP) or 2 mg/mL collagen. The platelet aggregation response was then measured by impedance aggregometry. Each pair of control and actin conditions was run simultaneously. The impedance area under the curve (AUC) was compared between control and actin groups under each activation condition using a paired t-test with significance at p<0.05. Preliminary results show ADP was no different between the control and actin groups (p=0.400, n=5). The AUC in response to collagen was significantly higher in the presence of actin compared to control (p=0.005, n=7). Exogenous muscle actin appears to increase platelet aggregation through the collagen but not the ADP activation pathway. Further investigation is required to better characterize this interaction. A better understanding of the mechanisms of actin on hemostasis could direct research into pharmaceuticals and therapies that could yield better outcomes for trauma patients.
 

The retina is made of mostly neurons and glia. In mammals when neurons degenerate in the brain or retina, they are not replaced. Studying other species, the Reh lab discovered a way to stimulate Muller glia in the retina, using the pro-neural Ascl1 transcription factor, to regenerate retinal neurons. My project focuses on another glial cell in the human retina: the astrocyte. Retinal astrocytes come from different progenitors in the brain and migrate, through the optic nerve, to the retina during development. There are two questions about astrocytes that are addressed in this project. When do astrocytes enter the retina? And can human retinal astrocytes be reprogrammed into neurons using the Ascl1 transcription factor? For the first question, based on immunohistochemistry(IHC) stainings done on sections of the human fetal retina I conclude that astrocytes enter the retina between 62-72 days of fetal development. For the second question, I hypothesize the Ascl1 transcription factor can reprogram human retinal astrocytes and the unique development and migration will affect the types of neurons they regenerate. My IHC stainings on cultures of retinal cells confirms that a lenti-virus I added to the culture with an astrocyte marker(Pax2) sufficiently targets astrocytes. I am currently working on isolating astrocytes from other cells in culture. Afterwards a lentivirus with the pro-neural Ascl1 transcription factor will be added to the astrocytes to begin reprogramming trials. Human retinal astrocytes have not been reprogrammed successfully before. If we can reprogram retinal astrocytes into neurons, it would potentially have implications for neural replacement in the retina. This would contribute to research in gene therapies for neurodegenerative retinal diseases such as: glaucoma, AMD and retinitis pigmentosa.

According to the Corruptions Perception Index, Nepal is ranked 117 out of 180 countries for corruption with 12% of public service users having paid a bribe in the last 12 months. While many studies have analyzed systemic corruption within Nepal, little research has attempted to measure the magnitude of systemic corruption, with particularly limited literature on systemic healthcare corruption. To estimate the prevalence of petty systemic healthcare corruption (i.e. between the public and low-level public officials or doctors), I designed a survey guided by prior United Nations Office on Drugs and Crime and European Commission research, and a series of interviews with Nepalis. Bir Hospital was randomly selected among 5 other public hospitals in Kathmandu, and I administered the survey to 41 participants over two days. 43.2% of respondents paid more than they expected, and 68.6% of these respondents felt this payment was outside of their cultural norms. Moreover, 41.7% of the respondents paid cash directly to a doctor, which interviews indicated is atypical. No correlation was found between income and how respondents rated their care; moreover, no statistical significance was found using a t-test between how respondents rated their care and whether they paid cash directly to a doctor. However, odds ratio analysis on whether a payment was outside of cultural norms and whether the desired care was received yielded a ratio of 1.23, indicating that these payments mildly increased the probability of getting the desired care. This, paired with interviews indicating distrust of the hospital system and prior research demonstrating corruption throughout Nepal suggests systemic healthcare corruption in the form of bribes is prevalent, but does not necessarily guarantee better care. Further research must be done to not only determine the efficacy of this survey method, but also the validity of this model.

Photovoice is a research methodology that uses photography as a tool for participants to express their thoughts on a topic of interest. Our project is specified towards marginalized communities and to give them a voice regarding issues each group faces. The main purpose of photovoice is to create change in our community by analyzing photographs provided by the participants.

Recent publications of photovoice projects at universities have yet to focus on a number of groups and their experiences on campus. We have decided to create a photovoice project specifically tailored to this area of interest.

This project focuses on four groups in the University of Washington Bothell campus: Students of Color, Muslim students, LGBTQ students, and first-generation students. 15 students participated in this study, with around 2 to 4 participants in each group. Our goal is to learn how these groups experience college/university differently compared to students outside of these groups. We asked participants to answer prompts that discuss how they experienced college academically, socially, and emotionally. Thus, we analyzed their experiences and formatted them into differing “codes” and “themes”.

Most themes in the project overlapped between the groups. The three notable themes were (1) The disconnect between faculty and students, (2) Expectations of what students should have and have access to, and (3) Struggling in finding ways to express oneself. Two more themes are to be created from the data we collected.

The project is still under development, and we are expecting to analyze these themes more in-depth. The goal of this project is to highlight disparities in the universities’ process of matriculation. Moreover, we hope to use this research to revise the assumptions and rules regarding resources and general student welfare to create more accessible resources and a streamlined transition from primary to secondary education.

Several factors influence students’ ability to access the disability accommodations necessary to complete coursework; one of the main factors, however, is the degree to which instructors are willing or able to accommodate their students’ needs. Crucially, students often need to discern if instructors will accommodate their needs before they can register for class. Students must navigate differences in communication styles and perspectives on what constitutes reasonable accommodation. Further, instructors’ knowledge of their own responsibilities, rights, and the process by which accommodations are accessed, is limited. Instructors are not made aware of their role in providing accommodations or the timeline for their involvement. They are often reluctant to provide assistance for fear of overstepping boundaries set by their institutions. The ambiguity of the instructor’s role in accessibility is actively causing harm. Multiply-marginalized students face a number of additional barriers: students who have less wealth have impaired access to healthcare, and are more likely to face poor healthcare outcomes. Further, the effects of marginalization ensure that students with intersectional identities are less likely to be wealthy. While these facts are rarely officially disputed, it is often difficult to determine what supports are missing, why they are missing, and how support can be provided to the benefit of all stakeholders. In order to establish how students and instructor perceptions differ from each other and from the recommended process, more data is sorely needed; this study utilized a basic survey to identify the areas in which instructors and students need further support. Most queries were designed to have quantifiable responses; freeform responses were evaluated to identify the most common topics and positionality respondents presented. Responses which directly oposed the most common positionalities were then quantified. Expected results largely aligned with known areas of need, specifically the need for more transparency and training surrounding accessibility. 

Racial and ethnic minority groups are less likely to seek or have access to mental health support services. Despite an increase in the quantity of mental health services nationwide, disparities in usage across racial and ethnic minority groups still exist, leaving resources underutilized and many individuals with unmet needs. Previous studies highlight both structural and non-structural barriers, such as income and stigma, which prevent certain racial groups from fully utilizing health care services. Studies often overlook health outcomes associated with immigrant status and overgeneralize findings to all populations. While scholars have studied the structural barriers to accessing mental health care services, there is a need to understand the cultural contexts preventing racial and ethnic groups from reaching out to these services. Using data from the National Health Interview Survey (NHIS), this study examines differences in access to and utilization of mental health care services of U.S born and immigrant populations from the years 2010-2020. The NHIS allows for the control and determination of effects of both structural and cultural factors such as health insurance coverage, financial instability, racial and ethnic characteristics, and migrant status to further understand the dynamics of access to and use of mental health care services. Foreign-born individuals are expected to show lower access to health care resources with varying levels of moderate mental distress. These differences are expected to vary for those who have spent more time in the U.S with more access to resources and higher levels of mental distress. Understanding trends related to mental health care can help develop better public policy responses and improve the promotion of health care services. This study will help address why services are underutilized and how to minimize health service inequalities among racial and ethnic populations. 

Our research group is exploring the feasibility of utilizing nanopore sensors for protein sequencing, whose compact size and ability to facilitate extremely long, uninterrupted reads of protein strands upstage the current procedure of using complex, expensive mass spectrometry (MS) devices. My project predicts the sensor’s raw signal data using a carefully tested combination of each amino acid’s volume and charge properties. Using my model to generate predictions for a specific database of proteins, I can compare the unknown raw signal to each of the predicted signals to single out the best matching/correct sequence. While the protein space of De Novo sequencing is vast (20 to the power of protein sequence length), this method effectively shrinks the protein space to a group of substantive, feasible sequences. Employing the current predictive model on a database of synthetic and natural proteins, when compared against an unknown protein’s raw signal, I found that, on average, the correct prediction consistently ranked within the 99th percentile of matches among a predicted test set of >20,000 sequences. Advancing single-protein sequencing can revolutionize protein research by enabling the identification of low-abundance proteins. Additionally, the increased sensitivity of the nanopore sensor could shed light on the so-called "human dark proteome," composed of approximately 3,000 human proteins that have not yet been identified despite genetic evidence of their existence.

Clearance of wound infections can be hindered by a bacterial biofilm; a complex extracellular matrix (EM) secreted by adherent bacteria that allows them to evade the host immune system and obviate antibiotics. A novel, synthetic peptide—known as an anti-α-sheet inhibitor—can disrupt biofilm stability by inhibiting the formation of amyloid fibrils, which contribute to the biofilm EM. This project aims to design and characterize alginate porous scaffolds that elute these synthetic peptides, for use as anti-biofilm wound dressings. The physical properties and peptide release kinetics of the scaffolds will be optimized for clinical applications, supported by in vitro efficacy studies with live bacteria. This project draws upon past work from the Bryers Research Group on engineering infection immunity and tissue scaffolds, in which biofilms are prevalent. Results of this project will provide an alternative approach to biofilm prevention, thus reducing the burden of biofilm-related infection complications.

Effective rapid tests have been developed to detect COVID-19 in nasal swabs. However, nasal swabs require a trade-off in which deeper insertion of the swab allows higher accuracy but causes greater patient discomfort. On the other hand, saliva can be collected non-invasively in large volumes. Therefore, this project aims to develop a point-of-care diagnostic device (“DiagnosDisk”) for the rapid and sensitive detection of SARS-CoV-2 nucleocapsid protein (NP) in saliva. The DiagnosDisk is a circular disk (25mm diameter) consisting of three layers, from top to bottom: a plastic adhesive capping sheet with a sample port, a hydroxylated nylon detection membrane, and an absorbent pad. Analytes are efficiently enriched by binding to anti-NP antibodies conjugated to temperature-responsive polymers (poly(N-isopropylacrylamide)) which aggregate upon heating. To prepare the sample, polymer- and gold-conjugated antibodies are mixed with the sample and bind to the target antigen, forming a sandwich immunocomplex. When flowing through the heated detection membrane (>37℃), the sandwich immunocomplexes aggregate and are captured on the membrane, producing a visual signal. I quantify the signal intensity on the membrane using ImageJ software. With this design, I hypothesize that the DiagnosDisk will have higher sensitivity than the lateral flow assay (LFA) by utilizing temperature-responsive polymers for sample enrichment and using larger sample volumes which increases the number of antigens that can be captured. From my initial testing, the DiagnosDisk enabled 2mL of buffer flow-through in 7.5 minutes, demonstrating a sample volume capacity 10-times that of most LFA. Next, I tested the DiagnosDisk with buffer samples spiked with 5ng/mL NP. Heated membrane pads produced signal intensities significantly greater than unheated membranes. I plan to apply the device to saliva samples next. Ultimately, by enabling rapid and sensitive detection of COVID-19 in saliva, the DiagnosDisk can help limit community transmission of the virus.

Ascidians are invertebrate chordates, with tadpole larvae that metamorphose into sessile adults. Molgulidae are a clade of ascidians where tailless larvae have evolved several times independently and our lab is interested in understanding the molecular mechanisms underlying the evolution of taillessness. There are two closely related Molgulidae species that are sympatric in Roscoff, France: Molgula oculata, a tailed ascidian and Molgula occulta, a tailless ascidian. The tailless M. occulta, have 20 notochord cells that do not converge and extend, called a "notoball", and have evolved a number of larval pseudogenes. During embryonic development, the Wnt planar cell polarity (PCP) pathway plays an essential role in regulating cell fate determination and other cell processes. Previous experiments have found that the Stolidobranch ascidian, Halocynthia roretzi, has a Wnt5 duplication, where Wnt5a is invovled in the notochord development whereas Wnt5ß is involved in muscle development. Bioinformatic analysis has been done on several developmental genes involved in notochord formation and the Wnt pathway which showed that most genes are highly conserved between M. oculata and M. occulta. We have identified two Wnt5 isoforms that may play a role in tail formation, and we are in the process of subcloning the 3'UTR regions to obtain gene specific RNA probes for in-situ hybridization. We expect to find Wnt5a expressed in the notochord and Wnt5ß to be expressed in the muscle cells. We are also cloning Dsh to examine gene expression. This information will allow insight into how the Wnt5 genes in M. occulta and M. oculata are implicated in notochord convergence and extension as well as whether the Wnt/PCP pathway contributes to the evolution of tailless larvae. 

Remote collaboration today rarely involves a single communication channel. Instead, teams frequently juggle a myriad of communication tools, such as video conferencing, group chat, and email. Each of these platforms provides different mechanisms for relaying information and media to ultimately meet the needs and goals of the team. While discussions occurring on different platforms are often related, existing tools used to support each type of communication are disconnected. To research how to bridge this gap and support seamless collaboration and communication across different platforms, we developed a toolkit that connects conversations between three of the most commonly used remote collaboration platforms: Slack, Google Docs, and Zoom, covering both synchronous and asynchronous modes of communication. We iteratively designed and implemented features such as adding information from Slack chat directly to Google Docs notes to build up meeting agendas and selecting specific snippets of Zoom meetings to be embedded into notes or sent to chat. We also plan to evaluate the effectiveness of our toolkit in helping streamline the transfer of information across different team communication sites and enhancing the remote collaboration experience for teams via subsequent qualitative user studies. Specifically, we will be conducting a week-long field study with existing teams, such as teams from industry, teams working on school projects, research groups, committees, etc. We will use a combination of experience sampling, diary study and post-study interviews to understand their experience. The results we expect to get from these exploratory user studies will help us answer the following questions: Which aspects of the tools work best for the users? Does the current UI and design make sense for how the user interacts with the toolkit? In which scenarios is the toolkit being used most effectively? These results will also guide us in designing additional features for the toolkit in the future.
 

Major Depressive Disorder (MDD) is one of the fastest growing causes of global disability and requires a variety of treatment options due to its multifactorial etiology. While it’s long been known that blood levels of the neurotransmitter GABA are predictive of MDD, recent studies have implicated the human microbiome as a potent GABA metabolizer that can affect circulating GABA levels. Our collaborators used bioinformatics to identify bacteria containing putative GABA-synthesizing genes (puuD) that are predictive of host GABA levels and depression scores. However, previous literature indicates that the puu operon is repressed under anaerobic conditions in E. coli and the putative PuuD proteins have low sequence similarity to the model PuuD enzyme. My project seeks to confirm the biochemical activity and substrate-specificity of these genes using in-vitro assays measuring both the putative PuuDs’ chemical reactivity and kinetics. Due to conservation in the active site structures between the putative proteins and the model PuuD, I expect that the novel PuuD-like proteins will have gamma-glutamyl hydrolase activity and substrate specificity for gamma-glutamylated-GABA. This work provides base-level evidence for the use of systems biology techniques to identify enzymatic function and lays the foundation for targeted therapeutics manipulating the microbiome for improved MDD outcomes.

Herpes Simplex Virus (HSV) causes lifelong infections due to the establishment of latency in peripheral nerves. The immune response to HSV infections involves CD8 tissue resident memory T cells (CD8TRM) to sense HSV reactivation, and the innate immune cells to mitigate viral replication and spread. Plasmacytoid dendritic cells (pDC) can produce type-1 interferons (IFN-I) and prime CD8 T cells, yet their role in human HSV infections is unclear. ChipCytometry is a highly multiplexed imaging platform useful in observing cell-cell interactions, cell distribution in a tissue, and cell phenotypes. Using Chip Cytometry, we analyzed a tissue biopsy from a genital herpes lesion with a panel of 24 markers to characterize immune cell infiltration during active HSV reactivation. We showed that CD123+/CD303+ pDCs were located near activated CD8 T cells, many of which were found in lymphoid like structures in the dermis. These lymphoid like structures formed in areas where large nerve bundles and blood vessels were present and were packed with T cells and other immune cells which expressed markers for activation, exhaustion, and proliferation. Further research into pDC functions in HSV infections and cell interactions in these lymphoid like structures may provide insight on mechanisms of immune cell surveillance and response of reactivating HSV infections.

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

Pseudomonas aeruginosa is a major cause of opportunistic infections in immunocompromised people. P. aeruginosa uses a cell-cell signaling mechanism called quorum sensing (QS) to regulate virulence functions and cooperative behaviors. QS in P. aeruginosa is regulated by two transcription factors, LasR and RhlR. These proteins control the production of extracellular proteases, “public goods” that benefit the entire population. These public goods create an incentive for individuals to cheat by availing themselves of the proteases without incurring their production cost. In fact, when P. aeruginosa is grown in media that requires QS for growth, cheaters emerge in the population and invariably carry mutations in lasR. We were intrigued by the clinical isolate E94, in which we observed the rapid emergence of cheating. E94 contains an inactive LasR via a transposon insertion. Transposons are DNA segments that move between genomic locations. E94 contains dozens of transposable elements. We hypothesized that transposon movement facilitates the adaptability of the E94 genome. I evolved cheaters from wild-type E94 in casein media, which requires QS for growth. I identified cheaters using a phenotypic assay. I then grew cheaters in non-selective media and identified revertant, protease-producing colonies. I used PCR analysis of the cheaters and their protease-producing progeny to determine if the transposon remains inside the lasR gene. I found no transposon movement from lasR upon cheater reversion to a protease-producing phenotype. We also determined that cheaters display no rhlR activity. We then hypothesized that cheating in E94 occurs by disruption of RhlR QS. We did not find mutations in rhlR itself and are performing whole-genome sequencing to identify other genetic targets. Understanding the mechanism of cheating and reversion in E94 gives us insight into the evolution of cooperation and conflict in populations and, potentially, a non-antibiotic approach to controlling bacterial populations a variety of settings.

