High throughput single cell/nuclei RNA sequencing (scRNA-seq/snRNA-seq) has been used to characterize the gene expression at the cellular level in disease control studies. Differentially gene expression analysis aims to emphasize the biological variation between samples without unwanted technical variation. Batch effect correction is occasionally performed where cells from each individual sample are treated as being generated from a batch. We analyzed snRNA-seq data from ApoE neutral and detrimental mouse models of Alzheimer’s disease (AD) to test whether batch correcting the data using all cells from each individual biological sample as representing a batch would result in 1) loss of disease relevant associations, 2) loss of biologically relevant cell types, and 3) reduced association between cell types and phenotypes. We performed scRNA-seq analysis of seven samples from two ApoE genotypes using Seurat workflow applying Harmony batch correction, using each sample as a batch, and without correction. Since we measured two disease-related phenotypes in mice from the two genotypes, we asked whether cell cluster membership associated with genotypes are also associated with unit changes in brain-diseases related phenotypes. After applying individual sample batch correction, we found differences in number of cell types (clusters) before and after batch correction, we showed loss of cell types/clusters specifically from the detrimental genotype, shrinkage of the differences in cell cluster membership across samples and reduced association between cell type membership, genotypes and phenotypes. In conclusion, batch effect correction should be applied consciously based on the experimental design to avoid over correction of biological variability and an appropriate design should help to avoid unwanted technical variation.

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. 

STEM education tends to avoid extensive discussion of ethics on topics such as climate change, treatment of disease, and nutrition. However, as textbooks set the foundation for curricula, it is crucial that they allow students to think critically about the ethics of performing science and societal impacts of biological research. Our work tests the hypothesis that introductory biology textbooks use predictable strategies to humanize science (e.g., inviting the reader to act like a scientist to develop a solution) and that some topics are more likely to have humanizing elements than others (e.g., some topics are better positioned to serve as launching points for instructors who seek to include humanizing elements in their class). Bringing in discussions of ethics into the science classroom is one strategy instructors can use to humanize biology. We define humanization as the act of positioning science in a social context and/or the act of discussing science through the lens of justice and/or injustice. Of the instances of humanization identified in these textbooks, ethics was mentioned in only 4.57% of these quotes. Ethics was discussed in relation to impacts on humans (41.0%), impacts on humans by humans (41.0%), and impact of humans (16.7%). Discussion of ethics was nuanced (43.6%), detailed (30.8%), and rarely included justice (10.3%) and equity/inequity (5.1%). Ethics was most commonly discussed in terms of biotechnology (38.7%), treatment of disease (12.0%), and environment (10.7%). These findings demonstrate that ethics is rarely made reference to in introductory biology textbooks, yet the field of biotechnology is at the forefront of biomedical innovation, thus having nuanced discussions about controversial subjects (e.g., CRISPR, GMOs, and eugenics) rather than only teaching what these subjects are is pertinent to the training of future scientists.

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.

Cytokines are molecules that mediate cell-to-cell communication in the immune system. Their sensing by immune cells drives their response and differentiation into different functional states, such as into memory or effector states for T cells, to form a proper immune response and combat infections or cancer. Signal transducer and activator of transcription (STAT) family proteins are transcription factors that act downstream of cytokine signaling to regulate the expression of genes driving differential cell functions. Different cytokines phosphorylate and activate different STAT family members to give rise to distinct cellular responses and phenotypic changes. However, there is substantial overlap between STAT members activated by different cytokines. For instance, type I interferon (IFN-I) activates both STAT1 and STAT3 signaling to drive genes associated with growth inhibition, cell death, and anti-microbial responses. Alternatively, Interleukin-2 (IL-2) primarily activates STAT5, but can mediate STAT3, to promote T cell effector differentiation and proliferation. Thus, how different cytokines work through a small common set of STATs to elicit distinct functional phenotypes requires further investigation. Therefore, my project seeks to probe T cell responses and STAT activity with different cytokines. To interrogate this, I will develop an assay to stimulate Jurkat cells, an immortalized T cell line, under various cytokines and analyze STAT1, STAT3, and STAT5 phosphorylation with flow cytometry. We expect Jurkats under IFN-I stimulation to have higher STAT3 and STAT1 phosphorylation than STAT5, but exhibit a more immunosuppressive phenotype, as STAT3 is known to inhibit STAT1-mediated gene expression. With IL-2 stimulation, we expect cells to have higher STAT5 activity with increased proliferation and enhanced effector function. By understanding the complexities of how our T cells differentiate and exhibit certain phenotypes from STAT activity, we can potentially tune the signal a cell interprets and the downstream cellular responses to effectively enhance effector functions, or prevent cell death.

 Subduction zones are regions where two tectonic plates converge, and one is forced underneath the other. They are the primary driver of plate tectonics, and a source of major earthquakes. These earthquakes occur at shallow depths where plates slide past one another quickly, but at greater depths, with higher pressure and temperature (PT) conditions, rocks behave more ductilely. The transition zone between brittle and ductile regions hosts slow slip events (SSEs), which accommodate motion between the plates during events that last months to years, as opposed to the seconds over which earthquakes occur. SSEs are an important mechanism for accommodating plate motion at depth, thereby affecting the occurrence of larger devastating earthquakes, but currently the processes which facilitate SSEs and the rocks that host SSEs are not well understood. By studying rocks formed in and ancient subduction zone from Santa Catalina Island in California, we can learn more about our own modern counterparts, as the rocks record the conditions where slow slip may have occurred. Using optical petrography and data from x-ray spectroscopy, I examine thin sections of epidote-rich blueschist from SSE PT conditions. Optical petrography allows me to characterize the mineralogy of this rock, and the x-ray data provide the chemical compositions of individual minerals. Using image-analysis software, I will pair these two datasets to estimate the bulk-rock chemical composition of my sample. These data will allow me to constrain the starting material (protolith) of this rock before it was metamorphosed in order to determine if it was originally a sedimentary or basaltic component of the subducting oceanic plate. Doing so will improve our understanding of the way in which rocks at those pressure-temperature conditions deform and chemically change to create the context in which modern SSEs occur.

Chimeric antigen receptor T-cell (CART) and T-cell receptor (TCRT) therapy are immunotherapies developed by modifying immune cells to target cancer. Producing CART and TCRT cells involves transduction with viral vectors that usually contain the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). As adoptive cellular therapies become more widely used in cancer care, adverse events (AE) and safety monitoring questions have been raised. We developed a PCR assay to target WPRE for use on archival formalin-fixed, paraffin embedded (FFPE) tissues with the goal evaluating CART/TCRT localization in patient tissues which are taken for routine clinical care. We designed WPRE primers to generate short amplicon for use on FFPE tissues. Limit-of-detection experiments confirmed that our PCR protocol detects CART/TCRT constructs for an institutionally-manufactured CD19 CART, and HA-1, and MAGEA1 TCRT products. The limit-of-detection concentration for the CD19 CART was 1000 copies of WPRE DNA/10uL and MAGEA1 at 100 copies/10uL. In fresh samples, we detected CART DNA at 0.0001% dilution (CAR-T cells into PBMC), and TCRT DNA at 0.001%. For extracted FFPE DNA from cells, we detected WPRE in all four product samples of various CART/TCRT admixtures confirmed by in-situ hybridization. We have also detected WPRE sequences in the FFPE DNA of a pre-clinical humanized mouse sample with MAGEA1 TCRT and a clinical lymphoma sample containing CD19 CART. We are in the process of testing our assay on archival patient samples. Detecting CART/TCRT cells in FFPE samples using a WPRE PCR assay is a novel yet simple technique suitable for clinical application in understanding AEs of immunotherapy. In detecting CART and TCRT in FFPE, we are now able to study trafficking of these immune effector cells when AEs occur or when post-treatment biopsies are available.

One challenge in developing point-of-care molecular diagnostics is achieving a high purity of the protein of interest. Immobilized metal affinity chromatography (IMAC) is a commonly utilized method to rapidly purify polyhistidine affinity tagged proteins. A barrier to accessing purification methods is the cost of materials such as columns manufactured by biotechnology companies at high cost. To address this issue, we have used an (RH)4-tag, a short peptide which consists of four consecutive arginine-histidine groups and can constitute an affinity fusion tag for IMAC due to the arginine interaction with the silica surface via an ion pairing mechanism. Further, the (RH)4 tag can reversibly bind with the silica surface, which allows for the elution of the fusion tag using L-lysine as the elution buffer. Therefore, the silica binding property of (RH)4 enables purification to occur without requiring high cost complex resins. To evaluate the efficiency of the purification process, we have chosen eGFP as the gene of interest of the fusion tag. The green fluorescence of eGFP can be utilized to quantify protein purification efficiency. We can conduct a chromatographic workflow using a column with a silica gel bead matrix, using purified samples of eGFP-RH4 diluted to different concentrations (from 10-100nM). The diluted protein samples are mixed with a silica particle suspension and incubated. The silica beads are removed by centrifugation at 5000xg, and the amount of each protein is derived from the difference between the initial fluorescence and the fluorescence of eGFP remaining in the supernatant. Preliminary results have shown the (RH)4 tag is functional when purifying eGFP on a silica based column and when purifying eGFP using IMAC. Further exploration includes finding an optimized buffer system and using developed methodology to purify different proteins of interest.

Arsenic toxicity is a major environmental problem, which causes various diseases including cardiovascular disorders. Several epidemiological studies indicated that arsenic causes cardiotoxicity, but the underling mechanisms remain unclear. The objective of this project is to determine the molecular mechanism of arsenic-induced cell death in cardiomyocytes. I hypothesize that arsenic trioxide induces ferroptosis, a unique form of iron-dependent cell death, in cardiomyocytes, which may serve as a novel therapeutic target for arsenic cardiomyopathy. Ferroptosis is a newly discovered form of cell death characterized by iron accumulation and lipid peroxidation, which is biochemically distinct from other cell death mechanisms such as apoptosis, necroptosis, and autophagy. I am examining whether arsenic trioxide induces ferroptosis in cardiomyocytes using propidium iodide/Annexin V assay along with ferroptosis inhibitors. I am measuring the levels of lipid peroxidation and cellular iron content in cardiomyocytes treated with arsenic trioxide. To understand the mechanism of arsenic trioxide-induced ferroptosis, I am assessing the cellular content of glutathione and the expression levels of glutathione peroxidase 4 (GPX4), a key suppressor of ferroptosis. Moreover, I am investigating the effect of arsenic trioxide on Bach1-heme oxygenase-1 (HO-1) signaling pathway, which regulates heme degradation and iron release. I expect that arsenic trioxide will induce ferroptosis in cardiomyocytes, associated with increased lipid peroxidation and labile iron levels. Expectations are that arsenic trioxide will induce glutathione depletion and GPX4 downregulation. Moreover, I predict that arsenic trioxide will activate the Bach1-HO-1 signaling to promote cytosolic iron accumulation. Together, this project investigates molecular mechanisms of arsenic-induced ferroptosis in cardiomyocytes, which may reveal a new disease mechanism for arsenic cardiomyopathy. Targeting ferroptosis represents a promising therapeutic strategy for arsenic cardiomyopathy and other complications associated with arsenic toxicity.

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.