Identifying risk factors for alcohol misuse among young adults is a critical public health priority given high rates of heavy drinking and alcohol-related consequences observed in this population. The field of behavioral economics has provided a set of quantifiable metrics that measure individuals’ demand for alcohol, which are important predictors of alcohol use, consequences, and response to treatment. Previous literature has also found that one’s self-reported drinking motives (e.g., drinking to cope with negative affect, to conform to peers, etc.) have important associations with drinking outcomes. Despite this literature, little is known regarding how one’s drinking motives relate to one’s demand. The study aims to investigate how different drinking motives may be differentially related to alcohol demand and whether birth sex moderates these relationships. The current study recruited 220 young adults (18-25 year-olds) from Washington state who report drinking at least twice a week and at least one recent heavy drinking episode (4+/5+ drinks for females/males). Participants completed online assessments that included the alcohol purchase task, which asked how many drinks they would hypothetically purchase and consume at various prices ranging from free to $20. Participants were also asked to report their birth sex and drinking motives (social, coping-anxiety, coping-depression, enhancement, conformity). I will conduct regression analyses to test for associations between drinking motives and alcohol demand, and to examine whether these associations are moderated by sex while controlling for age and discretionary spending. I hypothesize (1) stronger positive associations between coping motives and demand relative to other drinking motives, and (2) this relationship to be stronger for males. Results will improve our understanding of the relationship between drinking motives and demand between sexes and inform interventions focused on reducing alcohol misuse through alternate coping strategies or reducing demand.

Corepressors are proteins recruited by partner proteins to negatively influence transcription of genes. TPL is a corepressor from the model plant Arabidopsis thaliana, and while we understand a lot about how TPL works, there are still many mysteries remaining. My project aims to identify other proteins that work with TPL to form a transcriptional repression complex at a single-engineered promoter site. First, we created a synthetic repressor called dCas9-TPL that binds and represses the transcription of the RUBY reporter. The RUBY reporter is a visual marker designed to express throughout the entire plant, turning the green plant a bright purple. Our engineered RUBY line also carries two guide RNAs in its promoter with sequences not found anywhere else in the Arabidopsis genome. This allows dCas9-TPL to bind to and repress this particular gene and not affect the transcription of other genes. Visual screening of the repressed RUBY line showed these plants turn a faint whitish-pink instead of bright purple, signifying that the repression by TPL is working. I have identified the promising repressed RUBY homozygous line and have generated three mutagenized populations of 40,000 individuals using the chemical Ethyl methanesulfonate (EMS). The EMS protocol creates new point mutations allowing us to identify genes involved in repression that we can map through DNA sequencing. I will use visual screening to search for plants with bright purple organs, meaning that the repression by TPL is broken and that a putative TPL interactor may be mutated. By identifying regulators of corepressor function in plant biology, I hope to learn principles that can inform cellular engineering across many organisms and better understand why certain mutations associated with transcriptional repression cause developmental defects or diseases like cancer in humans. 

 Alzheimer’s disease (AD) is the most prevalent of neurodegenerative diseases, with over 6 million Americans suffering from the illness and cases increasing each year. TREAT-AD (TaRget Enablement to Accelerate Therapy Development for AD) is an NIH-funded, multi-institutional network that identifies and addresses new targets for AD drug development. Genetic targets of interest were identified via RNA-sequencing and proteomic analysis of post-mortem tissue from participants with AD. We tested the effects of inhibiting or overexpressing selected target genes hypothesized to increase the risk of Alzheimer’s. One such target is the CD44 gene, which regulates GABA receptor activity. To efficiently manipulate genetic expression, we used clustered regularly interspaced palindromic repeats (CRISPR) technology to enhance (CRISPRa) or repress (CRISPRi) genetic transcription in neural progenitor cells, stem cells in the process of differentiating into neurons. We used a cell line engineered to harbor CRISPRa and CRISPRi machinery. CRISPRa involves a catalytically inactive Cas9 protein fused to a transcriptional activator. In CRISPRi, the inactivated Cas9 is fused to a transcriptional repressor. This, along with the quantification of molecular pathways linked to Alzheimer’s, permits a window into the conditions that lead to earlier onset of AD, and thereby conditions that might be altered by new drug treatments. Here we report that underexpression of genes related to endosomal trafficking leads to changes in protein buildup that may be related to earlier onset of AD.

 Increased rates of opioid use have led to an alarming number of overdose deaths in Washington state. The National Center for Health Statistics reported 2,255 overdose deaths in May 2022, compared to 1,913 overdose deaths the previous year. Evidence-based interventions such as take-home naloxone, buprenorphine treatment, and HIV/HCV testing significantly reduce deaths related to opioid use. Unfortunately, opioid-related deaths most often occur outside medical settings, in communities where evidence-based interventions aren't accessible. To bridge this gap, our research team created recommendations for implementing evidence-based interventions by collecting and analyzing qualitative data (from 30 semi-structured interviews with first responders and mobile clinic staff). Few research exists integrating collaboration of community members in the research process. The voices of community members are often discussed in the research but left out in the decision-making process. Using a community-based participatory research (CBPR) approach, members from Research with Expert Advisors on Drug Use (READU), a newly formed group with lived substance abuse experience, were hired to co-conduct research on evidence-based interventions as described above. The recommendations developed by READU (co-researchers), alongside the research team, are currently being implemented to improve overdose response in first responder agencies in King County. This study aims to learn from the process of integrating CBPR into this research environment. READU members (n=8) participated in a 2-hour focus group to discuss topics relating to the research process and feelings/concerns about their experience. A thematic analysis approach is used to analyze the qualitative data from the focus group transcript to investigate reemerging themes. Based on the findings in this study, we expect to integrate the needs and suggestions for the CBPR approach (e.g., supportive environment, equitable participation, capacity building & empowerment, and improved programming & policy) from READU members into future community-based research design and process. 

Despite heavy exposure to Mycobacterium tuberculosis (Mtb), the bacteria that causes Tuberculosis (TB), some individuals show no evidence of infection and by defining these resistance mechanisms, we may identify novel treatment strategies. Among Mtb resistant individuals, our lab identified the Rab5a protein as differentially expressed as compared to controls with Mtb infection. By regulating vesicle trafficking, Rab proteins modulate a variety of cellular pathways including inflammatory signaling, antigen presentation, and autophagy, likely playing a role in Mtb clearance. We hypothesized that loss of Rab5a would alter IFN-êžµ gene expression. Monocyte-like THP-1 cells were electroporated with siRNA targetting Rab5a and yielded 70-90% knockdown at 24 hours versus scrambled siRNA control. Cells were then stimulated with DNA ligands for four hours before RNA analysis. Loss of Rab5a resulted in lower levels of IFN-êžµ gene expression after stimulation with Sheared Calf Thymus DNA (p=0.002, 53.9% reduction), Poly(I:C) (p=0.01, 42.8% reduction), supercoiled plasmid (p=0.03, 45.3% reduction), and cGAMP (p=0.008, 45.7% reduction). We conclude that Rab5a expression is required for Type I IFN production through the DNA-sensing pathway. By characterizing the pathways by which Rab5a modulates the macrophage Mtb response, we may identify host targets to augment protective responses that may serve as adjuncts to current TB treatments and vaccines.

In low- and middle-income countries (LMICs), human exposure to animals can represent a significant source of enteropathogens. The presence of domestic and non-domestic animals either in the vicinity or in immediate households within LMICs have the potential to contaminate soil and high-touch surfaces leading to enteropathogen transmission and infection. Among LMICs, access to water, sanitation, and hygiene (WASH) is limited and is responsible for contributing to the global disease burden. Lack of WASH regulations and implementation can compound transmission and infection rates of enteropathogens from animal exposures. The objective of this study is to determine the sources and quantities of household animal fecal contamination, an important exposure pathway for enteropathogens, in Ecuadorian households. Quantitative PCR (qPCR) assays were used to identify host-associated fecal contamination by detecting gene fragments of gut microbes that are specific to the feces of a given human or animal host. These microbial source tracking (MST) marker assays were used on environmental samples collected in Ecuador households including floor, mother and child hand rinses, and domestic water to assess the abundance and sources of household fecal contamination. MST marker targets included human feces associated markers (HF183 & HumM2), dog feces makers (DG37), ruminant feces markers (Rum2Bac), pig feces markers (Pic2Bac), bird droppings markers (GFD), and general bacteroidales markers (GenBac). The anticipated results are that the qPCR data will continue to indicate high levels of general and human-associated animal fecal contamination in animal-owning Ecuadorian households as previously observed in Phase 2 of this study. Findings from this study will highlight the need for the development and implementation of relevant public health interventions aimed at reducing animal exposures and improving overall hygiene practices to decrease the global disease burden among LMICs.

People embody the joy and pain of nature through their own experiences, and have been sharing and cherishing this interconnectedness from time immemorial through paintings, stories, songs, dance, and more. I am a migrant living in a foreign country. i was eating sushi that day…. is a solo dance and an ethnographic research project, exploring ways in which I connect to, resonate with, and embody nature and personal experiences through performance. The awe-inspiring habit of salmon migration sparks my curiosity about the complex and multifaceted nature of human migration. As anadromous fish, salmon spend their juvenile life in rivers, and migrate to the ocean where they spend their adult life. They return to the upstream rivers to reproduce when they reach sexual maturity. Salmon are able to precisely return to their natal river, and even to the very spawning ground of their birth. This creative research explores my questions around identity and the idea of belonging through examining migrating salmon, and my own migrating experiences. What is the definition of home? Is it where you were born, reproduce, and die? Or where you mature? Salmon migrate to optimize their chance of reproduction as it defines their success. What about humans? What are we migrating for? Are we ultimately going to return to where we were from? My creative process starts with producing a soundscore combining text, breathing, and waves. The choreography is inspired by and generated through filmed improvisations as I experiment with different ways to interact with the soundscore and the props, always keeping my research questions in mind. The piece is a product of my desire to understand and reify the fear, confusion, exhaustion, excitement, and hope in the process of migration.

Cardiovascular disease is the leading cause of death worldwide. A key reason driving the high mortality of heart disease is that the heart is unable to regenerate any muscle that is lost due to injuries like heart attacks. Furthermore, there are no current therapeutics that promote the creation of new muscle. However, in the past decade, scientists have attempted to address this issue by using stem cell-derived cardiomyocytes (iPSC-CMs) to replace lost heart muscle. A key limitation preventing using this therapy in humans has been that cardiomyocytes derived from stem cells remain immature relative to adult cardiomyocytes, and these immature cells cause several complications when transplanted into an adult heart. Identifying cardiomyocyte maturation regulators is needed in order to further develop this technology and translate it to patients. Previous studies from our lab and others have identified the RNA binding protein Muscleblind-like protein 1 (MBNL1) as a key factor controlling muscle maturation. MBNL1 expression increases as the heart matures after birth and it controls expression of many critical regulators of cardiomyocyte maturation, however, MBNL1 has never been studied directly for promoting iPSC-CM maturation. In this project, I am testing the hypothesis that increasing MBNL1 expression will improve the maturity of iPSC-CMs. I am using a genetically engineered stem cell line in which I can overexpress MBNL1 and an isogenic control line to test my hypothesis. I have found that MBNL1 expression naturally increases over time in iPSC-CMs. Additionally, I have validated the MBNL1 overexpression system in iPSC-CMs. Finally, I have used this system to test my hypothesis that MBNL1 will increase iPSC-CM maturity by measuring well-described transcriptional and structural hallmarks of maturity. Ultimately, this project will aid in identifying MBNL1’s role in controlling cardiomyocyte maturation, helping further develop stem cell-based therapeutics to repair damaged heart tissue in humans.

The Mongol Empire (1206-1368) under Chinggis Khan and his descendants was the largest continuous land empire in history, stretching across 5,000 miles of the Eurasian Steppe. During this period, referred to as the Pax Mongolica (lit. 'Mongol Peace'), the restoration of the Silk Road under a common administration allowed for the trading of goods between the Levant and Orient, and therefore the wider dissemination of technologies, cultures, and religions. This is juxtaposed with the great deal of brutality associated with the Mongol conquests, as evidenced by archaeological and textual records of mass killings during sieges, and modern scholarship regarding the Mongol Empire is tasked with balancing these perspectives. Witness to the Mongols is an upcoming sourcebook which compiles primary source material from all across the known world at the time, creating a complete picture of the life and legacy of Chinggis Khan, from his birth c.1185 to the fall of the last remnants of Chinggisid rule in 1502. Witness aims to compile, translate, and update scholarship for new audiences, both academic and at large, in order to communicate a more nuanced perception of Mongol imperial influence which, in my experience, otherwise propagates in Western education almost exclusively through the writings of Marco Polo. My specific contribution to this project concerns assisting in editing passages to ensure continuity between sources and revising introductory paragraphs and footnote annotations in preparation for publication. My particular fascination within the Mongol imperial sphere focuses on Mongol-Viet relations, especially in interpreting cultural dissonance regarding victory and failure of the Mongol invasions of Vietnam, as the successful defenses of Vietnam are widely celebrated to this day in folk tradition, though largely regarded in scholarship as Mongol victories for establishing a tributary relationship. As such, I am also tasked with investigating these threads for possible inclusion in Witness.

When the cell undergoes stress, it leads to an increase in protein instability and misfolded states that prevents proper cell functions. Small Heat Shock Proteins (sHSPs) are molecular chaperones that work to maintain a healthy proteome by associating with misfolded proteins to delay aggregation under stress conditions. Other chaperones and co-factors will then refold or degrade the misfolded protein client. HSPB5 is a human sHSP ubiquitously expressed throughout the body. A HSPB5 disease mutant, where arginine is mutated to glycine at residue 120 (R120G), is a defective chaperone associated with cataracts and desmin-related myopathy. HSPB5 comprises three domains but only one domain—the alpha-crystallin domain (ACD)—is folded. My research aims to understand the effect of the R120G mutation on the folded ACD’s structure. HSPB5 creates a dimer through electrostatic and hydrophobic interactions between ACD at the dimer interface. This network of interactions causes the dimer interface to be highly sensitive to electrostatics, working like a sensor for environmental charges. Wild-type HSPB5 is more positive at its dimer interface, likely facilitating interactions with negatively charged compounds. In the R120G mutant, the loss of arginines at the dimer interface site causes it to be less positive, hypothetically lowering HSPB5’s affinity for these compounds. Through site-directed mutagenesis, I obtained HIS-tagged cleavable constructs for wild type and R120G B5 ACD that allowed for easier purification. Using these constructs, I grew isotopically labelled N15 B5 ACD in minimal media and purified my protein sample through nickel affinity, size exclusion and anion exchange chromatography. Through NMR titration experimentation, I investigate how amino acid identity at the R120 site will affect ACD interactions with charged molecules in R120 mutant of HSPB5. Learning how mutations at the R120 site affect protein dynamics and client interactions will be a step forward in understanding the sHSPs’ overall chaperone mechanism.

Movements can be externally driven, based on responses to the environment, or internally driven, based on self-initiated triggers. We modeled these systems using saccades, which are rapid eye movements that shift the direction of gaze. We compared visually-guided saccades (VGS) to visible targets and memory-guided saccades (MGS), where a target was transiently presented and its location memorized before a saccade was made. As a result of aging, growth, and skeletomuscular damage, adaptations in brain circuitry are necessary to adjust motor output and maintain accuracy for both types of movement, but their corresponding mechanisms are not well understood. We hypothesize that VGS and MGS employ different adaptation mechanisms. The superior colliculus (SC) is the central structure in creating the saccade command signal, and prior research suggests VGS adaptation occurs downstream of the SC. To compare this to MGS adaptation, we recorded single unit activity from visuomotor neurons in the SC in four Macaca mulatta monkeys as they adapted their saccade amplitude. I analyzed this data, measuring the median number of action potentials during each saccade for both VGS and MGS; the “non-adapted” saccades, which had the largest amplitudes, were compared to the “adapted” saccades with smaller amplitudes. For VGS, neuronal activity remained constant as saccade amplitude decreased, but for MGS, the number of spikes decreased alongside the saccade amplitude, which indicates VGS and MGS adaptations employ different processes. Moreover, as visuomotor neuronal activity in the SC decreased during MGS adaptation, further research can be conducted to determine if MGS adaptation occurs within or upstream of the SC.

Myotonic dystrophy type 1 (DM1) is a genetic disease that causes many serious health conditions in a variety of tissues including skeletal muscle stiffening, weakness, and degeneration. DM1 is caused by a CTG repeat expansion mutation in the myotonic dystrophy protein kinase gene, DMPK. Expression of the mutated DMPK allele binds with the splicing regulator muscle-blind-like 1 (MBNL1), causing DM1 by sequestering and limiting its critical role in splicing mRNA. A main focus of the Chamberlain lab is the development of gene therapy to treat DM1, including increasing protein expression of MBNL1 to reduce the disease effects in muscle. Overexpression of MBNL1 in skeletal muscle could be beneficial but may have negative effects on cardiac tissue. The lab discovered that high, unregulated MBNL1 expression from gene therapy vectors in cardiac tissue can result in cardiac damage. In my study, I will focus on cardiac function when testing adeno-associated viral vector (AAV)-mediated systemic delivery of the MBNL1 gene to increase MBNL1 protein expression in muscle. Using analytical methods such as echocardiography and tissue histological techniques, I will determine whether it is possible to prevent MBNL1 protein production and its damaging effects in the heart while still expressing MBNL1 protein in skeletal muscle for therapeutic disease benefits.

Without proper genetic regulation, the creation and maintenance of cells within eukaryotic organisms such as yeast, plants, and humans is doomed to fail before it even begins. Protein corepressors are key to the genetic repression in all eukaryotic organisms and are vital for an organism to be able to properly coordinate their development and respond to environmental stimuli. In Arabidopsis thaliana, a model plant for genetic studies, the corepressor TOPLESS (TPL) is one of the main proteins that is used to repress the auxin pathway, which is essential to development and organ creation. Recently, the active domain of TPL has been pinpointed to an 18-amino acid long region named LIS1 homology (LisH) that is sufficient for activity. Previously, we found that the helix H1 of LisH in a plant corepressor functioned as a transcriptional repression domain in yeast. These observations suggest a broad conservation of mechanisms across kingdoms, suggesting this motif could be engineered to be a potent, short, and adaptable protein domain suitable for synthetic biology and therapeutics. My project aims to test the ability of the LisH protein domain to repress gene transcription in metazoans using mammalian cell culture. We will transfect human cancer cell lines with DNA encoding a dCas9-TPL fusion protein, which can be targeted to promoters of endogenous genes such as the cell surface antigen CD4, or synthetic constructs such as fluorescent reporter genes to detect differences in protein levels. Results of the project are expected to show that TPL and other foreign corepressors can function within the human cell just as efficiently if not more than human corepressors. Research into LisH's abilities will provide knowledge of its active domains and mechanisms in mammalian cells while also having the possibility to aid the scientific community by developing TPL as a rapidly deployable synthetic biology tool.

The dynamic expression of genes in an organism creates the biological complexity of life. Many are unique to a given lineage, while other genes are conserved and carry out the essential functions of life. Unsurprisingly, these essential genes are complicated to study, as interfering with their function often leads to death. One critical component of transcription is the multi-protein Mediator complex, which is found at every eukaryotic promoter where it helps coordinate the activation of gene expression. My project focuses on a core component of the Mediator complex, Mediator 21 (MED21). While MED21 is required for gene activation, my lab found that it also plays a role in the repression of gene expression, suggesting a complicated interplay between these two states. This can be challenging as many mutations in MED21 lead to lethal phenotypes. As an alternative, I hypothesize that using an integrase-based molecular switch to create a switchable MED21 will then allow me to differientate the role that MED21 plays in activation through the Mediator complex versus repression through the corepressor protein (TPL) in the model plant Arabidopsis. Integrases are capable of inverting DNA sequences flanked by unique sites, and I am engineering a switch that will turn off MED21 in certain tissues or in response to the addition of a chemical. By expressing an integrase protein from a lateral root-specific promoter, we can engineer a MED21 loss of function only in those specific cells while the rest of the plant is wild type and healthy. Future experiments include a switch from wild-type MED21 to a mutant form incapable of binding to corepessor TPL. This study will help us better understand the role MED21 has in repression versus activation, and also how state switching contributes to organogenesis.

 The primary goal of post-transplant care is to achieve the optimal balance of immunosuppression between infection and rejection. Physicians currently rely on laboratory markers such as changes in serum creatinine. Physicians use kidney allograft biopsies to detect acute rejection but biopsies are expensive, invasive, and susceptible to sampling error. This study investigates the use of donor-derived cell free DNA (dd-cfDNA) as a biomarker for kidney allograft dysfunction to allow for optimization of immunosuppression. This multicenter prospective study (Caredx) examines the correlation between dd-cfDNA levels and infection and rejection episodes in pediatric kidney transplant recipients. Dd-cfDNA is measured 3 to 12 months post-transplant and compared to the clinical outcomes of major infection events and biopsies. Data is collected from November 2019 to 2023 on 59 participants from Seattle Children’s Hospital, St. Louis Children’s Hospital, and Emory University. Using patient’s charts in EPIC I abstract patient demographics, transplant characteristics, laboratory, and kidney biopsy results. I assist with patient recruitment and retention by keeping track of when patients are within sample windows and ensuring their sample requests are sent. Preliminary data from the first 10 samples revealed that elevated dd-cfDNA levels were associated with viral infection or acute rejection episodes. We observed a decrease in the dd-cfDNA levels following treatment for BK viremia. Following data collection, we plan to submit an abstract to the American Society of Nephrology in May of 2023. Additionally, we will prepare a manuscript to submit to the Journal of Pediatric Transplantation in the Summer of 2023. Use of dd-cfDNA will enable early intervention and minimize damage to allografts leading to improved longevity and quality of life. Furthermore, the biomarker is less expensive, less invasive, and more accessible than reliance on transplant biopsies holding the potential to break down economic and physical barriers to health care.

Synthetic promoters are increasingly being used to control and fine-tune gene expression in multicellular organisms as part of engineering novel traits. For this genetic engineering project, we aim to build and activator system that when engaged leads to a statistically significant increase in gene expression compared to the unengaged system. Previously, our lab screened for promoters that are constitutively and ubiquitously expressed in Arabidopsis Thaliana. From this screen, multiple viable promoters were found, and a subset was selected for modification by adding sequences not found anywhere else in the genome (target sites). Our first experiments used the CRISPR-mediated gene regulation system to recruit repressors to create NOR logic gates (where a reporter is only expressed when two different inputs are absent) through the use of guide RNAs (gRNAs) complimentary the engineered target site. Currently, we are working to expand the capabilities of these synthetic promoters by swapping the repressor with an activator to see if we can boost expression from these promoters. Our activator construct has an enzymatically disabled Cas9 connected to an activation domain called EDLL, and an aptamer in the gRNA that attracts a second activation domain, VPR. We are currently generating transgenic plants carrying our synthetic promoters and activator constructs. I will quantify gene expression by measuring the levels of a fluorescent reporter under the control of our synthetic promoter. The use of both activator and repressor approaches will allow for the construction of increasingly complex genetic circuits. These circuits have wide potential applications across the fields of synthetic biology and metabolic engineering.

Lignocellulosic biomass, composed of cellulose, hemicellulose, and lignin, has become an attractive renewable carbon source for microbial bioproduction of value-added chemicals. Lignin is relatively difficult to depolymerize, and its enzymatic hydrolysate contains mostly aromatic compounds known to inhibit microbial growth when used as a carbon source. Pseudomonas putida (P. putida), a soil bacteria known for its tolerance to aromatics, has been engineered to catabolize lignin hydrolysate. Engineered microbes have also been encapsulated in hydrogels for on-demand bioproduction and exhibited enhanced tolerance to harsh processing conditions, i.e. freeze-drying and exposure to organic solvents. This research aims to create microbe-laden hydrogels from encapsulating engineered P. putida KT2440 cells in F127-bisurethane methacrylate (F127-BUM) hydrogels for robust on-demand bioproduction when using lignin hydrolysate as a substrate. To mimic growth rate inhibition that often happens in hydrolysate environments, we first examine if P. putida-laden hydrogels remain active when grown in a less-ideal medium, such as M9 minimal media (MM9) known to yield a slower microbial growth rate. We find that hydrogel-encapsulated P. putida containing a plasmid for heterologous expression of a green fluorescent protein (sfGFP) maintained its activity in MM9 continuous culture over two days as measured via fluorescence of the expressed sfGFP. This preliminary result on encapsulated P. putida growth and activity in a less desirable culture environment highlights the potential for utilizing aromatics-rich lignin hydrolysate in bioproduction as a more economical and renewable feedstock alternative.

FKBP51 is a protein that acts as a co-chaperone for glucocorticoid receptors and is active during the stress response. FKBP51 blunts glucocorticoid receptor signaling and can interfere with feedback inhibition of stress responses. Overall, increased levels of FKBP51 and its gene FKBP5 positively correlates with an increased risk of stress-related neuropsychiatric disorders. FKBP5 is expressed in serotonin neurons of the dorsal raphe nucleus (DRN), a brain region and system important to stress and anxiety responses. In order to investigate the function of FKBP5 in the DRN, new plasmids were generated to knock down or overexpress FKBP5, thereby changing FKBP51 expression in serotonin neurons. My first goal was to validate these plasmids using Neuro2A cells which endogenously express FKBP5. In order to do this, I cultured Neuro2A cells so that we could transfect the cells with either a CRISPR plasmid to decrease FKBP5 expression or an overexpression plasmid to increase FKBP5 expression. I used western blots to test for changes in FKBP51 protein, and that data was analyzed using integrated density in ImageJ. I found that the CRISPR knockdown plasmid successfully decreased expression of FKBP51 in cells and that the overexpression virus upregulated FKBP5. My second goal was to validate the CRISPR FKBP5 knockdown in vivo. Using Pet1-CRE mice that express Cre recombinase in serotonin neurons, we injected the CRISPR virus for a control virus into the DRN. I then used fluorescent in situ hybridization to look for changes in FKBP5 mRNA levels. I found that CRISPR successfully reduced FKBP5 relative to controls, indicating this virus is a viable way to reduce FKBP5 expression in vivo. This research is a clear step to better understanding stress-related neuropsychiatric disorders such as depression, anxiety, and PTSD.

Crowded farmworker housing conditions are associated with stress and adverse mental health effects. The H-2A program, administered by the Department of Labor (DOL), grants employers the ability to apply for foreign workers to perform temporary agricultural labor. H-2A employers are required to provide housing for workers at no cost. We sought to describe the distribution of H-2A worker housing and crowding within Region 10 (Washington, Oregon, Idaho, and Alaska). We defined crowding as the occasion when the reported total workers certified exceeds the reported total occupancy (the total occupancy capacity for all housing units per the employer H-2A application). Using data gathered by the DOL over 2020 and 2021, we examined 2,315 applications, representing 72,151 workers (54,873 in WA). Across Region 10, 22% (n=15,892) of all workers lived in mobile homes, 12% (n=8,476) in houses, 11% (n=7,832) in temporary worker housing, 9% (n=6,397) in stick-built homes, 8% (n=5,970) in labor camps, and 6% (n=4,054) in apartments. The definition of crowded housing was met by 24% of applications (n=567/2,315). A total of 36,101 workers (50% of total) lived in crowded housing conditions, with 10,569 workers in crowded mobile homes, 6,256 in temporary worker housing, 3,289 in houses, 2,931 in labor camps, 2,534 in stick-built homes, and 1,471 in apartments. Our results suggest crowding is prevalent in Region 10 H-2A worker housing and is especially of concern in mobile homes and temporary worker housing. Our work helps to guide efforts to prevent crowding and its adverse health effects among vulnerable workers.

Infection from Mycobacterium tuberculosis is the second leading infectious cause of death worldwide after COVID-19, with rates of tuberculosis infection greatest in low and middle-income countries (LMICs). Tuberculous meningitis (TBM) is one of the most severe forms of M. tuberculosis disease with over half of all cases resulting in death or neurological consequences. Recent studies in our lab have found that single-nucleotide polymorphisms (SNPs) in MUC5AC, a secretory lung mucin, are associated with increased TBM susceptibility, morbidity, and mortality. The purpose of my study is to identify the functional MUC5AC SNP. Four candidate SNPs were selected within the MUC5AC promoter region based on high linkage-disequilibrium scores across multiple global populations with a SNP in the MUC5AC promoter, rs28737416. I utilized molecular cloning techniques to combine a luciferase-expressing plasmid with isolated regions of the human MUC5AC promoter containing the SNPs of interest, and subsequently transformed this recombinant plasmid into competent cells. Next, I am performing in-vitro, site-directed mutagenesis to investigate how genotypic variation in each candidate SNP influences promoter function by measuring luciferase expression. I anticipate variants in at least one SNP of interest will reduce gene expression (measured by luciferase expression), indicating functionality. Characterization of this genetic mutation will provide insight into TBM susceptibility across populations and could inform studies of novel therapeutics to treat TBM.


This paper describes the expression of a non receptor tyrosine kinase, cymric (Uro-1), a HTK16-like (HydraTyrosineKinase-16) gene in Molgula oculata, an ascidian species with a tadpole larva and proposes that it plays a role in muscle development during the development of the tadpole phase. Cymric is also a member of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of non-receptor TKs, a group of similar tyrosine kinases that can be found across many invertebrate species, but whose function is not yet known. The role of cymric in the development of the tail in tadpole ascidians was suspected after a subtractive hybridization showed cymric is expressed in M. oculata, a species with a tail and not in Molgula occulta a closely related ascidian whose tadpole is missing a tail. In Situ hybridization shows that in M. oculata embryos, cymric localizes to primordial muscle cells. Through transcriptome and genome analysis using both ANISEED and NCIB we show that the tyrosine kinase of the cymric gene in the M.occulta is disrupted by a large retrotransposon insertion. This indicates that, although the mechanism is still not known, cymric is involved in determination of muscle cells. A better understanding of the role cymric plays in development in ascidian species could provide insight into its importance in the many other invertebrate species which also express SHARK proteins.

The drug rapamycin can increase lifespan in a variety of model organisms by repressing the activity of the mTOR complex, a cellular component required for growth and development. Other than one small study that looked at genetic variation in the effect of rapamycin on lifespan in fruit flies (Rohde et al., 2021), little is known about how natural genetic variation affects the response to rapamycin. Previous work by the Promislow lab, utilizing developmental time to indicate rapamycin’s affect, has shown that some strains of fruit flies are completely resistant to rapamycin while others are sensitive. These genotypic differences are also reflected in the metabolome, the complete set of small molecules and metabolites present within cells. Metabolome analysis of these strains revealed significant differences in metabolite concentrations between resistant and sensitive lines. Interestingly, when we treated sensitive strains with rapamycin, their metabolome profiles were like those of starved larvae. I hypothesize that rapamycin is affecting larval ability to take up nutrients and that the starved metabolome is a result of actual starvation. To test my hypothesis, I am designing a starvation assay to compare the death rate of sensitive and resistant larvae. Measurement of resistance to starvation is taken two days after rapamycin treatment by transferring larvae to nutrient deficient food. The duration of time for individual larvae to die is recorded, and the death rates between the two populations are compared. If my hypothesis is true, the sensitive larvae will have a higher death rate than the resistant larva. If death rates are similar however, that could mean that rapamycin does not cause a nutrient deficit and there is another explanation for its effects on the metabolome. This study will provide insights to the underlying mechanisms of sensitivity to rapamycin, and why it might differ between individuals.

The Pacific Northwest is home to a lush lowland forest that continually changes. Quantitative monitoring is essential in understanding forest health and climate change. The primary task was to help create a comprehensive, multi-year, dataset of floral phenology for Green River College’s learning forest; botany and ecology classes participated together in this endeavor. This quantitative data set allowed us to establish baselines for our forest so we could ask questions about how our forest changed over time. How do the native species compare to the invasive species? How many species are there, and which is most abundant? Using the ArcGIS Survey123 app, we measured floral species, abundance, GPS location, and phenology stages (buds, flower, seed/fruit, and leaves). The data was then analyzed using Qlik, an online visualization software. In 2022 alone, we compiled ~13,000 observations, estimating ~3.2 million inflorescences across ~140 species and are currently curating data from 3+ years. When looking at the floral expression in our forest from the last two dry summers, one native species (Wild Ginger) has disappeared, while many non-native and invasive plants were able to thrive. With our data set we can quantitatively look at entire plant families and monitor ecological change. A secondary benefit of this endeavor was to improve the educational capacity of our classes. Students were fully immersed in the forest and floral details. Photographs from students also helped create a repository for plant images of floral phenology stages which strongly aids in identification and higher level data quality. Our project helped undergraduates contribute scientifically while learning about their world. These contributions can help inform decisions on issues such as mitigating climate change or ecosystem preservation. Future directions include expansion of this to city parks and popular hiking trails to broaden our quantitative understanding of our ecosystems.

The alpine zone has been underrepresented in herbarium collections due to its difficulty in access and short growing season. Despite its underrepresentation, the alpine zone presents a unique opportunity to study climate change impacts due to species; limited ability to migrate to more suitable habitats. For this study, we are examining the Cascades Range from the Canadian border to Mount Adams. Our primary objective is to understand the distribution patterns of alpine species richness and how it is influenced by latitude and elevation. Our secondary objective is to see if these patterns in phytogeography correlate to species; life history characteristics of dispersal, pollination mode, and flower color. To research these questions, we created a species list of Washington's alpine plants using 50 Peaks Project data, historical herbarium records, and literature references. To assess latitudinal course patterns of species richness along the Cascades range, we created three relatively equal zones and scored the presence of each species in it. For statistical analysis, the total number of species per zone will be tallied and Chi-square analysis will be performed to test for significant differences in species richness. We will use regression analysis to quantify the relationships between latitude and the number of peaks, and latitude and average elevation. To compare life history traits across the three zones, we will analyze frequency distribution of those traits. Our preliminary results for latitudinal patterns indicate that the North Cascades have the most species while the Southern and Central Cascades are nearly tied. The final results from this study will inform the selection of future collecting locations and future analysis for species richness among peaks for the 50 Peaks Project. Preliminary Run through the Burke Herbarium, the 50 Peaks Project collects plant specimens to document diversity and distribution in Washington's Cascades Range alpine zone.

Students in a statistics course made concept models throughout the course. Previous studies suggest that making concept models is a useful way for instructors to assess learning. The growth and changes of these student-constructed models may serve as an indicator of how a student constructs and organizes their cognitive structure. But, little is known about how much impact the Covid-19 disruption has had on a student’s cognitive restructuring in STEM courses. Therefore, we ask the following research question: What factors best explain the changes in the student-made models about statistics as representations of their cognitive structure? To address this research question, we analyzed concept models from three assessment points and two quarters of the same course, one taught before the Covid-19 disruption and the other during the Covid-19 disruption, to estimate the impact that the distributions had on their cognitive structures. After converting the 180 student model into graphs, we calculated the number of concepts in each model to build a linear model to compare a quarter taught before and during the major disruptions. First, we created a box and whisker plot that shows model growth across two different quarters, pre and during, and then we created a model to explain how the disruption term influenced the rate of concepts being added to student models at three different assessment points of the course. Our preliminary results revealed that students in the emergency online environment added concepts to their models at a significantly slower rate. Future directions and limitations will be discussed. This work is important because it helps teachers and professors understand how college students are learning in different environments.

The dramatic increase in antibiotic-resistant Pseudomonas aeruginosa infections makes it necessary to find new approaches for treatment. P. aeruginosa employs a communication system called quorum sensing (QS) that uses density-dependent accumulation of small diffusible molecules to mediate the production of factors that benefit the entire bacterial community; creating a potential drug target. P. aeruginosa has two QS systems: las and rhl. A previous analysis of the laboratory strain PAO1 revealed that mutations in the las system result in individuals that act as social cheaters that reap the benefits of communally produced goods without expending energy on their production. lasR mutants have a fitness advantage when cultured in a medium requiring the activity of the community goods for survival. I set out to better understand the rhl QS system. I was interested in determining if ΔrhlR mutants act as social cheaters in co-culture with rhlR-competent strains. To make that determination, I screened 12 clinical isolates from the Early Pseudomonas Infection Control (EPIC) study for the ability of their isogenic ΔrhlR mutants to grow in QS and non-QS selective media and their ability to persist in coculture with the parent strain. I also competed these strains against isogenic ΔrhlR mutants by growing them in coculture and using flow cytometry to determine the relative final frequencies of the parent strain and ΔrhlR mutants. Finally, I aimed to determine if rhlR mutants arise spontaneously from the parent strains in a long-term growth experiment by sequencing and functional analysis of the mutants. My preliminary work demonstrates that a small subset of the ΔrhlR mutants dramatically increase relative to concentrations of EPIC strains in coculture and mutations in rhlR do arise in vitro; however, their exact functional effects are still to be determined.  Understanding ΔrhlR cheater dynamics may provide therapeutic targets for antibiotic-resistant P. aeruginosa infections.