With the human life expectancy doubling in only the last 100 years, science has opened us up to this new found idea of longevity and healthy aging. Congruently, people living with HIV have longer life expectancies and can expect to live as long as those who do not have HIV. Healthy aging strategies to navigate living with HIV have only recently developed. The purpose of this study is to explore the associations between physical activity and cardiorespiratory fitness (VO2 peak) in older adults living with HIV. We will conduct a secondary analysis of cross-sectional baseline data of older adults living with HIV, who are participating in the HEALTH randomized clinical trial (ClinicalTrials.gov NCT04550676). This secondary analysis focuses on data involving waist-worn actigraph GT3X/1 monitors, home based actigraphy, and graded exercise tests involving VO2 max that are collected from the ongoing HEALTH clinical trial. We will analyze VO2 max and accelerometry data at baseline between decades of people in their 50, 60, and 70s. This sample is approximately 50 older adults with HIV. We will use descriptive analysis, Pearson correlation coefficient, and Chi-squared test. VO2 max is a valid measure of cardiovascular fitness and aerobic endurance based on the maximal oxygen consumption of individuals during an exercise protocol. This project cultivates an awareness around healthy aging strategies and bio behavioral interventions with a population that has low cardiorespiratory fitness. Both cardiorespiratory fitness and physical activity have been poorly measured using self-report methods in older adults. This secondary analysis uses gold standard objective measures (VO2 Max) to show data points with relation to variability in exercise. This data empowers people living with HIV to engage in strategies to improve their physical function and suggests the need for more bio behavioral interventions to be defined in order to facilitate healthy aging.

Eukaryotic DNA is compacted into cells by wrapping DNA around nucleosomes. Nucleosomes are composed of core or variant histone proteins. Unlike core histones, histone variants enable specialized chromatin functions such as gene transcription or inheritance. Given their crucial and widespread functions, core histones and their variants are typically evolutionarily conserved in sequence and function. Therefore, lineage-specific differences in histone repertoires present unique opportunities to understand their functional consequences on genomic organization and biological processes.

Two such changes, both likely selectively advantageous, have occurred in budding yeast and flies. In budding yeast, the histone variant H2A.X, involved in DNA repair, entirely replaced core histone H2A. A crucial SQ motif on the C-terminal tail of H2A.X is required for its DNA repair mechanism. In flies, the loss of H2A.X and a fusion of the SQ motif from H2A.X with a conserved variant H2A.Z, which is implicated in gene expression, gave rise to a unique H2Av variant. We are recreating the histone H2A compositions of flies in yeast to determine the functional consequences of these evolutionary re-arrangements. Specifically, in S. cerevisiae, we moved the SQ motif or the entire C-terminus of H2A.X to H2A.Z, creating a fusion histone similar to the D. melanogaster H2Av. Therefore, the H2A repertoire in these engineered yeast resembles a fly's. Upon treatment with DNA damage agents, these “Drosophilized” yeast show reduced DNA damage repair efficiencies similar to mutant yeast with no SQ motif. Thus, we hypothesize that the abundance and/or localization of the SQ motif is critical for efficient DNA repair in yeast. Furthermore, we will test if processes like sporulation, meiosis, or resistance to stressful conditions that require DNA damage repair are affected in our “Drosophilized” yeast. These experiments will reveal the biological consequences and potential advantages of the unique yeast H2A repertoire.

In this study, we explore the potential of using data from the DECam Deep Drilling program for asteroid science. The program, originally intended for supernovae and variable star science, produces valuable data that can be used to discover and analyze asteroids. Our focus is on the COSMOS fields, which are observed 5 times in each of the g, r, and i filters every 3 nights. We employ Heliolinc to match the data with known asteroids and discover 296 independent rediscoveries of known asteroids, as well as new discoveries that require further analysis. To analyze the known asteroids, we use forced photometry based on JPL ephemerides to measure their colors. Our results show that the average (g-r) color gets bluer and (r-i) gets redder with increasing distance from the sun. We use this information to develop a distance color metric that combines the colors to maximize sensitivity to distance. Additionally, for some of the best measured objects, we determine their rotation periods using Lomb-Scargle analysis. Our preliminary results demonstrate the potential of the DECam Deep Drilling program for asteroid science, particularly in analyzing asteroid colors and rotation periods.

Automatically, we consider genes as existing solely to serve the host. Aiding in host survival allows the host to reproduce, causing these genes to propagate in the next generation. However, the existence of selfish genes, which ensure their own survival at the cost of their host, brings this conventional wisdom into question. One such type of gene is called a toxin-antidote (TA) system, which express a molecular toxin and its antidote, the latter of which prevents death, but actively kills hosts that express toxin without co-expressing antidote. The aim of this project is to develop a mechanistic understanding of one invertebrate TA system called PEEL-1/ZEEL-1, which expresses an evolutionarily novel, transmembrane toxin protein PEEL-1, and a transmembrane antidote protein ZEEL-1. Data shows that PEEL-1 co-opts a protein called PMPL-1 to kill animal cells; however, PEEL-1+PMPL-1 do not kill yeast. By identifying why PEEL-1 doesn’t kill yeast, we gain insight into PEEL-1’s toxin mechanism. One hypothesis is that it kills by inducing osmotic stress, which yeast may be invulnerable to due to their cell wall designed to resist osmotic stress. To test this, we’ve expressed PEEL-1, PMPL-1, and PEEL-1+PMPL-1 in separate yeast cultures, chemically degraded their cell walls via Zymolyase treatment, and screened for growth. Preliminary data showing that the digested experimental culture (co-expressing) experiences little post-treatment growth compared to the digested controls (non co-expressing) supports the osmotic stress-related mechanism of PEEL-1 toxicity. Our work in identifying PEEL-1 toxicity will define the first mechanism of an animal TA system and guide our understanding of the different ways that protein-driven cell death can develop in nature.

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.

Botryllus schlosseri is a non-indigenous species of colonial tunicate commonly found in many marinas of the Puget Sound. Colonies of Botryllus can grow asexually through the process of blastogenesis (also called ‘budding’) which involves the formation of buds which mature into new zooids on a weekly cycle. Apoptosis and oxidative stress are involved in the final stage of blastogenesis, a time when the system resorbs the zooids and the newly matured buds take over. Although nickel has been found to be genotoxic and can induce many aspects of the cellular stress response in animals, little research has characterized these effects on marine invertebrates. In this study, we performed acute exposures of nickel chloride at sublethal concentrations in B. schlosseri collected from the field to identify changes to blastogenic timing, DNA damage, apoptosis, and the oxidative stress response. These data will establish the in vivo tolerance and stress responses of B. schlosseri to nickel exposure. Furthermore, these data will inform future studies which seek to use nickel as an immortalization agent in cell culture with the goal of generating the first continuous cell line for any marine invertebrate species.

 Reconstructing past environments can help us understand plant community evolution over time. For example, plant silica (phytoliths) can help us reconstruct canopy openness. Phytoliths are formed when plants uptake monosilicic acid from the surrounding soil through their roots and deposit it as opalized silica in and around cells; they have been used as a tool in paleoecology because they are well-preserved in the fossil record. For phytoliths formed in the outermost layers of leaves (epidermis), there is a relationship between morphology and light availability. A previous method established this correlation using modern soils in Costa Rica to apply to sites in the Eocene-Miocene of Argentina. However, it is unclear whether this model can make accurate inferences in other geographic regions. Here, we expand the method using modern phytolith samples from the Southeastern United States to generate a dataset and apply it to fossil phytolith assemblages from the North American Great Plains Region to reconstruct changes in vegetation during Oligocene-Miocene grassland expansion. For this work, we use an optical microscope to observe and count the phytolith assemblages to reflect a range of vegetation types in North America. We focus on phytolith morphotypes representing silicified epidermal pavement cells and measure their size and shape using ImageJ. We expect a linear trend between LAI (Leaf Area Index, the quantified relationship between morphology and light availability) from phytoliths and observed LAI which can be used to form the model for North American environments and applied to the fossil phytolith record of the Great Plains Region. Expanding on this method could make its use more widespread and lead to similar research in other regions of the world. Current models suggest the persistence of closed forests through this entire interval, a result we wish to further test using this updated model.

One of the most striking adaptations in land plant evolution is the specialization of reproductive structures. LEAFY (LFY) is well characterized in flowering plants (angiosperms) as a transcription factor initiating the development of the flower, which contains the reproductive organs. LFY also regulates the first cell division of the zygote (diploid phase) in mosses (a type of non-vascular plant). The fern Ceratopteris richardii is a type of non-flowering plant that is midway phylogenetically between mosses and flowering plants, in the sister clade to seed plants. With a lab-friendly, independent haploid phase (gametophyte), transgenic protocols and a reference genome, C. richardii is ideal for studying LFY’s functional evolution. Previously, targeted silencing of the two fern LFY homologs CrLFY1/2, demonstrated that it maintains the identity of the apical stem cell in gametophytes. To further characterize the function of fern LFY, I study the effects of its over expression on gametophyte development. To that end, I record gametophyte development using bright-field and fluorescent microscopy. Preliminary results suggest that overexpression of CrLFY may affect development of the sperm-producing gametangia (antheridia) in fern gametophytes, with more antheridia found in transgenic plants late in development. Given that antheridia continue to be produced in wild type gametophytes in the absence of fertilization, I test the hypothesis that CrLFY overexpression causes delayed fertilization (by a yet unknown mechanism) and that increased antheridia represent a secondary effect. Here, I experimentally delay fertilization by withholding water needed for sperm release (“flooding”), and compare the number and pattern of antheridia on transgenic and wild type gametophytes with and without flooding. Functional characterization of LEAFY in a fern, and of other master regulators of development more generally, contributes to a better understanding of the evolution of land plants via the potential repurposing of ancestral genetic pathways into novel functions.

A grand challenge in the field of mass spectrometry proteomics is the problem of peptide sequencing. The dominant approach to this problem uses a database search; however, with the use of machine learning, peptide sequencing can be solved without using a database. Casanovo is a recently-developed, state-of-the-art machine learning model that solves this problem. However, Casanovo is trained on biased data, because most mass spectrometry proteomics experiments digest proteins using trypsin, which preferentially cleaves after lysine and arginine. This can result in incorrect predictions for data that was not generated using trypsin. Hence, we hypothesized that using a non-tryptic dataset to refine an existing Casanovo model would produce more accurate predictions on non-tryptic data. I constructed an unbiased dataset by downsampling from preexisting data, yielding a set of peptides with a uniform distribution of n-terminal amino acids. After splitting the data and fine-tuning Casanovo on an unbiased training dataset, I used the model to predict on an unbiased validation set. I also applied Casanovo to two non-tryptic datasets: antibody sequencing data and immunopeptidomics sequencing data. For the latter dataset, an antibody binding affinity tool, NetMHCpan4.0, was used to determine the binding probability of the predictions to test plausibility. We demonstrate that the fine-tuned model, Casanovo_ne, increases performance when predicting on non-tryptic data. Applied to the uniformly distributed validation set, Casanovo_ne predicts more accurately than the original Casanovo model, and predicts more uniform terminal amino acid distributions. Additionally, Casanovo_ne predicts more peptides that are likely to be MHC binders than a database search strategy. Finally, Casanovo_ne accurately represents the digestion rules for most non-tryptic enzymes. As future work, we will modify Casanovo to take as input the identity of the digestion enzyme, alongside each spectrum. We hypothesize this approach will further improve Casanovo’s performance for samples prepared with alternative enzymes.