Alveolar macrophages are vital immune cells, residing in the distal lung parenchyma. They provide the first line of defense against pathogens like mycobacterium tuberculosis (Mtb). They help initiate immune response within the lung and respond to viral infection. Mtb is the pathogen causes tuberculosis. This disease is fatal, and is the leading cause of death among infectious diseases globally. TOLLIP is a ubiquitin-binding protein that is involved in regulating innate immune responses by interacting with many receptors and the transport of endosomal cargo. Currently, TOLLIP’s role in TB pathogenesis is unknown, however, mice without the TOLLIP gene (Tollip-/-) have more severe Mtb disease. The goal of my project was to measure effects of adding mycolic acid and integrated stress response inhibitor (ISRIB) to macrophages of Tollip-/- and B6 Wild Type mice in vitro. I conducted the Bronchoalveolar Lavage (BAL) technique to extract macrophages from the lung via the trachea of B6 Wild Type and Tollip-/- mice. After macrophages were harvested, I plated them in mycolic acid and ISRIB in according wells with media. The cells were then infected with tuberculosis in the animal BSL-3, and data was collected through enzyme-linked immunosorbent assay technique and critically analyzed. Results illustrate that in both B6 Wild Type and Tollip-/- macrophages, adding ISRIB and mycolic acid results in an increase in production of the cytokine TNF, but Tollip-/- had a higher increase. Furthermore, the Tollip-/- samples had high variation in amount of TNF produced while Wild Type had minor amounts, raising further questions about how TOLLIP-deficient alveolar macrophages react when mycolic acid or ISRIB is added. Ultimately, the findings from this research, which is better understanding what specifically stimulates certain cytokines to help immune response to tuberculosis, is essential, and this is a small stepping stone in the broader goal of developing therapeutics for tuberculosis.

Despite a rise in egalitarian views over the past decade, the trend has recently slowed down and scholars are struggling to understand why. Past research shows an unexpected trend of young millennials, specifically men, reverting to traditional gender norms. Childhood family instability is proposed as one possible explanation, but it has not been examined empirically. The goal of this project is to understand how one form of family instability, divorce, impacts men’s view on love, marriage, and gender norms. The research is driven by two questions. First, how does the experience of divorce affect a father's view on gender? Second, did the father’s post-divorce view on gender affect the son’s attitudes, and, if so, how were these attitudes transmitted from father to son? The data will be collected through individual semi-structured interviews with divorced fathers who had custody of their son and the sons of these fathers who are at least 18. The study expects that children of divorce will reject egalitarian views because they have observed their parents separate through their attempt to uphold egalitarian views. While this project only examines males, future research can expand this study through interviewing single mothers and their female children. This project will help address the puzzling phenomenon of why there is a declining trend toward egalitarian gender views.

Many genes involved in different cell differentiation processes are known, however it is more challenging to know the history of transcription in each individual cell within a new organ. To fill that gap, I am building and testing a genetically-encoded recorder that uses two serine integrases to follow expression in developing roots of the model plant Arabidopsis thaliana. An integrase is a protein that binds to unique DNA regions, called integrase sites. Once bound, the integrase can perform an inversion or excision of the DNA between the integrase sites, depending on how the sites are oriented relative to one another. In a one-integrase design, we put a constitutive promoter between two fluorescent reporter genes, flanked by integrase sites. When the integrase is expressed under the control of a promoter for a developmental gene, there is a switch between the reporters only in the cells that are undergoing that developmental program. I am now using molecular cloning techniques to build a recorder that can track the expression of two different genes, using two different integrases. My integrase-based recorder will provide insights into when and where genes are “switched” on and off to promote cell specification. In the future, reporter genes can be replaced by genes that control development, and, in this way, we can engineer plants with different root structures. Control over root architecture could lead to plants that are more resilient to heat and drought.

Engineered genetic circuits provide an environmentally friendly path to chemical industries, including fine chemicals and therapeutics. To effectively modulate genetic circuits, a programmable tool to control multiple genes is necessary. CRISPR-mediated gene activation (CRISPRa) is an emerging tool suitable for this purpose. In CRISPRa, a nuclease-deficient dCas9 protein is used to deliver a transcriptional activator domain (MCP-SoxS) upstream of genes of interest. A complementary guide RNA (gRNA) enables dCas9 recruitment to any DNA target. Despite the programmability of CRISPRa, the number of genes that can be simultaneously regulated remain unexplored. In this work, we aim to experimentally investigate the number of gRNAs limitation in the chemical bioproduction context. First, we designed CRISPRa circuits with an increasing number of guide RNAs encoded on plasmids constructed with a scalable and high-throughput technique via Golden Gate Assembly. CRISPRa circuit performance was then evaluated by simultaneously regulating multiple fluorescent proteins as a proxy for multi-enzyme cascade in biosynthetic pathways. Increasing the number of gRNAs was found to decrease CRISPRa activity, suggesting competition of CRISPRa components. Furthermore, we applied the constructed circuits for metabolically engineered pathways in P. putida regulating production of p-aminocinnamic acid (pACA), a precursor for polymer synthesis vital in photovoltaic and biomedical applications. Bioproduction of pACA in P. putida was enabled by simultaneously regulating 9 heterologous genes. The outcome of CRISPRa circuits will be analyzed via High-Performance Liquid Chromatography (HPLC).The implication of this work will allow us to construct large scale CRISPR genetic circuits and optimize multi-gRNA CRISPR circuit integrations into other systems such as non-model organisms and cell-free systems, which will expand metabolic engineering capabilities and chemical productions beneficial in a wide range of biosynthetic applications.

 As climate change worsens, flooding from extreme weather and sea-level rise continues to threaten coastal populations and energy infrastructure. Small, isolated island states like Puerto Rico have weaker electrical grids and are especially vulnerable. Coastal habitats such as mangroves and coral reefs buffer shorelines and offer natural protection against these storms. To identify where habitats reduce the risk of flooding and erosion, we used a spatial model that takes in biophysical data and estimates an exposure variable for every 250m of coastline. The model shows that habitats safeguard 250,000 people living on vulnerable coastlines. Most of these people live in major port cities with substations and fuel terminals that deliver power to the entire island. As urbanization and global warming further degrade coastal habitats, Puerto Rico loses its best defense against tropical storms. Our results highlight the importance of sustainable development planning, especially as the island invests in its renewable energy transition. The spatial model can help prioritize which vulnerable communities receive resilience funding and where to avoid siting tourism to preserve ecosystems. Our model also reveals degraded habitats that could be targeted for ecological restoration. In future projects, we will apply the model to other states to explore relationships between communities, energy, and climate across multiple land and seascapes.

Current smart home devices understandably prioritize the needs of a primary user/owner, who is also typically the purchaser of the device and a corresponding subscription plan. Yet smart home cameras and other smart devices with microphones, location tracking, and other spatial sensing capabilities invariably impact the privacy of people nearby, such as family, friends, guests, neighbors, and domestic workers. We refer to these affected nearby people as adjacent users (or adjacent subjects) because they may interact with smart devices but do so with relatively little or no direct awareness, consent, access, control, or benefit. Although work in privacy and security has begun to address the privacy needs of adjacent users, there is little design research that has responded with either concrete interventions like proposals or prototypes. We present a novel speculative design of a smart home camera called Arca with a physical camera prototype and a mobile application. A significant insight of our empirical and design research is that the most common issues with smart camera privacy is the interpersonal tensions and conflicts stemming from inadequate disclosure, consent, autonomy, and transparency from primary owners. Whereas traditional privacy/security research often focuses on harms from improper disclosure of personally sensitive information, our research suggests that many adjacent users do not necessarily mind being recorded, they do mind the lack of “communication,” “respect,” and “professionalism” from primary users. Furthermore, our studies reveal that even if our specific privacy modes and access sharing features are not regularly used, they may nonetheless function as mechanisms to facilitate better, more open conversation between primary and adjacent users. We continue our work with the goal to enhance adjacent user privacy and experience with privacy-sensitive camera features and reduce tension between adjacent users and primary users.

Smartphone detection of anemia using patient photos has the potential to provide a non-invasive method of measuring hemoglobin levels, introducing the possibility of increasing the accessibility and cost-effectiveness of current practices. While traditional methods of anemia detection require a complete blood count by a trained healthcare professional, smartphone detection instead relies on the user to take a high quality picture of their fingernails. However, it currently lacks the ability to provide feedback to the user on the quality of their image. For example, an overexposed image or one with low fingernail visibility can lead to inaccurate predictions of hemoglobin levels. We propose that machine learning classification methods can analyze these patient images to estimate the image quality and predict the effectiveness of smartphone detection of anemia for a given image. With various classical machine learning models, we demonstrate and compare the capabilities of each in classifying images of patients’ hands as being of “good” or “bad” quality (or on a more granular numerical scale) when given features of the images. Preliminary results show that a logistic regression model reaches 91.4% accuracy labeling images when compared to empirically assigned labels, and we expect iterative models to achieve improved performance. When completed, we would propose that this classifier could be used in the field to identify if patient image is of high enough quality to produce an accurate measurement of hemoglobin levels in real-time, providing feedback on the phone to adjust or correct the image-taking process.

Ophthalmic drug administration has been increasingly prevalent in recent years, with eyedroppers being utilized to administer costly medication like that for glaucoma. There haven’t been many solutions addressing eyedropper instillation for those with preexisting conditions like arthritis, who often deal with a host of problems when administering them: producing the necessary force to distill a drop, aiming the drop into the eye, and contamination of the eyedropper tip. We are testing the question of whether accessible eye drop aids can significantly improve eyedrop compliance and distillation for the elderly. Solutions to eye drop administration can save money and make the overall process easier for many patients. Existing solutions on the market seem to address the issue of contamination using apparatuses that press onto the lower eyelid, but there is still much to be desired with the force and aim required. Many require the use of gripping or squeezing, motions that many elderly patients can’t apply as much force with. I propose a couple of solutions to these problems in the form of eyedropper aids that each make use of a few different methods, including translating the motion, applying the force with different limbs, and even mechanizing the force required. Through a quantitative study, I hope to eventually test these prototypes through an ophthalmology clinic among a wide variety of elderly. Assessing these prototypes through both questionnaires and observation, I hope to notice an increase in effectiveness from previously existing apparatuses. We will use a survey to ask a variety of questions to around 100 elderly patients with varying expertise in eye drop instillation. The survey will ask whether the tool was more useful, easier, how hard it was to assemble, and we will also monitor quantitatively whether the accuracy of drops actually instilled was better. This work hopefully saves patients money from medication cost from a reduction in wastage, allows for better administration of medicine, and eases the process of distillation.

The glymphatic system, which is primarily active during sleep, is a network of astroglial perivascular channels within the brain that allows for cerebrospinal fluid (CSF) influx and exchange. Glymphatic exchange plays a crucial role in the clearance of amyloid, a hallmark in the development of Alzheimer’s. Recently, a bidirectional relationship between Alzheimer's disease and sleep has also been suggested with amyloid deposition associated with mid-life sleep disruption. However, the mechanistic link between sleep disruption, particularly over chronic time scales, and the development of Alzheimer’s pathology remains unclear. This study investigated whether chronic sleep disruption, similar to that experienced in aging population, impacts downstream Alzheimer’s-related neuropathology. We hypothesized chronic sleep disruption will result in decreased glymphatic function and increased amyloid plaque burden. This experiment utilized a chronic sleep disruption model using Lafayette Sleep Fragmentation chambers, where mice underwent either chronic sleep disruption every two minutes during normal sleeping periods (daylight hours) or normal sleeping conditions (sham) from 10 weeks to 18 weeks of age (n=120). After eight weeks of sleep disruption or sham exposure, glymphatic function was assessed by dynamic in vivo near infrared imaging following stereotactic CSF tracer injection. Animals were perfusion fixed, cryosectioned, and glymphatic function was further assessed by measurement of fluorescent cerebrospinal fluid tracers in brain tissue. Aquaporin-4 localization, amyloid plaque deposition, and markers of astroglial and microglial activation were assessed by immunofluorescence. The collected data demonstrated that sleep disruption significantly increased neuropathological outcomes. The measured impact of glymphatic function was also correlated with these downstream pathological effects. These findings could be an indicator of interactions between neurological disease progression and an inflammatory expression after sleep disruption. They can also shed more light on the complex relationship between Alzheimer’s disease progression, the glymphatic system, and chronic sleep disruption.

Traumatic brain injury (TBI) is a leading cause of death and disability worldwide and has been established as a risk factor for neurodegenerative diseases such as Alzheimer’s disease (AD). Neurofibrillary tangles (NFTs), aggregates of intracellular tau, are hallmarks of AD and are observed in the post-TBI brain; however, the mechanisms that contribute to tau aggregation and accumulation are not well understood. One key mechanism that may contribute to this tau aggregation is decreased clearance by the glymphatic system, a perivascular pathway that clears solutes, including tau, from the brain. PS19 mice with tau pathology were crossed with Aqp4-/- mice lacking the astroglial water channel aquaporin-4 (AQP4) or Snta1-/- mice lacking perivascular localization of AQP4. Behavioral tests were performed on the PS19:Aqp4 transgenic crosses at 4 or 6 months of age. Brain tissue was collected and stained for markers of phosphorylated-tau (p-tau) pathology. Sham or mild TBIs were performed on PS19:Snta1 transgenic crosses at 3 months of age. I performed behavioral testing at 4 months (1 month post-TBI) or 6 months (3 month post-TBI). Brain tissue was collected and stained for markers of p-tau pathology. I imaged this immunostained tissue and quantified the pathological tau burden. I observed that Aqp4 deletion was sufficient to exacerbate tau pathology in PS19 mice at 6 months old, in the absence of TBI, and more advanced tau pathology was observed in PS19+Snta1-/- mice at 6 months old (3 months post-TBI) compared to PS19+Snta1+/+ that also received a TBI. Loss of AQP4 or loss of perivascular AQP4 promotes tau pathology in a mouse model of tau pathology. These studies may provide a mechanistic basis for the vulnerability of the post-traumatic brain to tau aggregation and neurodegeneration and suggest that targeting glymphatic dysfunction may be useful in the prevention and treatment of neurodegeneration.

The application of engineered origami structures has become increasingly popular for the past decades. Among the variety of origami patterns, the Resch pattern recently began to reveal its potential in the field. It is a planar tessellation composed of pre-defined polygons. Some of its interesting properties feature controllable morphability and self-supporting reformability. However, its static and dynamic response to an external load remains to be unveiled. Therefore, this project aims at studying the Resch-patterned origami structure’s folding behavior, associated stored potential energy, and impact mitigation capability. We first constructed its kinematic model in order to accurately predict the folding motion of a tessellation. The model was then augmented with a torsional spring embedded in each crease line to predict the force-displacement relationship under external loads. We fabricated a series of prototypes with polymers and conducted static compression tests to compare and calibrate the kinematic model. The force-displacement curve generated from the kinematic model was fitted to the experimental result, that the two curves shared a very similar profile. As for the physical model, it demonstrated consistent force-displacement and energy dissipation properties over cyclic compression-expansion tests. After studying the fundamental behavior of the Resch pattern, we performed dynamic impact tests on our physical model to explore its potential for impact mitigation. A cylindrical weight was dropped on the center of our Resch pattern at its natural posture, and by tracking the motion of the impactor, we determined the energy and momentum dispersed in the impact. In summary, the Resch-patterned origami structure’s unique properties exhibit great potential for impact-mitigating structures for deployable panels with repeated loads. We envision that the energy handling mechanism of the Resch pattern investigated herein can be employed in numerous engineering structures, including lightweight deployable architecture.

In the era of Internet of Things, the operation of various kinds of sensors or devices on the shelves in warehouses or supermarkets often requires batteries or complex wire management. To address this issue, we propose a charging solution utilizing the near-field wireless power transfer (WPT) with multiple relay resonators, also known as a multi-hop WPT system. In this study, we designed and simulated several coil geometries for the WPT system to ensure high efficient power can be delivered from a transmitter to a receiver through various coil hopping configurations. After evaluating the trade-off between the coupling coefficient of coils in parallel and series as well as the design complexities, we constructed many unified coils in one geometry. We then measured the coil-to-coil estimated efficiency using the scattering parameter obtained through a Vector Network Analyzer on a foam board shelf. Our results show the efficiency range from 9% to 81% in the worst and best hopping configurations, respectively. Furthermore, we proposed a power efficiency optimization approach to improve the worst hopping configuration by up to 80%. We anticipate that the success of this work will significantly reduce the staff cost associated with the maintenance of wire management and charging systems on each shelf. It will also simplify the assembly process and enhance the accessibility of smart shelves while potentially mitigating environmental impact by reducing battery usage.

Starting in European folklore, antisemitism has gone largely unnoticed in fairy tales. Antisemitism present within our modern fantasy standards stems in large part from European folklore, further manipulated by rising antisemitic propaganda in the late nineteenth century, coalescing in Nazi propaganda, and internalized as stereotypes by storytellers, thus leading to a double standard of otherizing and assimilation in Western culture. As folklore evolved into the current fantasy genre, these stories transferred antisemitic and stereotypical of villainous Jewish characters into our modern-day media. Antisemitic caricatures populate our media, and gentiles, or non-Jews, often don’t realize it because of how pervasive these stereotypes have become in the main body of Western culture developed over the millennia. Drawing connections to the villainized images of a Jewish-coded character throughout fantasy stories and films, this paper uncovers the systemic and institutional problems Western media pushes onto an indifferent public, where thinking critically about our media doesn't extend to antisemitism. From case studies including Tolkien's work, Barbie movies, and the Harry Potter series, this paper identifies antisemitic stereotypes that have become impossible to avoid in media. Through widespread acceptance of these stereotypes, we perpetuate the misconceptions of our predecessors and otherize the Jewish body. Through perpetuation of hateful stereotypes in modern media and a lack of acknowledgement of ongoing antisemitism, we normalize the person of Jewish descent as the villain. Spotting these stereotypes is the first step in overcoming this imagery. Subversion becomes the next step to undo the damage to Western culture and modern media; reinterpreting and rewriting antisemitic stories as aspirational stories is a way to subvert the ideas of what a hero and a villain can look like.