By 2050, the global human population is expected to reach 9.7 billion. Supporting this rapid growth will challenge global food systems, increasing the demand for healthier affordable foods such as poultry-sourced products (meat and eggs). Improving the accuracy of genotype-to-phenotype predictions for farmed animals could enable better breeding strategies and management practices that are crucial to meeting this goal. By characterizing regulatory genomic regions within various tissues, epigenetic factors which dictate specific cellular phenotypes can be pinpointed: improving phenotype prediction accuracy. To this end, we genetically sequenced (whole-genome bisulfite sequencing) samples of reproductive tissues (magnum, shell gland, isthmus, and ovary) from farmed groups of chickens (G. gallus). Through bioinformatics, we functionally annotated regulatory patterns of DNA methylation to identify tissue-specific epigenetic variation across the female chicken reproductive system. To tackle this, we utilized CGmapTools software to comparatively analyze tissues in a pairwise manner. In order to quantify each pairs’ differentially methylated regions (DMRs), all methylated regions were intersected and filtered through a statistical t-test. We found the pairwise comparison analysis between isthmus and ovary tissues to show the highest number of significant DMRs, being 51% hypermethylated in the isthmus. On the other hand, a comparative analysis between isthmus and shell gland tissues showed the lowest number of significant DMRs, being 54% hypermethylated in the isthmus. Upon annotating and conducting enrichment analyses on the DMRs, we learned that genes related to ECM-receptor interactions and focal adhesion were most prominent. Results of this research will aid the FAANG consortium (an international effort to improve farmed animal production) in improving genotype-to-phenotype predictions, hopefully enabling more sustainable genomic selection practices and genome-enabled management in the future of agriculture.

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.

Vision issues can significantly hinder a child's academic performance and overall quality of life. Despite the availability of existing vision screening and therapy methods, up to 25%-30% of school-aged children struggle with undiagnosed or untreated vision problems, primarily due to financial limitations, logistical issues, and a lack of prioritization of vision care. Untreated pediatric vision problems can lead to social issues and long-term systemic failures. Therefore, it is crucial to provide comprehensive vision care that ensures the diagnosis of all types of visual impairments and appropriate treatments. To address these challenges, computer programs and software applications have been developed as new screening and therapy approaches. This study examines the relationship between pediatric vision and academic performance in school-aged children and evaluates the feasibility of using technological advancements in this medical field. I have conducted a meta-analysis review that involved analyzing and synthesizing data from various studies to explore the link between pediatric vision and academic performance, as well as studies that evaluated the efficacy of using computer programs and software applications as screening and therapy approaches for children's vision issues. Based on the preliminary results, I found that untreated vision problems in children can negatively affect academic performance and overall quality of life. I also discovered that technological advancements have the potential to improve access to vision care and treatment for children, particularly for those who face financial or logistical barriers. I hope that these findings will raise awareness about the importance of early detection and intervention in pediatric vision care, and encourage policymakers and healthcare providers to prioritize this issue. My research also highlights the potential benefits of using technology to improve access to vision care for children and suggests that further studies are needed to explore the efficacy of different technological interventions.

Methanotrophs are prokaryotes that naturally consume the potent greenhouse gas methane for energy. Through metabolic engineering at an industrial scale, these microorganisms hold potential to mitigate the contribution of methane emissions to global warming. In particular, Methylotuvimicrobium buryatense can sustain robust growth both in nature and experimental settings; it is a promising engineering candidate. To develop a robust metabolic engineering platform using M. buryatense, biologists require a deeper understanding of the genetic mechanisms by which it functions. Here, I present an open-source software tool designed to interactively explore the transcriptome of M. buryatense. By integrating bulk RNA-seq datasets collected from experiments over the past decade and applying an array of unsupervised machine learning clustering algorithms, we cluster genes by their expression profiles in differing growth conditions. These gene clusters are annotated with gene ontology (GO) terms using statistical enrichment analysis to assist in functional interpretation of the clusters and the genes that comprise them. To enhance domain-expert researchers’ ability to explore and drill-down into specific queries, I unify these cluster-specific analyses in a web-hosted tool using interactive data visualization techniques centered on a ReactJS frontend and Azure Cloud backend. With both exploratory and query-focused use cases, this software tool can support M. buryatense biologist workflows for predicting functions of hypothetical proteins, showcase new or confirming putative regulatory processes, and generate new experimental hypotheses from the presented transcriptomic trends.

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. 

The Developmental Origins of Health and Disease (DOHaD) hypothesis evaluates how the prenatal environment affects health after birth. The placenta is a multi-faceted organ that sustains life during human development and is key to evaluating the DOHaD hypothesis. Glial Cells Missing Transcription Factor 1 (GCM1) is a transcription factor that plays a critical role in placental development. Our goal is to understand the downstream effects of GCM1 on various genes necessary for placental development by evaluating gene expression after GCM1 knockdown. The BeWo choriocarcinoma cell line is a model of placental syncytiotrophoblasts cells which undergo a cell fusion process called syncytialization in the placenta to form multinucleated cells that help exchange nutrients. Previously, we knocked down GCM1 in full-term primary placental cells that spontaneously syncytialize and assessed gene expression using RNA sequencing. We identified 10 differentially expressed genes. Based on those findings, we hypothesized that GCM1 plays a greater role during early pregnancy leading us to repeat the GCM1 knockdown in BeWo cells. BeWo cells were treated with 20µM, 50µM and 100µM forskolin (FSK) for 48hr to induce syncytialization which was confirmed via qPCR of syncytialization markers GCM1 and Syncytin-2 and through fluorescence microscopy. GCM1 expression increased 3.15, 1.3, and 1.2 fold respectively after treatment with 20µM, 50µM, and 100µM FSK, whereas Syncytin-2 increased 78.1 fold after 50µM FSK treatment. We then performed an siRNA knockdown of GCM1 in unsyncytialized BeWo cells with two concentrations of siRNA (25nM and 50nM) for 24hrs and observed a 70% and 80% reduction in GCM1 expression, respectively. Next steps include optimizing the siRNA procedure for syncytialized BeWo cells and comparing these results to our previously conducted experiment. Overall, this will improve understanding of how GCM1 coordinates gene expression in the placenta during pregnancy.

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.

Justice-centered approaches to equitable computer science (CS) education frame CS learning as a means for advancing peace, antiracism, and social justice rather than war, empire, and corporations. However, most research in justice-centered approaches in CS education focus on K--12 learning environments. In this position paper, we review justice-centered approaches to CS education, problematize the lack of justice-centered approaches to CS in higher education in particular, and describe a justice-centered approach for undergraduate Data Structures and Algorithms. Our approach emphasizes three components: (1) ethics: critiques the sociopolitical values of data structure and algorithm design as well as the underlying logics of dominant computing culture; (2) identity: draws on culturally responsive-sustaining pedagogies to emphasize student identity as rooted in resistance to the dominant computing culture; and (3) political vision: ensures the rightful presence of political struggles by reauthoring rights to frame CS learning as a force for social justice. Through a case study of this \emph{Critical Comparative Data Structures and Algorithms} pedagogy, we argue that justice-centered approaches to higher CS education can help all computing students not only learn about the ethical implications of nominally technical concepts, but also develop greater respect for diverse epistemologies, cultures, and experiences surrounding computing that are essential to creating the socially-just worlds we need.

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.

Lung-resident type 1 conventional dendritic cells (cDC1s) are critical for initiating immune responses against foreign respiratory viruses and endogenous assaults like cancer cells. Importantly, the cDCs of each tissue display unique phenotypes and functions dictated by the environment they populate. However, our understanding of the factors that regulate cDC1 development and function in the lung is minimal. The cytokines, FMS-like tyrosine kinase 3 ligand (FLT3L), and granulocyte-macrophage stimulating factor (GM-CSF) are essential for the development of tissue-resident cDC1, but in in vitro systems, these factors are insufficient to generate cDC1s with a lung-specific phenotype. RNAseq analysis of lung cDC1 identified a clear enrichment in genes associated with transforming growth factor beta (TGF-β) signaling, a factor also known to be enriched in the airspaces of the lung. TGF-β has been previously reported to maintain DC quiescence in the lung, however, our data suggested it may also play a role in differentiation and lung specification of these cells. Based on this, I hypothesized that adding TGF-β to in vitro bone marrow (BM) cultures alongside established cytokines FLT3L and GM-CSF would generate cDC1s phenotypically and functionally similar to in vivo lung cDC1s. The addition of TGF-β, but not GM-CSF, to optimized FLT3L-BMDC cultures resulted in development of cDC1 which highly resemble lung cDC1. The development of a high throughput in vitro system that differentiates functional lung cDC1s will not only inform the in vivo factors required for development of these cells but also provides a useful platform for interrogation of lung cDC1 function and a stepping-off point to begin models for human lung cDC1 production and study.

 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.

With rising greenhouse gas emissions, both emissions reductions and greenhouse gas capture and conversion are necessary to mitigate the impacts of climate change. Though carbon dioxide comprises the largest proportion of global greenhouse gas emissions, methane causes over 80 times more global warming per unit than carbon dioxide. While methane can be converted catalytically at high temperatures and pressures, bacteria called methanotrophs transform methane into biomass at ambient temperatures and pressures. Engineering these organisms to consume methane globally can help slow climate change. At the Lidstrom Lab, I study the relationship between the methanotroph genome and metabolic regulation. Through this work, I have focused on finding genes that facilitate responses to environmental conditions and am now looking at genes that impact overall growth rate. I design and construct mutant strains, eliminating genes that appear to use the cell’s energy unnecessarily based on transcriptomics data. We expect that eliminating such genes will allow the cells to devote more energy to methane consumption and growth, improving growth rates under low methane conditions. This work is being extended to experiments in a bioreactor to observe how selected mutations and growth conditions impact growth rate at low methane. For this work, I am setting up the bioreactor to maintain the intended reaction conditions and utilizing a gas chromatogram to monitor methane consumption over time. These experiments reveal how the methanotrophs grow under specific conditions. I am also analyzing the results of the experiments using Python, MATLAB, and Excel. Long-term, this research will help prepare methanotrophs for deployment in the field to consume methane in areas of atmospheric methane release including landfills and agricultural sites.

The Lieber lab is exploring chemotherapy-drug-free in vivo expansion systems that involve knock-out a gene in hematopoietic stem cells (HSCs) by precision genome editors. Part of this system is an immunotoxin that targets non-transduced HSCs and eliminates them, thereby allowing for the selective expansion of edited HSCs. The goal of my project is to test one of our target proteins CD49f, integrin α6, which has been reported to be expressed on primitive HSCs. In my studies, using flow cytometry with a PE-conjugated anti-CD49f antibody, I have confirmed that CD49f is present on 63% of human CD34+ cells, a cell fraction that is enriched for HSCs. PAI Life Sciences then set out to generate a CD49f-targeted immunotoxin. If the immunotoxin works, CD34+ cells should be highly susceptible to the conjugated immunotoxin. I then tested the immunotoxin on tumor cell lines and primary human CD34+ cells. Measured by flow cytometry, nearly 100% of HEK293T, HeLa and MDA-MB231 cells expressed CD49f. Next, I incubated three cell lines with increasing concentrations of the CD49f-peptide-saponin immunotoxin, ranging from 0.5pg/mL to 10µg/mL. The cell viability and total cell number was measured three days later by two methods: i) flow cytometric quantification, using DAPI staining and ii) counting cells in a hemocytometer after Trypan-blue staining. In tumor cell lines, MDA-MB-231 and HeLa cells, only at the highest immunotoxin concentration a ~58% decrease in total cell numbers was found. The number of viable cells was ~ 4.1% lower than that of untreated controls. Then, I performed cytotoxicity studies with primary CD34+ cells. At the highest concentration tested (10µg/mL), around 6.7% cells were killed by the immunotoxin. Overall, the data indicates that the cytotoxic activity of the new CD49f-targeted immunotoxin is suboptimal. Future steps will include the design and testing of new immunotoxin conjugates.