Titus Livius, better known as Livy, was a Roman historian writing at the beginnings of the Augustan age and the fall of the Roman Republic. Livy wrote his magnum opus, Ab Urbe Condita, as a moralizing history in response to the moral degradation and civil strife he experienced during the collapse of the Republic. The first seven chapters of Book I outline Roman prehistory before Romulus’s reign and contain several imitations and allusions to archaic poetry and Classical historiography. Livy himself believes that the subject of his writing would have been better suited for poetry than prose. This project aims to provide quantitative evidence for Livy's claim by quantifying and analyzing instances of literary devices of earlier poetry such as alliteration and etymologizing to determine whether a poetic influence is implicit in Livy’s prehistory of Rome. Preliminary results based on the quantity of alliteration present in Livy's writings suggest some influence by earlier poetic sources. However, it is difficult to substantiate any conclusions on statistical grounds due to fragmentary literatures. Instead, this project also proposes further hypotheses about source accessibility and poetic inclination that may explain the quantitative phenomena.

 When homeless youth turn 26 years of age many of the organizations and resources they have been utilizing for survival, such as food and shelter, become unavailable as many organizations no longer consider them youth. The purpose of this study is to identify gaps in the transition period for homeless youth moving towards adulthood to explore ways to make the transition more efficient and accessible. I conducted a systematic literature review among databases and ten studies are included. Restrictions on studies included were publication dates after the year 2000, publications in English only and studies done in the United States. This study includes data from both currently and previously homeless youth from ages 18 to 30. Several areas are identified as gaps that affect the ease of transition for homeless youth including lack of consistent support in case management or mentorship, preparing the youth ahead of time for exit out of youth programs, housing models being utilized at time youth are aging-out at twenty-six years old, and how the youth values themselves. Recommendations for programs such as a housing first model that collaborates with mentoring and community support and youth preparation programs that begin at least a year before a youth turns 26 years old are discussed in this study.

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

Following the enactment of Florida’s Parental Rights in Education Act (2022), the country is faced with an increase in anti-LGBTQIA2+ bills in state legislatures (e.g., SB 30; MO SB134). With this onset of hostile policy, it has become increasingly important that members of the LGBTQIA2+ community are supported in their identity. Given this climate, our research team has aimed to understand how we can further support LGBTQIA2+ youth in school environments. Research shows that LGBTQIA2+ affirming sex education can support a variety of health outcomes for young people (e.g., Snapp et al., 2015). What is less agreed upon is the active ingredients of what makes sex education affirming for LGBTQIA2+ youth. Therefore, our research team is examining the question: What are the necessary components involved in delivering LGBTQIA2+ affirming sex education? To answer this question, we will conduct a comprehensive literature review (CLR: Onwuegbuzie & Frels, 2016) to assess sex education curricula that affirm LGBTQIA2+ identities. An integrative framework that employs both narrative and systematic styles will be applied to ensure a reflective research lens. Our primary research modes consist of empirical articles and educational policies influencing LGBTQIA2+ sex education. Preliminary findings show a necessity for more comprehensive preservice teacher training that resists heteronormativity and cisgenderism (Goodrich & Barnard, 2019; Naser et al., 2022) and covers a larger range of topics (Naser et al., 2022). Results from this study could inform teachers in presenting a sex education that affirms LGBTQIA2+ identities along with educational policies that creates curriculum standards for inclusion. Discovering elements of sex education that affirm LGBTQIA2+ identities could create a foundation for educators to utilize, which could enhance the mental and physical health of youth with these identities. Additionally, sex education that affirms LGBTQIA2+ identities can combat anti-queer public policy through consciousness-raising and ultimately grassroots organizing.
 

Schools across the country struggle with the issue of meal debt and lunch shaming practices used to discourage the accumulation of negative meal balances. These practices include dumping students’ trays in the trash upon non-payment, requiring students to work off their debt, or publicly shaming them. Although lunch shaming was banned in Washington State in 2018, the impacts of this policy shift have not been studied. High-poverty schools shifted to government-subsidized universal school meal programs (USMP) to address students’ food insecurity. Studies show USMP significantly increase the meal participation of students who were already enrolled in free lunch programs. While scholars speculate stigma might explain this phenomenon, it has not been studied directly. This study explores the role of stigma in school meal participation by studying two populations; students who are considered non-poor with meal debt and students who are enrolled in free and reduced priced meal programs. This study considers the role of stigma as a barrier to school meal participation by studying meal debt and students at the eligibility margins in free lunch programs. This study addresses existing gaps in the literature through a combination of in-depth interviews and observations of schools ineligible for government-subsidized USMP. In-depth interviews with school administrators expand understandings of school-level cultures, mealtime procedures, and experiences with meal debt. Observations at a single school over a 2-month period reveal how students experience mealtime, how kitchen workers execute meal debt policies, and the dynamics between students, their peers, and authority figures in the lunchline. Findings show that stigma is reproduced in the lunch line and act as a barrier to student meal participation. 

Polychlorinated biphenyls (PCBs) are lipophilic environmental pollutants previously used in industrial and consumer products that are being found in fatty compartments of many aquatic species consumed by humans today. The gut microbiome is increasingly recognized to influence the metabolism and biotransformation of many substances including those linked to neurodevelopmental disorders (NDDs) such as PCBs. To test the hypothesis that the gut-brain axis mediates PCB neurotoxicity, we studied how maternal PCB exposure modulates gut microbiome in offspring. C57BL/6 mouse dams were randomly assigned to receive either vehicle (peanut butter and peanut oil mixture) or the Fox River PCB mixture at a daily oral dose of 0.1, 1.0, or 6.0 mg/kg body weight. To study changes in the gut microbiome, shotgun metagenomic sequencing was used to sequence the DNA of the large intestinal contents of male and female pups collected at postnatal day (PND) 28 and PND 35 (n>5 per group). The sequences were then aligned against a curated database (includes RefSeq, cell-cultured, and mouse-specific high-quality metagenomically assembled genomes) used for downstream analysis, and also the Kyoto Encyclopedia of Genes and Genomes Orthology groups (KEGG KOs) to predict functional changes of the microbiome. The analysis confirmed that maternal PCB exposure produce gut dysbiosis in the offsprings. Female pups were found to be more susceptible than males at both ages and for both sexes, PND 28 groups were more susceptible than PND 35 groups. Functional predictions of the microbiome also showed PCB-mediated increase in pathways involving ammonia production and cytochrome oxidases, but a decrease in sugar transport systems (which all may negatively influences the brain) in an age-, sex-, and PCB dose-dependent manner. In conclusion, maternal exposure to PCBs produced gut disruption in gut microbiome and may be implicated in developmental neurotoxicity through the gut-brain axis.

Osteoporosis, a polygenic disease characterized by low bone mineral density (BMD) and increased fracture risk, is the most prevalent bone disease impacting over 200 million people worldwide. Because of the associated financial burdens and reductions in quality of life, there is an urgent need to determine the genetic causes of osteoporosis. The WNT family of proteins has been implicated in numerous developmental and disease pathways, with WNT16 specifically being linked to osteoporosis risk. WNT proteins contain 24 conserved cysteines, and mutations involving half of these cysteines are associated with human diseases or disrupted development in animal models. While modifications in cysteines 1-6, 8-9, 11-12, and 24 in different WNT proteins have been defined in vivo, the impact of cysteine 10 (c10) alteration remains unknown. Wnt crystal structure suggests that c10 creates a disulfide linkage with cysteine 11 and resides in a region that directly interacts with Frizzled receptors to initiate WNT signaling pathways. My hypothesis is that loss of c10 in WNT16 will result in altered BMD indicative of elevated osteoporosis risk. To test this, I outcrossed CRISPR-generated somatic zebrafish mutants harboring mutations at the wnt16 locus that target the c10 position, and isolated wnt16^w1012 mutants. Sanger sequencing and sequence alignment revealed a three amino acid deletion at Cys214, corresponding to c10 (p.Cys214_Gly216del). Analysis of micro-computed tomography scans showed significant decreases in wnt16^w1012 mutant centrum length, which matches wnt16 knockouts. My data indicates that wnt16^w1012 mutants phenocopy wnt16 knockouts, suggesting that c10 plays an essential role in WNT16 secretion and/or activity. My data further suggests that mutations that alter c10 have potential to contribute to osteoporosis pathogenesis. My ongoing studies are focused on further characterizing musculoskeletal phenotypes in wnt16^w1012 mutants and understanding the consequences of the mutation on protein structure through computational modeling.

It is well-established that mutations have impacts on an organism’s fitness; however, the fitness effects of mutations are not static, and can vary depending on environmental contexts, such as the species in which a mutation is found. Evolution of the same gene in different species could thus lead to the evolution of different phenotypes, as different species would favour different sets of mutations. If that gene could be exchanged between species, it could lead to increased evolutionary possibilities, as high-fitness genotypes that require a prerequisite deleterious mutation in one species could become accessible if the mutation is not deleterious in another. Our research examines how the presence of two bacterial hosts, Escherichia coli and Klebsiella pneumoniae, could affect the evolution of an antibiotic resistance-conferring TEM-1 β-lactamase gene located on a conjugative plasmid. If different hosts confer different mutational effects to TEM-1, the process of horizontal gene transfer (HGT) that allows mutations to be shared between species could open up more mutational possibilities than those accessible in either single-species population alone. We tested this hypothesis through three rounds of experimental evolution in the presence of the antibiotic cefotaxime, where we evolved two single-species E. coli and K. pneumoniae populations and one multi-species population where HGT was simulated with a shared plasmid pool. We are now reconstructing the genotypes found in all three populations after each round to assess how much antibiotic resistance they confer in both species, and hope to see if the genotypes acquired under HGT treatment provide higher resistance compared to the single-species populations. Our results have practical implications for the predictability and nature of antibiotic resistance development in the real world, a current global health crisis, and potentially motivate further study in predicting resistance emergence in clinically encountered multi-species populations.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with a mortality rate of ~30%. In ~80% of cases, MCC development is attributed to the integration of Merkel cell polyomavirus (MCPyV) DNA into the host’s genome, leading to the expression of viral oncoproteins and tumorigenesis. Developing treatments that sustain immunity against MCC is imperative to address recurrent and/or progressive disease. In many cancers, tumor-infiltrating B cells have been associated with better prognosis and response to immunotherapies. However, the mechanisms by which B cells contribute to tumor immunity in humans have been difficult to resolve in part due to the inter-patient heterogeneity of tumor-specific antigens. The shared nature of MCPyV tumor antigens in MCC allows for MCC-specific B cell responses to be studied across patients. Using DNA-barcoded and fluorescently labeled viral oncoprotein tetramers, we analyzed the transcriptome, proteome, and receptor repertoire of MCC tumor-infiltrating B cells in 12 patient samples at single-cell resolution. From paired heavy and light chain sequences, we cloned 8 antibodies from B cells specific for the MCPyV oncoproteins to confirm binding to MCC-specific antigens. Transcriptomic and proteomic analyses of MCPyV-specific B cells revealed heterogeneity of intra-tumoral B cell responses. Interestingly, we found that the absence of MCC-specific germinal center (GC) B cells in MCC tumors associates with disease progression: ~80% of patients with no detectable GC B cells had MCC progression within a year post-surgery, whereas patients with detectable GC B cells remained progression-free a year after surgery (n=12, p=0.0043). These results suggest strong synergy between B cells and T cells may regulate tumor growth, as B cells rely on signals presented by T cells to differentiate into GC cells. Our long-term objective is to identify B cell phenotypes associated with anti-MCC responses to develop therapeutics that boost cancer-specific immunity.

 The Circumgalactic Medium (CGM) refers to the vast outer regions of gas within the gravitation influence of a host galaxy. The CGM contains a significant amount of gas flowing into and out of the host galaxy; transitions such as HI and OVI are frequently observed in absorption along quasar sightlines that pierce the CGM within close projected impact parameters. Absorption feature properties such as column density and central wavelength can provide insight into the content and kinematics of the CGM. Our research team, known as the Werk SQuAD (Student Quasar Absorption Diagnosticians), is currently assessing the two-dimensional distribution of gas in the CGM around galaxies with a redshift value of less than 0.65. We use measured gas column densities from the Hubble Space Telescope’s Cosmic Origins Spectrograph quasar absorption spectra and a program called GALFIT to measure galaxy inclination and azimuthal angles, to help us understand these kinematics relative to their orientation to the background quasar. All host galaxies have redshifts measured spectroscopically by the Werk SQuAD as part of the CGM2 survey. In particular, the galaxy inclination angle, combined with the quasar azimuthal angle and absorption-line column densities provide a measure of the extent to which gas flows concentrate along the major or minor axes of galaxies (along the disk of the galaxy or flowing near or away from the galactic center). Here, we examine trends in the CGM with quasar azimuthal angles and ion column densities. Ultimately, our goal is to further understand the physical interdependence of the gas that flows through the CGM and galaxy evolution.

Massive stars place powerful constraints on stellar evolution and are observed in a menagerie of exotic evolutionary phases. These objects play a crucial role in regulating their environments. They drive the chemical evolution of their host galaxies, and set the energy balance of their surroundings via feedback processes. Due to the importance of massive stars, placing constraints on their evolution serves as a key to understanding galactic ecosystems. Notably, stellar variability is a powerful probe of the poorly-constrained physics of massive star evolution. In particular, variability studies on ensembles of evolved massive stars can significantly constrain stellar evolution. We aim to understand the variability of hot massive stars through a census of these objects. We accomplish this using data from the Gaia mission, cross-matched with light curves from the Zwicky Transient Facility (ZTF). We expect to characterize the evolution of massive star variability timescales and amplitudes along the main sequence and beyond. Our results will place key constraints on the evolution of massive stars. 

River otters (Lontara canadensis) are high trophic level opportunistic feeders. Their diet, examined through scat, provides key information about local ecosystems. Previous studies have observed a diet dominated by fish, including studies in California (90%), Utah (96.5%), and North Dakota (83%); however, my preliminary results on otter diet at the mouth of the Snohomish River highlight a greater variation in observed prey. The meeting of the Snohomish River and Possession Sound creates a salt wedge estuary with extensive tide flats, providing access to diverse freshwater and saltwater prey species. From 2012-2015, river otter scat samples were collected, dissected, and identified by students at the Ocean Research College Academy (ORCA). Averages over the three years showed that fish were the dominant prey type in fall, winter, and summer; however, crustaceans dominated the river otter diet in the spring. In this study, I dissected scat samples from fall 2022 through spring 2023 from 2 sample locations in Everett Marina. In the more recent data, fall 2022 has followed the pattern of these historical studies, but the winter samples have indicated a shift of fish and crustaceans. In fall 2022, 5/25 samples showed 50% or fewer fish bones, while winter 2023 showed 11/13 samples with less than 50% fish bones. Given that otters are described as opportunistic feeders, this shift in winter diet, relative to previous years, may suggest a significant environmental shift. Future research must examine physical environmental features such as changes in river discharge and/or water temperature. Identifying specific fish species in samples may also reveal prey availability throughout the seasons.

Phytoplankton production depends on a number of factors including nutrient availability, water chemistry variables, and light penetration. Previous studies have shown light penetration to be important for submerged aquatic vegetation, the primary producers that support the marine food web and the ecosystem. Possession Sound is a productive sub-basin of the Salish Sea with complex influences on local water chemistry and primary productivity. Given the significance of primary production in a salt water estuary, this study looks at the seasonal relationships between water chemistry and light penetration, measured by photosynthetically active radiation (PAR) in the Possession Sound estuary across three sites. I collected Seabird CTD and YSI EXO profile data as well as PAR sensor results, in Possession Sound from July 2022 through March 2023. Accompanied with historical data collected by past Ocean Research College Academy researchers, I analyzed site dependent relationships as well as the seasonal relationships. Preliminary analyses showed PAR decreasing with higher salinity and turbidity, but increasing with temperature. Limited connection was observed with dissolved oxygen. Studying the relationships among light penetration and water chemistry allows us to better understand the complex relationships among the key factors determining seasonal primary production.

In estuaries and marine environments, eelgrass (Zostera spp.) is considered a keystone species as many marine species depend on the beds for food and shelter. Eelgrass beds also trap sediment, stabilize substrate, reduce wave energy and reduce coastal erosion. The degradation of eelgrass beds can instigate a decline in those species that rely on the eelgrass beds. Eelgrass is traditionally mapped using aerial photographs or diving surveys, which can be time-consuming and ineffective. Another strategy is to use boat-mounted Acoustic Doppler Current Profiler (ADCP), to create transects of eelgrass beds through backscatter data. The purpose of this study is to assess the application of an ADCP to map local eelgrass beds in Possession Sound and determine its advantages over single-beam sonar. I used a boat-mounted RD Instruments Workhorse 600kHz ADCP during transects of a Possession Sound eelgrass bed. Raw data was filtered using Excel to isolate backscatter data for analysis and visualization. These results were compared to the results from a single-beam sonar system. It is expected that the height of the eelgrass and extent of the beds will be seen through the visualizations, even in winter and early spring surveys. A multibeam sonar like an ADCP will be more efficient in mapping a greater area of eelgrass beds as well as providing greater clarity in visualizations than a similar single-beam sonar through the ADCP’s emittance of multiple sound impulses at once.