Multiple myeloma (MM) is an incurable malignancy of the B-cell lineage, characterized by neoplastic, monoclonal expansion of plasma cells in the bone marrow. Remarkable progress has been made in the treatment of MM with the anti-CD38 monoclonal antibodies such as Daratumumab and Isatuximab, which can kill MM cells through the induction of complement-dependent cytotoxicity (CDC). The CDC efficacy of Daratumumab and Isatuximab is however limited by membrane complement inhibitors, including CD46 and CD59, which are upregulated in MM cells. We recently developed a small recombinant protein (Ad35K++) capable of blocking CD46 and sensitizing tumor cells to anti-CD20 mAb triggered CDC (e.g Rituximab and Ofatumumab). Here we tested Ad35K++ in combination with Daratumumab and Isatuximab. We show that Ad35K++ increases the CDC efficacy of Daratumumab and Isatuximab on Burkitt’s lymphoma and MM cell lines (MOLP8 and SUDHL-8). Ad35K++ salvaged the efficacy of Daratumumab and Isatuximab at subtherapeutic (“low”) doses in MM lines. Daratumumab and Isatuximab treatment of MM lines (without Ad35K++) resulted in the upregulation of CD46/survival of CD46 high MM cells which escaped a second round of Daratumumab and Isatuximab treatment. Escape was reduced by combining Daratumumab and Isatuximab with Ad35K++. We also tested a small recombinant protein that targets CD59 (rIYD4) in combination with Daratumumab and Isatuximab on MM cells. rIYD4 also increased CDC killing of MM cells by Daratumumab and Isatuximab. The combination of Ad35K++ and rILYD4 additively enhanced the CDC effect of Daratumumab and Isatuximab. Studies with patient MM cells will be reported. Overall, our data demonstrate that Ad35K++ and rILYD4 are efficient co-therapeutics of Daratumumab and Isatuximab and could be used to improve the treatment of multiple myeloma.

Alcohol use disorder affects 5.8% of adults in the U.S., incurring a yearly cost of $249 billion annually. The repeated and prolonged use of alcohol creates a variety of physiological and behavioral issues. Acute alcohol withdrawal syndrome encompasses seizures, delirium tremens, and in some cases, death. The dependence and withdrawal cycles lead to neuroinflammation, worsened withdrawal symptom severity, and impaired neuromodulation. The striatum plays an important role in the chronic aspect of addiction. This project focuses on how alcohol alters microglial function in this key brain region. At the Neumaier lab, we found that alcohol withdrawal increased the expression of genes involved in the unfolded protein response (UPR) in striatal microglia, the brain’s immune cells. The UPR is activated when there is an increase of misfolded proteins in the endoplasmic reticulum; which contribute to impaired cell function. The UPR is a protective mechanism in moderation, but when left unchecked, has been shown to increase cell death. We predict that removing CHOP will lead to a decrease in withdrawal symptoms and regulate the need to consume alcohol. Knocking out CHOP involves breeding CHOP fl/fl |Cx3cr1 CreER /eYFPI mice. We observe changes in the offspring via emotional behavioral tests after a 5 week period of CIE (chronic intermittent alcohol exposure). A second cohort’s brains are analyzed using cryosectioning and immunohistochemistry (IHC). My role is preparing the brains for analysis. The first step is removing and perfusing, which prepares the samples for cryosectioning. This is my other specialty, which is freezing and slicing the brains. Lastly, I conduct analysis with IHC, and look for physiological changes in the striatum. We anticipate our findings will have a positive and large impact on treating alcoholism. Our hope is to reduce the stigma surrounding addiction, and instead offer compassionate and scientifically based care.
 

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 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.

The brain is one of the most complex and vital organs in our body. While thousands of people get treated for concussions, our knowledge of the impacts of brain injury is fairly limited. Brain injury caused by concussions occurs in about 1.9 million youth. Coupled with anxiety, this type of injury can become further complicated, which is why a holistic approach is necessary when treating patients, and even more critical when considering pediatric cases. There is research evaluating concussions and anxiety independently, but there is little research analyzing the joint relationship between the two. The Care4Kids study at the Seattle Children's Research Institute is one site in a multi-site study that examines post-concussive symptoms in children between the ages of 11-18; in conjunction with this work, I present the findings of a literature review evaluating the intersectionality between anxiety and concussions, asking the question, “What are the effects of pediatric sports-related concussions on anxiety?” The methodology for this review primarily focuses on synthesizing previously conducted research studies and reviews to present a comprehensive picture on the current discussion in research involving anxiety and concussion, and also its impact in the scientific community. In this review, I analyze the various aspects of pediatric sports concussion symptoms, focusing specifically on the manifestation of anxiety in post-concussive children, the degree of this anxiety faced across all ages, and a comparison of the short and long-term effects. With the findings from this literature review, we can gain a more comprehensive understanding of the relationship between anxiety and concussions in children to better predict and detect concussive symptoms in the future in order to ultimately provide children with efficient and conclusive post-concussive care.

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.

In flowering plants, LEAFY (LFY) is a key floral developmental gene; homologes of LFY exist in nonflowering plants, where this developmental regulatory role cannot exist. In an effort to understand what role LFY serves in a nonflowering plant I am studying the model fern Ceratopteris Richardii, as ferns produce spores not flowers. My goal is to determine if the two fern LFY homologues, CrLFY1/2, play a role in the reproductive or vegetative development of ferns. To investigate this function, I am characterizing the phenotypes of transgenic plants that overexpress one or both of the fern copies. To that end, I record data on key developmental markers across the diploid generation of the fern, or sporophyte. Given that LFY overexpression has been associated with early flowering and leaf compounding in flowering plants, I anticipate early reproductive development and/or abnormal leaf development in my transgenic ferns sporophytes. Preliminary results suggest that transgenic ferns are producing spores earlier than controls, and are not displaying all expected markers of vegetative development. Here, I present data on the vegetative development and appearance of reproductive structures of C. richardii ferns overexpressing one or both of two gene duplicates, CrLFY1 and CrLFY2 to help elucidate the functional evolution of the this important developmental regulator of flowering.