Unique relationships between photosynthetic active radiation (“PAR”), turbidity, chlorophyll, and dissolved oxygen (DO) can impact primary productivity levels and directly relate to the health of an estuary. Previous studies show that microbial processes depend on PAR and dissolved oxygen. To some extent, this can govern the rate of near-surface mixing, affecting turbidity and the amount of chlorophyll produced. My preliminary results using data from Possession Sound, a salt wedge estuary, show similar patterns. By examining the previous Ocean Research College Academy’s (ORCA) profile data from 2015-2022, I observed that an increase in chlorophyll corresponded with an increase in turbidity. Further, if there is an increase in chlorophyll, there is an increase in dissolved oxygen. From the fall of 2022 to spring of 2023, I collected additional profiles, including PAR data, from three sample locations in Possession Sound. My results show that when PAR is high, chlorophyll levels and turbidity are also high. However, PAR values are inversely proportional to dissolved oxygen concentrations. This means there are correlations among chlorophyll, turbidity, DO, and PAR values with depth. However, PAR can be affected by many factors, so it is difficult to say that any one of these parameters directly relate to PAR values with depth. Future research can examine different parameters affecting PAR with depth, as availability of sunlight and nutrient levels can also affect PAR data.

Neurological trauma, from a spinal cord injury (SCI), can have devastating effects on the quality of life of individuals, often resulting in a significant loss of motor function and accompanying bowel and bladder complications. Recent research has combined use-dependent physical rehabilitation, which is the current gold standard of treatment, with the delivery of neuromodulators or electrical stimulation to improve motor-recovery outcomes after SCI. However, little work has been done to understand the scope of psychological changes that occur after SCI, or how these changes may, in turn, affect an individual's ability to recover from the trauma. To fill this gap, our project examined changes in motivation levels after a chronic cervical contusion of the spinal cord that produces impairments in forelimb-motor function. We also studied how subsequent motor recovery altered motivation. Our experiments were conducted in an adult rodent contusion model of chronic cervical SCI. We used a skilled forelimb reach-and-grasp behavioral task to assess the motor performance and injury severity of rats. To assess motivation, we recorded the duration that rats attempted to complete the reach-and-grasp task over a thirty-minute time window. Motivation levels and motor performance were assessed pre-and post-SCI and before, during, and after therapy. Our results show that motivation levels were significantly impacted by the SCI, with motivation loss positively correlated to injury severity. Surprisingly, despite significant motor recovery, motivation levels continued to remain low months after the injury. These results provide new insights into the effect of SCI on psychological factors, which will inform future investigations and the design of therapies targeting neurological trauma.

In this study, we present a dexterous implementation of the Rubber Hand Illusion (RHI) in virtual reality (VR). The RHI is a classic perceptual illusion in which a sense of embodiment of a non-self object is elicited by synchronously and congruously stroking both a visible non-self object (i.e., a rubber hand) and the subject’s actual hand, hidden from view. While powerful, the classic RHI experiment is constrained by physical reality. Here, we present a new VR-RHI implementation that integrates Unity’s collider-based physics system and SteamVR’s hand pose estimation algorithm to achieve real-time rendering of real-world collisions. This enables precise visuotactile concordance and thus induction of the RHI over a virtual hand. Data from healthy, right-handed human VR-RHI participants (n=17) demonstrated a strong, bounded, linear correlation between VR render offset and proprioceptive drift till a certain threshold. We have designed and validated a new gaze drift metric that uses integrated eye-tracking hardware and SDK support for gaze-object collision to allow gaze-based self-localization. Based on preliminary results, we believe using gaze may refine the proprioceptive drift metric by minimizing the required movement of the subject’s body and contralateral hand while self-localizing after RHI induction. In addition, we have implemented a new feature of the experiment to separate the visual and tactile sensations by showing the subject the actual hand location rendered in the virtual environment during the induction. During these trials, the subject is aware of the offset, but preliminary results suggest that we are still able to induce the illusion. Furthermore, we have also implemented a new induction method where we use movement to induce the illusion rather than tactile sensations. Finally, we have improved the experiment protocol by automating data collection and experimental loops so that the experiment can run without a third party.

We are researching adeno-associated viral (AAV) vector based gene therapy for the rare genetic muscle disorder GNE myopathy (GNEM). This disease manifests as progressive skeletal muscle atrophy leading to restricted ambulatory ability and loss of fine motor skills. GNEM is caused by defects in the GNE gene, negatively impacting the sialic acid biosynthesis pathway. The sialylation of muscle fibers does not occur without this pathway, causing a loss of muscle function due to sialic acid deficiency. AAV gene therapy can be used to alternatively express the GNE gene by directly targeting muscle or utilizing liver expression. Sialylated glycoproteins secreted by the liver can be taken up by muscle and the sialic acid recycled. Our lab plans to advance GNEM AAV gene therapy by combining liver- and muscle-specific promoters to reduce the dose of AAV required for sialylation of muscle fibers. We have cloned six tandem promoters and three control promoters that will be tested in human liver cells (HepG2) and murine myoblast cells (C2C12). The tandem promoters combine different regions of liver and muscle promoters. A double (firefly and renilla) luciferase mechanism is used to indicate promoter strength, where the amount of luminescence from firefly luciferase is measured. Our lab is currently recording the promoter strength and level of transfection in the cells. The ratio between these expressions indicate the overall strength of the promoter. We are also cloning these tandem promoters into AAV vectors with the intent of testing the vectors in mice. The aim of our research is to produce a promoter resulting in the expression of the GNE gene in both muscle and liver cells, increasing sialic acid production to aid patients affected by GNEM using a minimal dosage.

In 2020, inclement weather spoiled over 1,800 pounds of produce at the UW Farm - roughly 15% of annual production. The resilienncy tunnel, our design for a greenhouse is intended to protect crops and extend the production season by multiple months, eliminating the need to quickly harvest spoiling crops in extreme weather using single-use plastic bags to deliver to the UW Food Pantry. The execution and operation of this project will notably contribute towards action VI of the UW Sustainability Action Plan, involving a target that 35% of campus food is from local sources by 2025. Our team shares the ambitious goal of designing an agricultural structure and landscape that sustainably and respectfully gives back to the community and surrounding areas with three key pillars: Resilience, Renewability, and Reciprocity. The Resiliency Tunnel, a 1500 square foot greenhouse and 600 sf educational space, will enable the UW Farm to grow produce with higher nutritional value in greater quantities and ensure that this produce reaches food-insecure populations. The UW Farm supplies dining halls, the UW Food Pantry, and the greater community with produce each year. As the climate changes, however, the increasing discrepancy between high demand during the academic year and high production during the summer growing season results in inadequate supply during the spring and fall. A greenhouse facilitates resilience against these conditions through crop protection and temperature control. Our proposal incorporates passive thermal design, a solar array, and rainwater catchment integrated with the structure and the landscape. 

Southeast Asian communities in the United States and in Asia have a preexisting notion that whiteness equates to beauty and that fair complexions are standard. The colorist mindset is implicitly taught through the media, magazines, workplaces, and most commonly the average Asian family household. Teachings of anti-Blackness are introduced from a young age by advertising the use of whitening products, avoidance of the sun, or not being allowed to wear any clothing that exposes skin because of the risk of tanning. This generational upbringing negatively impacts the way young Asian Americans view themselves, view others, and continues the cycle of internalized colorism and anti-Blackness. My research explores how cultural upbringing, generational trauma, and family dynamic influences the understanding of anti-Blackness being something ingrained into Southeast Asian communities. Through conducting interviews and surveys, I examine how colorism is represented and inherently taught within these communities and the ways it has negatively impacted younger generations in terms of self-image. Rather than presenting statistical research, my research presents in the form of a magazine consisting of stories, original art, and portraiture. By incorporating my skills as a photographer and designer, my research encourages audiences to engage on a more interpersonal level to confront the ways colorism exists in our communities and how we can begin a journey of healing and unlearning our prejudiced biases. The results of this project are displayed through a collaborative, ongoing magazine where the interviews are transcribed as articles with photography and original artwork connecting to the theme of colorism and anti-Blackness. This magazine uses the research collected from the interviews and surveys to produce a design piece that shares experiences, allows audiences and participants to be vulnerable, and explores how socio-political topics can be translated through art/design.

Currently, nearly 10% of Americans have Type 2 Diabetes (T2D) and a further 34% have prediabetes - placing it among the most common chronic diseases in the United States. The capacity of the brain to elicit sustained remission of diabetic hyperglycemia in rodent models of T2D following intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) is now well established. Previously, we reported that prolonged (>24 hr) hypothalamic signaling by extracellular signal-related kinases 1 and 2, members of the mitogen-activated protein kinase (MAPK) family, is required for the sustained antidiabetic action of FGF1. Recent studies have now revealed that in addition to FGF1, FGF17 also stimulates the MAPK/ERK signaling pathway, which promotes oligodendrocyte proliferation and overall neurocircuit remodeling. Based on this observation, we hypothesize that FGF17 would have potent antidiabetic actions by activating hypothalamic MAPK/ERK signaling and remodeling hypothalamic neurocircuits. To test this hypothesis, cohorts of adult male C57BL/6J wild-type mice recieved a single icv injection of either FGF17 or saline vehicle. Analysis revealed that FGF17 induced sustained hypothalamic MAPK/ERK signaling, though the magnitude of this induction was less than that obtained with FGF1. We then injected FGF17 or a saline vehicle icv in a cohort of adult, male, diabetic ob/ob mice (diabetic and obese due to mutation in the leptin gene) and took daily measurements of blood glucose, food intake, and body weight. We found that FGF17 induced a transient reduction in food intake and body weight, but was able to normalize diabetic hyperglycemia for 30 days. This data shows that FGF17 is able to induce sustained activation of hypothalamic MAPK/ERK signaling and has antidiabetic actions. This is significant because sustained remission of hyperglycemia can reduce the risk of developing associated chronic diseases such as cardiovascular disease, diabetic nephropathy, and diabetic retinopathy.v Currently, I am performing immunohistochemical studies to identify the specific hypothalamic neurocircuits that FGF17 acts upon to produce these effects.

The mammalian immune system can mount distinct responses depending on the type of pathogen. In the intestine, infections with large parasites trigger a “type 2” immune response. This culminates in fluid secretion into the lumen and increased smooth muscle contractility that pushes the pathogen, intestinal parasites, through the digestive tract and out of the body. Epithelial tuft cells detect worms through their brush-like microvilli projecting from the cells into the lumen and express specific receptors that detect parasites. Once a worm is detected, tuft cells produce IL-25. Binding of IL-25 to receptors on ILC2s causes intestinal ILC2s to proliferate and secrete key cytokines that bind to receptors on intestinal epithelial stem cells and cause a remodeling of the intestinal epithelium. While tuft cells have previously only been associated with parasitic infection, a significant expansion of tuft cells was also found in the distal small intestine of tumor-bearing APC-min mice. In colorectal tumors, IL-25 was found to promote intratumoral ILC2s and suppress anti-tumor immunity, which led to increased tumorigenesis. We used immunofluorescence microscopy to visualize and quantify Flare25, a reporter of Il25 transcription, in tumors and healthy tissue of APC-min mice. The role of IL25 is therefore important to understand, however antibodies for IL25 have failed to detect the protein, so finding another way to visualize it would help determine the role IL25 has in the “weep & sweep” and tumorigenesis pathways. Here we used CRISPR to insert DNA encoding a hemagglutinin (HA) tag at the N-terminal of the IL-25 protein. The presence of the DNA insertion was confirmed through sequencing and genotyping, however we could not detect HA by immunofluorescence microscopy. These studies expand our analysis of Il25 mRNA in the small intestine, but further work is needed to understand why we could not detect HA-tagged IL-25 protein. 

In regions with high HIV prevalence, periods of pregnancy and postpartum are associated with higher risk for HIV acquisition. The WHO recommends oral tenofovir (TFV)-based pre-exposure prophylaxis (PrEP) as an effective method of reducing HIV acquisition risk for pregnant people. Although most pregnant patients in Kenya with identified HIV risk factors accept PrEP when offered, >50% discontinue PrEP within the first 30 days.​ Few studies to date examine factors that contribute to PrEP discontinuation in this population and there are no existing intervention studies aimed at improving PrEP adherence during pregnancy/postpartum. We utilized data from the ongoing Mobile Women and Children (mWACh)-PrEP study, a randomized control trial testing a two-way short messaging service (SMS)-based platform to facilitate communication between peripartum patients taking PrEP and remote nurses. In the parent study of 379 HIV-negative, cisgendered Kenyan women taking PrEP, 188 were randomly assigned to the intervention (mWACh-PrEP) group. The purpose of our sub-analysis was to evaluate message contents and identify frequently discussed topics. System messages were tagged into categories based on subject. Of weekly automated messages, 42.1% were responded to by participants, with an average response time of 87 minutes; topics of responses included PrEP concerns (55.1%), antenatal concerns (22.4%), maternal health concerns (17.8%), and infant concerns (17.2%). A total of 366 spontaneous messages were received, with >50% of participants sending at least one spontaneous message; topics of spontaneous messages most frequently included antenatal concerns (22.9%) and PrEP concerns (19.7%). These results indicate that a two-way SMS based intervention provides important support to pregnant and postpartum patients taking PrEP; the mWACh-PrEP system could be an effective solution not only to promote PrEP adherence within this population, but also to increase access to maternal healthcare.

When discussing ways to alleviate the effects of poverty, the focal point of policy decisions tend to center on improving material poverty. However, there is a growing recognition that poverty includes temporal constraints as well as material. Time poverty is defined as the lack of discretionary time available for rest, socialization and leisure once accounting for time spent at work and during necessary, committed activities. This study was designed to identify a relationship between time poverty and wellbeing in college students, as well as if race could be a suitable predictor for time poverty. I hypothesized that students with higher perceived and actual time poverty will report lower scores in wellbeing, and that this disparity will also be seen with students of colour. Through a Qualtrics questionnaire, 161 participants completed a modified version of the national American Time Use Scale (ATUS) designed to understand how people spend their daily time. They then answered questions about their perceived general wellbeing, then questions centering their perceived time poverty. The results from a regression analysis suggested that only perceived time poverty (M = -.0068, SD = .64919) negatively predicted wellbeing, R^2 = .325, F(5,150) = 14.41, p < .001, where higher scores predicted lower wellbeing (β = .564, p < .001). Actual time poverty and race were not found to predict well being, nor was there a significant interaction between race and time poverty on wellbeing.​ The results of this study likely stem from low reliability of the materials and indicate a need to create a smaller-scale measure for time poverty in specific demographics (e.g. college students). However, the effect subjective time poverty has on wellbeing supports past literature, and reinforces the importance of improving time constraints.

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

 Birth control is an important tool to prevent unwanted pregnancies. However, many people who might benefit choose not to use birth control, contributing to a range of negative outcomes, including unwanted pregnancies and sexually transmitted infections. Many factors affect people’s perceptions of birth control options, and ultimately influence their use of contraceptives. Previous research shows racial differences in rates of birth control utilization, but little work has explored why these racial differences exist and if and how culture contributes to birth control attitudes and utilization cross-racially. Furthermore, most previous research in this area focuses exclusively on race, typically reporting only on Black, Hispanic, and White women, with little or no intersectional analysis. The present study investigates how culture influences birth control attitudes, considering both a broader range of racial categories and the impact of multiple intersecting identities on culture. Our goal is to gain insight into the health care decision processes of intersectionally marginalized patients. We accomplish this with a cross-sectional qualitative study. We first pre-screen potential interviewees to recruit participants with diverse cultural backgrounds. Selected participants will participate in structured 1:1 interviews, answering questions about how their cultural background (including race, ethnicity, religion, and family) influences birth control attitudes. We will conduct thematic analysis to determine what aspects of culture impact birth control attitudes. Shedding light on how culture influences the perception and use of birth control provides insight into a broader range of patient populations, allowing for improved contraceptive counseling and education in the medical setting. Recruiting a diverse sample will illuminate the lived experiences of individuals who are typically excluded from research and scholarship, allowing future advancements in birth control to be more representative and sensitive, with the knowledge of all cultural experiences in mind rather than just a select few.

A primary stressor of college students is financial insecurity. Research estimates that 36% of college students are food insecure, 36% lack reliable housing, and 9% experienced homelessness in 2018. Sources of financial stress for college students typically include housing, tuition and other academic expenses, credit card debt, familial responsibilities, and employment status. Furthermore, research has also shown that psychological stress correlates with decreased sleep quality. During periods of intense stress, an individual may have more frequent disturbances during sleep, sleep for fewer hours, and adopt later waking times (Galambos et al., 2013). This study investigates the relationship between financial insecurity and sleep quality of students attending a 2-year college in Washington state. More specifically, this study aims to determine the impact of a sleep wellness workshop on the sleep quality of college students. Data collection will occur through an online Qualtrics survey before and after the sleep wellness workshop. The Qualtrics survey includes modified Sleep Quality Scale questions, the Beck Anxiety Inventory (BAI), and the Perceived Stress Scale (PSS). It is hypothesized that experiencing financial insecurity will positively correlate to poorer sleep quality. It is predicted that this relationship is mediated by stress and that the sleep wellness workshop will improve sleep quality in college students. Understanding factors that contribute to poor sleep quality is vital as sleep plays a key role in maintaining proper cognitive functioning. The results of this study are intended to improve current resources, as well as implement new resources, concerning sleep quality and stress of college students.

This study examines the impact of ease of public transport access on unemployment. Commuting is a significant barrier to work because of its time commitment, expense, and reliance on car ownership. The average commute time in the United States is increasing, rising by 10% from 2006 to 2019. This effect is especially prominent in major cities. As workers travel further to reach their jobs, suburbs far from work centers continue to grow, and affordable urban housing is becoming a rarity, public transportation ridership has seen a decline. Past studies focused on a singular city area showed significant differences in employment between those reliant on public transportation and those with automobiles. With my study I want to see if this applies on a nationwide scale. This study uses a linear regression run on the 2017 US Demographic Census data using the software RStudio for now, as more research is conducted alternate sources may be used. The impacts of variables such as race, economic health of the county, geographical differences, and immigration are isolated to determine the extent to which ease of access to public transport is a cause of unemployment. Tests run on the coefficients of the regression determine statistical significance. A second regression run on the 2020 US Demographic Census determines whether the COVID-19 pandemic had an effect. With the rise in remote workers, it is possible that the impact of ease of access is mitigated. A negative correlation between the ease of public transportation access for a county and its unemployment rate is expected. This study could shed light on another cause of unemployment and the impacts of car-reliant transportation.

 Cystic fibrosis (CF) is a progressive, life-threatening disease, that results from the formation of thick mucus that builds up in the lungs, digestive tract, and other parts of the body. It leads to severe respiratory and digestive problems as well as other complications including opportunistic infections and diabetes. CF is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Recently, the initiation of highly effective CFTR modulators including Trikafta (a combination of the medications elexacaftor, tezacaftor, and ivacaftor) has significantly improved the quality of life and life expectancy of patients with CF. However, recent clinical studies have shown that CF patients taking Trikafta have an increased risk of developing obesity and diabetes, though the underlying mechanisms remain unknown. In addition to being expressed in peripheral tissues, the Cftr gene is also expressed in the brain in the arcuate nucleus (ARC), a key brain area involved in metabolic regulation. To begin testing the hypothesis that Trikafta predisposes to metabolic syndrome by altering the activity of signaling of neurocircuits that regulate metabolism in the ARC, I investigated the ability of Trikafta to activate neurons in the ARC of mice (based on histochemical detection of c-Fos, a marker of neuronal activation). Following a single intracerebroventricular injection of Trikafra, compared to vehicle-treated mice, I found that mice treated with Trikafta had significantly increased activation of neurons in the ARC. I am now conducting studies to identify the phenotype of the neurons in the ARC that are activated by Trikafta and predict that successful completion of these studies will advance our understanding of the pathogenesis of obesity and metabolic impairment induced by Trikafta and inform the development of strategies that can avert these deleterious side effects.

To study genetic factors and the molecular mechanism underlying the development rate of mouse embryos, two mouse subspecies were studied. I have collected mouse embryos from crosses between C57BL6/JxCAST/EIJ (B6xCast), CastxB6 (the reciprocal cross), and intercrosses within either strain to examine the effects of maternal and paternal genomes on the developmental rate. I used mouse embryos every 24 hours at gestational stages E11.5-E14.5. Staging by the Embryonic Mouse Ontogenetic Staging System (eMOSS) is used to estimate the actual developmental stage based on limb bud morphology and compared to the gestational stage based on the estimated time of conception by observation of a vaginal plug. I also used gDNA to determine the sex of each embryo to investigate whether sexes play a role in developmental rate. I observed an interesting pattern that the mouse subspecies and the source of each parental genome affect the developmental rate of embryos. I observed embryos with at least one Cast parent, have faster development than B6xB6 starting at gestational stage E13.5. The developmental rate is independent of the sex of embryos or the litter size. Single-cell transcriptomic analysis is ongoing to determine the genes and mechanisms behind the change in developmental rate of mouse embryos. It is important because the result will contribute to human development in early stages, and help to solve problems such as preterm or underdeveloped infants.

Uridine-5'-triphosphate (UTP) is a precursor for RNA synthesis. Ura3 catalyzes the conversion of orotidine-5'-phosphate (OMP) into uridine monophosphate (UMP) and is commonly used as a selection marker to characterize mutation rates of S. cerevisiae. URA3 can be positively selected for by growing cells in the absence of uracil and can be selected against by growing cells in the presence of the toxic fluorinated UTP precursor, 5-Fluoroorotic acid (5-FOA). While mutations in URA3 make up the majority of 5-FOA-resistant mutants, mutations in a small number of other loci can also cause this phenotype. We whole genome sequenced the 5-FOA-resistant mutants with a wild type URA3 and identified mutations to URA6 in each of these individuals. URA6 is an essential gene that encodes an enzyme that catalyzes the conversion of uridine monophosphate (UMP) into uridine-5'-diphosphate (UDP). Here, we describe 41 non-synonymous mutations to URA6 that permit growth in both the absence of uracil and in the presence of 5-FOA. It remains unclear how the URA6 mutants can maintain a functioning UTP synthesis pathway while remaining resistant to the toxic fluorinated precursors. We hypothesize that these mutations alter the protein structure in a manner that decreases the affinity for fluorinated substrates while maintaining the affinity for UDP. To test this, we will evaluate the structural changes to URA6 resulting from these non-synonymous mutations. Our goal is that our findings can benefit our understanding of the UTP biosynthesis pathway and encourage further investigation of the mechanisms involving fluorinated substrate analogues.

The ocean and atmosphere interact through air-sea exchanges of heat and energy across the air-sea interface. These air-sea fluxes have important implications on global weather and climate patterns. Because estimation of covarying turbulent variations is not feasible in Numerical Weather Prediction (NWP) models, the turbulent air-sea exchanges are typically estimated using bulk air-sea flux algorithms based on state variables. However there are large differences in the values estimated by different NWP and even when they agree, without a reference data set, it may be that all NWP are equally biased. For this project, I used in situ observations collected by Saildrone Uncrewed Surface Vehicles (USV) in the central tropical Pacific to assess bulk flux estimates from multiple atmospheric reanalyses including NCEP Climate Forecast System Reanalysis (CFSR), ECMWF Reanalysis v5 (ERA5), NCEP/NCAR Reanalysis 1 (NCEP1), and NCEP/DOE Reanalysis 2 (NCEP2). Preliminary results, based upon hourly, spatially-interpolated, co-located values that are then made into 24-hour “daily” averages, indicate that all of the reanalyses had a strong correlation with USV observations for net heat flux and net SWR, but the correlation was much weaker (0.5 to 0.7) for other flux components and very weak (~0.25) for the net longwave radiation for NCEP1 and NCEP2. The root mean square errors for the 24-hour-averaged differences were 55 to 66 W/m^2 for solar radiation and 20 to 30 W/m^2 for latent heat flux. In my analysis of my results, I looked at the differences region by region for each of the flux components and state variables as well as for each of the products. As Saildrone technology becomes more widely used and more intercomparison studies such as this are conducted, observations from Saildrones could eventually be integrated into NWP models, possibly improving forecast accuracy.

Glutamine synthetase (GS) is a highly regulated enzyme that catalytically converts glutamate to glutamine which is associated with ammonia assimilation. One of the effects of dysregulation in the GS inter-conversion process is hyperammonemia, which can cause death or brain damage. GS is conserved across all prokaryotes and eukaryotes. Among enzymes, glutamine synthetase's ability to polymerize is still a structural mystery and the functional characteristics of its self-assembling filaments remain unknown. The aim is to understand the occurrence of filament formation in GS and the effects on enzyme activity. We hypothesized that filaments may influence the association of GS substrates or allosterically regulate GS. I purified the GS of Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Helicobacter pylori by using Ni-column and size exclusion chromatography (SEC). Then, I examined the GS of pseudomonas under different buffer conditions (Mg2+, Co2+) using negative staining. Under Magnesium (10mM) conditions, the known dodecamer structure of GS was observed. Under Cobalt (10mM) conditions, the filament was being induced. To better investigate the structural mechanism of filament formation we turned to cryogenic electron microscopy (Cryo-EM). The next step is to create a model of the filament interface of GS and identify the residues involved. This research has broad implications in the field of metabolic engineering, as understanding the structure and the role of filament formation in GS could help develop new therapeutic targets in metabolism.

The aim of the present study is to characterize the morphology of the pharyngeal airway by quantifying the volumetric features in the following four regions: naso-, velo-, and oro- pharyngeal spaces and retroglossal space. Using these volumetric features we will observe how fat composition and the volume of each region of interest play a role in obstructive sleep apnea. A fat composition in the different pharyngeal structures, including the tongue body and base, soft palate, and pharyngeal wall, were quantified as well. Eight 7-8 months old Yucatan minipigs of both genders were sedated and placed in prone to have magnetic resonance imaging (MRI) using dynamic TFE-sequence with synchronized physiological monitoring. The volumes of each region of the pharyngeal airway spaces from MRI images were acquired through anatomical segmentations (VISTA-sequence) and 3D reconstructions using Radiant-DICOM Viewer and ITK-SNAP software. The cross-sectional areas were measured separately and averaged for each region as well. The fat composition was measured using MATLAB for 3D analysis. The script digitized the DICOM image and then parsed giving a relative fat composition for each individual pixel in 3D format. The total volume for each region was computed and fat composition of the tongue base was 13.14%. These results showed that the volume of the 3D segmentations for each region of interest of the pigs with greater weight, displayed more implications within the pharyngeal airway due to the larger fat composition in comparison to pigs that were of normal weight. These results will be later used for further analysis of specific details regarding if there is a certain threshold of fat composition to the onset conditions of obstructive sleep apnea. The measurement of the fat compositions in the tongue body, soft palate, and pharyngeal wall are ongoing.These volumetric results provide the baseline for the following study on the consequences of pharyngeal morphology by the tongue base volume reduction or enlargement. The fat composition characterizes the adipose tissue distributions in the different pharyngeal structures. Together, these data will help to understand the mechanism which regulates the upper airway patency.

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

A feature of the criminal legal system of concern to scholars is court-issued monetary sanctions — the fines, fees, and costs imposed on people convicted of crimes. Prior research suggests these debts exacerbate inequality and perpetuate disadvantages for the affected individuals, families, and communities. While previous studies have found that monetary sanctions negatively affect family members of the debt-burdened individual, the impact of these debts on parent-child relationships remains unexplored. Attempting to bridge this gap in knowledge, this study examines monetary sanction’s influence on the nature of parenting. Scholars understand parents play a vital role in shaping and providing emotional and material resources for their children. Given this, it is important to understand the impact of monetary sanction debt on parents’ ability to provide these resources and ultimately how monetary sanctions impact child well-being. Utilizing interview data from a previous multi-state study of over 200 individuals with legal debt and children, supplemented by an analysis of interviews of similar individuals, this study examines how stress and material deprivation resulting from monetary sanction debt reduce the emotional and material capacity of parents to address their child’s needs. Preliminary analyses show parents frame the impact of debt as generating financial and emotional stress for them, and they attempt to minimize its direct effects on their children. In some cases, it is clear that parents are unable to provide important resources for their children, and the stress of debt negatively affects their relationships. These preliminary results suggest one additional avenue as to how monetary sanctions affect individuals. As other researchers have discovered, the impact is not simply resident with the person in debt but has an additional negative reach that should be understood as we look to reform the practice of monetary sanctions.

Zoom classes or online meetings may be seen as temporary resolutions to COVID-19 restrictions. However, in the mental health field, telehealth continued to represent 20% of outpatient visits when the emergency restrictions of COVID-19 eased in late 2020 (Gentry et al., 2021). Telemental health is a form of online mental health services that can be used to deliver Evidence-Based Practice (EBP) to clients without in-person meetings. Despite the strength, one major barrier to implementation of telemental health services is that some youth may be less engaged in the session (AlRasheed et al., 2022). Limited research indicates that practical organizational support such as trainings, seminars, additional funding for clients, and technology support for clients may improve client engagement, along with a positive organizational climate that promote providing telemental health services (Oetzel & Scherer, 2003). The current study expands on past research to examine the relationship between organizational support and youth engagement in telemental health services among Community Mental health providers (N= 173) who participated in a Washington State-funded EBP training initiative (CBT+). As a part of CBT+, the providers attended a Cognitive Behavioral Therapy for youth training and follow up support and completed self-report surveys. Using survey questions regarding organizational support and youth engagement, we conducted 12 linear models to examine the relationship between organizational climate and practical organizational support with six measures of youth engagement. Results indicated that a positive organizational climate is associated with greater perceived effectiveness of therapy (β = .39, p = .000). The finding suggests that, when perceived effectiveness is low, we encourage organizations to tailor strategies to improve organizational climate for telemental health services, even after COVID-19 restrictions loosen. I will present additional findings and implications regarding practical organizational support in the poster presentation.

Microtubules are dynamic polymers of 𝛼- and 𝛽-tubulin subunits instrumental in the organization and division of chromosomes during mitosis. There is an intrinsic structural polarity to microtubules due to the orientation of 𝛼𝛽 heterodimers in the microtubule lattice, so that there is a fast growing plus end and a slower growing minus end. Kinetochores are protein complexes that assemble on chromosome centromeres and attach to microtubules. Proper chromosome segregation relies on kinetochore attachment to the plus ends of microtubules. Kinetochores are thought to initially bind the microtubule lattice and plus end attachments are then achieved by the action of plus end directed motor proteins or microtubule disassembly. While the plus end attachment is essential for mitotic fidelity, it remains unknown if the kinetochores themselves have an intrinsic polarity preference. Using total internal reflectance fluorescence microscopy, we have found that individual kinetochores assembled on centromeric DNA have a strong preference for binding the plus ends of stabilized microtubules in the absence of motor proteins and ATP or microtubule dynamics. Furthermore, using optical trapping we are able to measure the rupture forces of kinetochores on both ends of microtubules and have found that the observed preference for plus ends is matched by a greater binding strength at plus end tips. These results together give insight into how kinetochores could efficiently form plus end tip attachments and how they likely play a part in cell cycle regulation by using tension to sense a correct attachment. A better understanding of the specific mechanisms of kinetochore microtubule binding is valuable for understanding control of mitotic progression and could potentially inform more targeted anti-cancer therapies that focus specifically on dividing cells without impacting regular cell function.

The emergence of SARS-CoV-2 (CoV-2) and the rapid growth of the global pandemic has necessitated more than ever the need for fully comprehensive studies of viral pathogenesis. Previous studies of CoV-2 highlight the importance of its viral proteins in transmission and adaptability into human hosts, but few have delved into the role that viral RNA structure plays in emergence and pathogenesis. Viral RNA structure is a known contributor to host-jumping replication as seen in related coronaviruses (CoVs), Flaviviruses, and Alphaviruses. Given this phenomenon and that all human CoVs originated from bat CoVs, we hypothesize that genomic and structural differences in the human CoV-2 RNA compared to the bat RatG13 CoV RNA, the believed progenitor of CoV-2, may have contributed to the species-jumping event from bats to humans that caused the emergence of human CoV-2. To test this hypothesis, I used a CoV-2 replicon system to compare replication of the wildtype (WT) replicon versus a mutant replicon (RaTG13-N/3’UTR(syn)) that contains a firefly luciferase reporter gene and synonymous mutations from the 3’ end of RaTG13. In preliminary replication experiments, RaTG13-N/3’UTR(syn) had notably higher replication than the WT replicon in primate fibroblasts. Based on this result, I used electroporation techniques and Renilla luciferase assays to determine the replication kinetics involved in differential replication of these viral replicons in human versus bat epithelial lung cells. I anticipate increased replication of the RaTG13-N/3’UTR(syn) replicon in bat cells compared to human cells when compared against the WT which would entail a mechanistic study of how RNA structures contribute to viral fitness in the two species using genomic and proteomic approaches. If this data supports our hypothesis, this could enhance our understanding of CoVs to allow us to target and survey zoonotic viruses that threaten species-jumping into the human population.

Werner Syndrome is a rare autosomal recessive disorder characterized by premature aging and heightened susceptibility to cancer. The condition is caused by a loss of function in the Werner protein (WRN), a helicase that is essential for maintaining genomic stability by assisting in DNA replication and repair. While much is known about WRN's role in the cell, the mechanisms underlying the acceleration of aging in its absence remain unclear. Recent studies have suggested that aging is associated with a reduced ability to maintain heterochromatic silencing of repetitive DNA sequences in the pericentromeric regions of the genome, also known as satellite repeats. Abnormally high expression of pericentromeric satellites can have a wide range of consequences on the cell, including DNA damage and changes in global gene regulation. Moreover, DNA damage can result in the appearance of satellite DNA fragments in the cytoplasm, leading to pro-inflammatory responses. This study seeks to determine whether WRN-deficient cells transcribe higher levels of satellite RNA and have elevated levels of satellite DNA in the cytoplasm. I focus on SAT II DNA, a class of pericentromeric satellites, using a pair of human fibroblast cell lines, with or without WRN. I isolate, purify and reverse-transcribe RNA from these cells and use qPCR to quantify the levels of SAT II RNA. Additionally, I fractionate WRN-deficient and control cells into cytoplasmic and nuclear fractions, isolate their DNA, and use qPCR to quantify the levels of SAT II DNA in the cytoplasm versus the nucleus. Preliminary results suggest that WRN absence leads to increased expression of SAT II RNA in Werner-depleted cells compared to controls, which may indicate that satellite heterochromatin is not being properly maintained. These findings may impact our understanding of aging and cancer predisposition, along with the mechanisms by which heterochromatic silencing regulates gene expression and cellular function.

Intermediate metabolism in cells has generally been studied without considering the arrangement of enzymes within the cell. However, recent developments have shown that many metabolic enzymes form organization systems that are made up of oligomers stacking linearly into filaments. The enzyme phosphoribosyl pyrophosphate synthetase (PRPS) makes a precursor required for all de novo nucleotide synthesis in cells, and therefore plays an important role in cellular metabolism. This project aims to characterize the PRPS protein in Xenopus tropicalis and Giardia lamblia. We hypothesize that PRPS from X. tropicalis will have similar biochemical and structural properties as compared to human PRPS, while PRPS from G. lamblia will have different biochemical and structural properties. This hypothesis is supported by the small evolutionary difference between PRPS from humans and X. tropicalis as compared to the large evolutionary difference between PRPS from humans and G. lamblia. This difference would be especially interesting to examine from the perspective of filament formations in the PRPS protein. So far, we have created the Xenopus tropicalis and Giardia lamblia plasmids by cloning. Test expressions of the X. tropicalis yielded protein expression in E. coli cell strains C43 and RIL, while test expressions for G. lamblia have been successful in C43, BL21, pLysS, and Rosetta cell strains. This demonstrates that both X. tropicalis and G. lamblia PRPS can be expressed in E. Coli strains. An analysis of X. tropicalis will allow us to test how filament formation changes with only small evolutionary differences in PRPS. It could also be used for further research in vivo using frog eggs that act as a singular cell system. If it is confirmed that the G. lamblia protein is different from human PRPS, PRPS in G. lamblia could serve as an antibiotic target since current methods of treatment for the organism are very harmful to the human microbiome.

Antiretroviral therapy (ART) prevents the progression of human immunodeficiency virus (HIV) by suppressing viral load, limiting transmission. Of the ~20 million people receiving ART, 30-40% do not maintain adequate medication adherence, resulting in treatment failure and drug resistance. Monitoring HIV medication adherence improves the efficacy of ART but requires bulky and expensive instruments that are not widely accessible at the point-of-need (e.g. doctor’s office or patient’s home), so a rapid and accessible diagnostic alternative is necessary. Our group developed the REverSe TRanscrIptase Chain Termination (RESTRICT) enzymatic assay to provide rapid and inexpensive measurement of HIV drug adherence by measuring antiretroviral drug activity indicated by fluorescence. However, one current limitation of RESTRICT is the need for trained operators to complete multiple precisely timed steps required in the enzymatic activity assay. We aim to create a 3D-printed microfluidic device that will automate the liquid handling steps required for RESTRICT via precisely tuned capillary action for rapid and user-friendly measurement of antiretroviral drugs. To that end, we first demonstrated a proof of concept by creating a microchip with a controlled 15 minute liquid delivery time and consistent liquid extraction. Through optimization of 3D-printing methods, channel geometry, and surface treatment, we created a microchip designed to deliver liquid in 14.64 minutes that ran experimentally in 19.12 ± 2.33 minutes. In the future, we will demonstrate the feasibility of RESTRICT run on-chip and fluorescence measured off-chip by testing clinically-relevant drug concentrations using the controlled liquid delivery time and liquid extraction methods developed. By creating an automated and rapidly fabricated microfluidic chip for therapeutic drug monitoring, we hope to achieve a hands-off device that removes external manipulation to increase the accessibility of RESTRICT-on-a-chip for point-of-need settings without specialized equipment or highly trained operators.

People living with epilepsy (PWE) often have a poorer quality of life (QoL) compared to the general population. Anti-seizure medicines (ASMs) are used to control seizures in PWE but are often associated with side-effects that lead to reduced adherence. Poor adherence is associated with reduced seizure control which can also negatively impact QoL. A rat model of acquired epilepsy was used to evaluate how poor adherence to the ASM, perampanel (PER), impacts an animal’s engagement with environmental enrichment, which is provided to promote species-specific behaviors and general well-being. Cardboard enrichment was provided to single-housed male Sprague Dawley rats with acquired epilepsy and I scored their level of engagement at the start of each day: i.e.,1 being no engagement; 4 being completely engaged. Animals were observed for 8 weeks: 4 weeks without PER and 4 weeks with PER in a fully adherent (100%) or variably nonadherent (50%) dosing paradigm (10 mg/kg/day, p.o.). I recorded data on the number of days till first engagement with the enrichment, days till max, and max enrichment score. After compiling the data, I found no significant differences in the max enrichment score or days till max score amongst the 100% or 50% treatment groups. Interestingly, when compared to their pretreatment baseline, fully adherent rats took less time to initially engage with their enrichment, i.e., 6-9 days, compared to 9-12 days, respectively. In contrast, nonadherent rats did not show a similar improvement in their enrichment behavior when treatment was initiated. These results suggest that fully adherent rats were more willing to interact with their enrichment whereas, poor medication adherence may have a direct negative impact on QoL. Further investigation is necessary to determine if this is due to a difference in seizure control between the groups.

Increasing mental illness among college students continues to be a critical issue. College life is often a time of great transition, contributing to and potentially exacerbating students' rising stress, anxiety, and other mental struggles. Poor sleep habits may develop during college, and poor sleep quality can amplify stress. To explore how institutions might help students struggling with stress, we conducted a small pilot study examining whether a sleep-focused workshop would reduce stress levels in students at a Pacific Northwest community college. For this pilot study, I helped generate an online pre- and post-workshop survey of demographic questions, the UCLA Loneliness Scale, and the Perceived Stress Scale (PSS). Our results indicated that participants' workshop attendance reduced their stress levels. Before the sleep workshop, participants scored an average of 20.27 (SD=7.41, range=2 to 30) on the PSS, while two weeks after the workshop, the average PSS score had reduced to 16.58 (SD=7.65, range=4 to 27). These results suggest benefits of a sleep-focused wellness workshop for college students. In our present study, we replicated the pilot study. I examined student stress in correlation to sleep quality and assessed these factors through an online survey of demographic questions, sleep quality questions, the Beck Anxiety Inventory, and the PSS. We distributed this survey to participants before and after a sleep workshop run by the Bellevue College psychology department. I hypothesized that poor sleep quality correlates to higher student stress levels. I also hypothesized that the intervention of a sleep workshop would reduce stress levels. I hope that our results provide insight into the utility of wellness workshops for students and whether they are an avenue to help students manage stress and improve their sleep quality, which could improve student mental health by lowering stress and anxiety and improving quality of life and education overall.

Chinese tumultous history has seen political uprisings and cultural changes, which often saw many centuries of history to be lost to time. The most recent revolution comes in the form of industry, elevating China from a rural agrarian society to an economic powerhouse. In just the last three decades, China sped through progress faster than any country before it and the effects of that speed can be seen in its social, historical and cultural landscape. Having spent two years in the city of Suzhou, China, the experience of seeing the progress of the country my parents left and the one I lived shows a clear difference which made me understand the necessity of cultural preservation in the face of industrialization. Suzhou transformed from a Heritage city to one of industry and while city life flourishes, our way of living and culture becomes an imitation of the West. Under the bright city lights at night that depict Suzhou’s modernity, I came to see the emptiness of a city that fully embraced urbanization and commercialization with little respect for its original inhabitants. As the city grew younger, I walked near empty streets, abandoned skyscrapers, and shopping malls full of Western brands that illustrated the irrelevancy of history in the face of progress. Painting becomes a way to examine historical roots and values of Chinese art, searching for a resolution between China’s past and present and asking the viewer to reflect on the impacts of industrialization.

Schizophrenia is highly heritable and has been associated with many brain structure abnormalities, including significant differences in cortical thickness, surface area, and gray matter volume. It is widely believed that abnormalities in early neurodevelopmental processes contribute to the differences in brain structure observed, and many genetic risk variants associated with schizophrenia influence brain development. Further research is required to understand whether genetic mechanisms underlie the typical pattern of cortical thickness and surface area deficits in schizophrenia or deviations from normal trajectories of cortical thickness and surface area development. Using data collected from 406 participants, including 246 individuals with schizophrenia, 70 unaffected relatives of schizophrenia patients, and 90 healthy controls, the current study will test for associations between polygenic risk for schizophrenia and expected brain measures of cortical thickness and surface area using a Regional Vulnerability Index (RVI) which quantifies the resemblance between an individual’s brain scan to expected brain deficit patterns in people with schizophrenia. Along with that, we will be testing for associations between polygenic risk for schizophrenia and the typical development of cortical thickness and surface area using centile scores which benchmark an individual’s neuroanatomical measurement in the context of the normative age and sex-related trajectories. We hypothesize that polygenic risk for schizophrenia will be positively associated with cortical thickness and surface area RVIs and centile scores for cortical thickness and surface area across all diagnostic groups. Understanding the relationship between genetic risk for schizophrenia and alterations in brain structure and development may facilitate early detection of schizophrenia and aid future efforts to prevent the onset of psychosis in vulnerable individuals.

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

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

Sindbis Virus (SINV) is an alphavirus that is transmitted between birds via mosquitos and causes disease in humans after spillover events. While many alphaviruses have potential to cause severe disease such as Venezuelan Equine Encephalitis Virus (VEEV), SINV is known to cause less life-threatening but still severe and debilitating chronic illness predominantly associated with fever, arthralgia, and myalgia. Given its lower morbidity, SINV often serves as a model system for infectivity and pathogenesis studies of alphaviruses. Macrophages are of particular interest for studying pathogenesis as they are important targets of alphavirus infection. Our aim is to identify viral mutations that contribute to differential replication between avirulent and virulent strains of SINV in macrophages. To test this we exchanged sequences between SINV strains Girdwood (avirulent) and AR86 (virulent) to construct reciprocal chimeras which will be tested alongside parent clones. We observed a significant difference in replication between parent AR86 and Girdwood in macrophages and predict that specific RNA structural differences contribute to this variable replication in macrophages, and we are currently mapping determinants of this phenotype. This work could improve our understanding of host-viral interactions of alphaviruses by discerning the mutations required to shift from avirulent to virulent strains which will play an important role in predicting mutations that contribute to greater pathogenicity in human infections.

In the era of the internet, numerous individuals participate in fan communities on online social platforms to pursue their interests. Despite all members sharing genuine passions, these communities exhibit varying levels and types of engagement, with some attracting millions if not billions of active users daily, while others show little to no activity. Additionally, users tend to engage in discussions on certain topics more frequently in specific communities. This research aims to explore the reasons behind this difference by investigating the interaction between community size and openness. While previous works focus on the impact of either size or openness, this study develops hypotheses on how their interaction affects member engagement. A survey study is outlined to test these hypotheses, examining members' sense of belonging, level of hostility, and variation of opinions in four distinct communities representing different interactions between community size and openness: large and open, large and closed, small and open, and small and closed. The survey will be distributed among TFBoys fans on Sina Weibo in the Spring of 2023. The expected results will provide insights into how community size and openness interact to influence member engagement in online fan communities, offering recommendations for online social platforms to design features that promote member engagement and contribute to scholarly knowledge in Computer-Mediated Communication and Social Media Studies.

In a liberal and democratic society, unjust policies can be prevented by the people through voting or political action. People in a free society have a default political responsibility to prevent unjust policy in these ways. However, people under a non-democratic regime do not have the means to actively engage in political affairs and therefore do not causally contribute to unjust policy. This paper analyzes the protests against the zero-COVID policy employed by the Chinese government since the outbreak of the COVID-19 pandemic. I use three theoretical approaches to examine to what extent people have a moral responsibility to oppose unjust policy. First, people can follow the heroic example of Antigone in Sophocles' tragedy and mount heroic resistance at great personal cost. They can appeal to higher moral values and take up rebellion against the authoritarian government. Second, individuals can honor their social roles despite the state's rules. Doctors should treat the patients even though the zero-Covid policy demands them not to do so. Finally, individuals can seek to preserve truth and inner freedom. This is the approach with the least cost. People should seek truth and be aware of the lies made by the government. In the meantime, they need to protect themselves from being punished by the authoritarian government. These are three models of moral responsibility. I argue that sticking with the truth is the minimum moral requirement for individuals under authoritarian regimes.

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

Previous studies have indicated that the neurohypophyseal hormone, oxytocin (OXT), reduces body weight in high fat diet (HFD)-induced obese (DIO) rodents, nonhuman primates and obese humans through reductions in food intake and increases in energy expenditure. Findings from recent pre-clinical and clinical data indicate that the long-acting glucagon-like peptide 1-receptor (GLP-1R) agonist, semaglutide, reduces body weight, in part, through reductions of food intake. Based on these findings, we hypothesized that the combined treatment of OXT and semaglutide would produce an additive effect to evoke weight loss in DIO rats. To test this hypothesis, rats were fed a HFD for approximately 5 months prior to being implanted subcutaneously with a 28-day minipump that infused OXT (50 nmol/day) or vehicle (VEH; saline). Data from animals that received escalating doses of the incretin hormone, glucagon-like peptide-1 (GLP-1) and semaglutide was analyzed over the 32-day infusion period. Daily energy intake and body weight were tracked for 32 days. Body composition was assessed immediately prior to onset of treatment and near the end of the treatment period using Quantitative Magnetic Resonance (QMR; EchoMRI 4-in-1). OXT (50 nmol/day) alone and semaglutide (3 nmol/kg) alone reduced body weight by approximately 9.9±1.1% (0.05<P<0.1) and 4.3±1.4% (P<0.05), respectively (N=6-7/group), relative to pre-infusion baseline, while GLP-1 at either dose (1 or 3 nmol/kg, SC) was ineffective (P=NS). However, the combination of OXT and semaglutide (3 nmol/kg) produced a more pronounced reduction in body weight (14.1±1.4%; P<0.05) compared with either treatment alone (N=6-7/group). These effects were associated with reduced energy intake, adiposity and adipocyte size (P<0.05). Together, these findings support the hypothesis that the combination of OXT and the GLP-1R agonist, semaglutide, produces an additive effect to evoke weight loss and body adiposity in DIO rats by reducing energy intake. Our findings will create a preclinical infrastructure upon which future studies are undertaken to address whether oxytocin may be used as an adjunct with other treatment approaches, including low doses of the FDA-approved GLP-1R agonist, semaglutide, to evoke weight loss in humans with obesity.

Many with clinically diagnosed Alzheimer’s disease (AD) dementia during life show comorbid neuropathologies at autopsy. We sought to develop and evaluate a composite brain pathology score (BPS) in the Adult Changes in Thought study. We derived BPS using nine standard neuropathological indicators such as Thal phase, Braak stage, and CERAD score, using confirmatory factor analyses. We compared BPS to the AD Neuropathologic Change (ADNC) score, which quantifies neuropathologic changes that underlie AD. We performed non-nested modeling to compare BPS and ADNC scores’ associations using the last cognitive score (memory, executive function, language, and visuospatial ability) prior to death, adjusting for age at death and sex. Non-nested models were compared using Adjusted-R², Davidson-MacKinnon J-test, and Cox–Pesaran tests. We focused on people whose last cognitive data were≤2 years prior to death. We compared ADNC and BPS’ associations with last known clinical diagnosis (no dementia vs. any dementia), adjusting for age at death and sex, by considering area under receiver operator characteristic (ROC) curves. Sample size was 886 with mean age of death of 89 and 57% female. A bifactor model fit best, with residual correlations for Thal phase and CERAD score, and for LATE stage and presence of hippocampal sclerosis. The BPS explained more variance for each cognitive score and was superior and statistically significant compared to the ADNC score in non-nested model comparisons. The BPS was more strongly associated (Odds Ratio=3.2) with dementia diagnosis than the ADNC score (Odds Ratio=2.0). The area under the ADNC and BPS’ ROC curves were 0.74 and 0.78, respectively. The difference between the areas was statistically significant (p-value ≤ 0.0001). We demonstrate an approach to developing a composite BPS which incorporates multiple forms of pathology. Future efforts will focus on co-calibrating and harmonizing BPS in other autopsy cohorts.

About one-quarter of photosynthetically fixed carbon is cycled through the marine microbial community in the form of metabolites, the intermediate compounds or products of metabolic processes. Marine heterotrophic bacteria are largely responsible for consuming these metabolites as a source of carbon, energy, and nutrients, yet little is known about transporter affinity and uptake kinetics of bacteria for abundant substrates. Homarine is a small, nitrogen-containing, zwitterionic metabolite that is produced by the cyanobacterium Synechococcus as well as some diatoms and haptophytes, where it is thought to function as an osmolyte. Dissolved homarine is present in the ocean at very low concentrations (~1.1 nM in Puget Sound). I hypothesize that these low concentrations are the result of high affinity bacterial transporters for homarine. Homarine can be used as a sole carbon and nitrogen source for OBi1, a marine bacterium isolated from Puget Sound. In this study, I investigate the uptake kinetics of homarine by OBi1 in the lab using the Michaelis-Menten model. I compare the uptake kinetics of OBi1 to similar homarine uptake experiments in the Salish Sea in June 2019. I expect that OBi1 will have a high affinity for homarine uptake and will take up homarine at nanomolar concentrations. I also anticipate that the marine microbial community in Puget Sound will have similar uptake kinetics to those observed with OBi1. Understanding the uptake kinetics of homarine by marine bacteria sheds light on the cycling of homarine in marine environments like Puget Sound and can help us understand the processes that keep the dissolved homarine concentration so low.

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

Recent studies and news sources have brought attention to the possible health risks associated with cooking on a gas stove due to the release of high concentrations of pollutants, such as NO₂, particulate matter (PM), and other chemical pollutants. But currently, there is little information on how individuals can monitor their own indoor air. Several low-cost air quality sensors are available, but little data to show if these work for home environments. Low-cost sensors are important because they can be more accessible to more people due to their affordability. Two brands of low-cost air quality sensors were used in this study to measure PM and various chemical pollutants: the TSI AirAssure and the Vaisala AQT530. We conducted several kitchen experiments that involved cooking on the stove or the oven while monitoring pollutant levels on these sensors. I found that pollutant levels rose significantly when cooking on both appliances. One experiment revealed that within minutes of cooking in the oven, NO₂ levels went above the recommended EPA standard of 100 ppb per hour. I also did a comparison experiment between the Vaisala AQT530 and a high-quality sensor from the Puget Sound Clean Air Agency at the downtown air quality station (10^th and Weller). Results showed moderate correlations between the sensors for NO, NO₂, and CO concentrations and a strong correlation for PM concentrations. We will continue to evaluate the sensors by doing a direct comparison of these sensors with standard regulatory methods in a test kitchen. The goal is to derive correction equations that can be applied to the data received from these low-cost sensors. My findings in the kitchen and sensor comparison experiments will provide valuable information on when pollutant levels impose health risks and information on the level of accuracy of the data retrieved from these devices.

Healthcare providers in rural areas face clinical challenges vastly different from their colleagues in urban areas. Researchers and providers have suggested different approaches, but it remains unclear how to best provide care in rural areas. My research contributes to rural medicine by examining how healthcare providers in Kodiak, Alaska, provide reproductive health in an under-resourced clinic, island, and state. I sought to answer three questions: First, what health disparities do providers and patients experience? Then, how do care providers and receivers navigate these barriers and work under these conditions? Lastly, what are the best practices, and how can they be supported in the future? To investigate these questions, I utilized clinical observation and ethnographic interviews carried out in 2022-2023 with reproductive healthcare providers. The data revealed three main barriers, including geographical challenges, a lack of general and specialty providers, and an overall lack of resources and necessary facilities. Providers commonly navigate these barriers by sending patients to Anchorage that has more resources and specialty physicians. Although this practice solves some immediate problems, many providers recognize that it can perpetuate systemic inequalities and inevitably lead to inaccessibility to care. As such, providers use numerous methods to keep patients on the island, including a rotating schedule of visiting providers and a close-knit, collaborative unit of providers. Overall, my research exposes the barriers to providing care in a specific rural and underserved community and highlights successes and the best strategies used by their providers to overcome these challenges. Additionally, it provides evidence that to support rural communities and their providers, we must first understand each communities unique experiences and circumstances before creating interventions tailored to that specific community. Therefore, my research can be used to inform health interventions and thus improve the quality and delivery of care in rural and underserved communities in and beyond Alaska.

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.

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.

 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.

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.

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.

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.

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.

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.

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.

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.
 

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.

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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. 

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.

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.

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.

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.

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 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.

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. 

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.

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.

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.