Over the past year, I have conducted movement and personal-narrative-based epistemological research interrogating the gender semantics of manhood versus “male-hood" within our Western society. Borne of the progressively queer politics of gender that define all transgender men as men, as well as the freedom of identity and expression among all individuals that queer theory permits, this research has intended to discover the identity of “man” disconnected from the previous phallic definition. I have observed, physicalized, and self-referentially disrupted forms of masculinity in our society through embodied movement explorations and semantic restructurings, with specific ties to masculinity within my own life and upbringing. More recently this research has primarily focused on the pressures that make manhood an assumed consequence of maleness and what it means to interfere within that predestined process, as well as the associated consequences of doing such. From these investigations, I developed three iterations of physical performance works, the third of which will be presented at this symposium. Each work has come with different conclusions and has revealed areas lacking understanding and/or definition. So, though I am presenting a finalized work, this research remains quite unfinished and never intends to be considered so. Due to the personal nature of this research, this work is only directly applicable within my lived experience and I hesitate to generalize my research to claim any relevance to lives other than my own. My motivation behind this research has always been one of personal discovery, but by sharing it, I hope to expand this research and bring it into a larger conversation than myself. 

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 Svoboda Diaries, preserved in 61 volumes, are a unique record of life in 19th-century Iraq. Working as an officer in the Lynch Company, a British steamship venture on the Tigris River that operated between Baghdad and Basra, Joseph Svoboda (1840-1908) kept his diaries in English for 40 years (1862-1908). Along with the historical information they preserve, these diaries reflect the evolution of Joseph Svoboda’s writing style and vocabulary, and contain a variety of different spellings signifying the diverse linguistic environment of Ottoman Iraq. The presenters of this paper have been working as a team, editing and digitizing Svoboda Diaries as a part of the ongoing Svoboda Diaries digital humanities project. Our team currently follows a detailed pipeline that describes six steps: digitization, transcription, editing, Arabic translations, final proofing, and publishing. We have transcribed the scanned diary pages line by line, and systematically proofread one another’s work. Team members recently developed a framework of proofreading in order to identify and indicate illegible and damaged portions. The orthographical, language-related and cultural issues presented by these diaries have pushed us to be creative and methodical in how we collaboratively edit the Svoboda Diaries for digital preservation and publication, and signify potential applications for research across diverse disciplines. By documenting the research and experience Svoboda Diaries Project has amassed over the course of a decade, this group presentation contributes to the literature about accurately preserving and making accessible primary documents, and provides guidelines to navigate the ambiguities of editing handwritten historical texts. The paper also discusses future prospects for the Svoboda Diaries Project, such as the introduction of TEI coding that will enhance digital representation of this source material.

On the surface, career warrior Larry Alan Thorne seems a likely candidate to be an anti-hero. He fought in three unpopular wars, voluntarily joined the Waffen-SS twice, and was convicted of treason in his homeland, Finland. A carjacker and three-time prison escapee, Thorne, né Lauri Allan Törni, has become a celebrated folk hero and declared a legend by many. He was awarded Finland’s highest military honor, an Act of Congress was passed to grant him American citizenship, John Wayne portrayed a fictionalized version of him in the iconic 1968 film The Green Berets, and he is the only known Waffen-SS soldier buried in Arlington National Cemetery. My research explores the question, “How is public memory of Larry Thorne a case study of historical revision and rehabilitation?” My presentation analyzes numerous primary and secondary sources, including depictions of Thorne in movies, music and social media, as well as personal correspondence with experts including Michael Cleverley, author of the leading English language biography of Thorne, and Professor Oula Silvennoinen of the University of Helsinki, who co-wrote a Thorne biography in Finnish. This research project reveals the significance and nature of a decades-long historical revisionist campaign with regard to a figure with a dubious past, and how those efforts to shape the public story of Thorne involve, at times, potentially misleading narratives. The current personality cult of Larry Thorne is a case study of historical revision in public memory, and his story is still being written and rewritten.

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.

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. 

CCR5 is a co-receptor required for HIV to infect the body. Several AIDS patients were completely cured after receiving hematopoietic stem cell (HSC) transplants from donors with a mutation in their CCR5 gene making it functionally inactive. However, HSC transplantation is a very risky and expensive procedure, making it inaccessible for most AIDS patients in developing countries. Our aim was to develop a technology that introduces CCR5 gene mutations in the HSCs of AIDS patients in vivo by a single intravenous injection of a gene transfer vector. The vector delivers a genome editing enzyme (base editor) targeted to the CCR5 gene. CCR5-gene edited HSCs will give rise to CCR5-negative HIV target cells, thereby blocking HIV infection and providing life-long protection. We tested three strategies to functionally inactivate CCR5 expression: i) creating a premature stop codon, ii) eliminating the ATG start codon and iii) mutating splice acceptor sites to skip exons. We employed an advanced adenine base editor version (ABE8e) and an early version of cytidine base editor (CBE) delivered with helper-dependent adenovirus vectors (HDAd5/35++) that efficiently infect HSCs in vivo. The base editors are directed to specific target sites by single guide RNAs (sgRNAs). We screened a small library of sgRNAs and identified two species (sgSTOP2 and sgR5-1) that mediated the highest on-target editing rates and CCR5 down-regulation. HDAdAd5/35++ vectors were produced using these sequences. In a test cell line infected with these vectors, 50% of CCR5 alleles were edited. This blocked HIV infection in 40% and 95% of HDAd-ABE8e-sgSTOP2- and HDAd-CBE-sgR5-1-infected cells, respectively. We concluded that the HDAd-CBE-sgR5-1 vector is more efficient in blocking HIV infection and will further improve this vector and test it in primary human lymphocytes and HSCs in the context of HIV infection. This approach has the potential to provide a technically simple HIV/AIDS therapy.

HIV-2 represents approximately 1-2 million of the 38.4 million people living with HIV (PLHIV) worldwide. Existing guidelines and recommendations for the treatment of HIV-2 infection are largely derived from in vitro data and small-scale clinical studies, and are often extrapolated from studies of people living with HIV-1. Furthermore, of the two most common HIV-2 groups (A and B), only a few examples of drug resistance in group B have been reported in literature. As a result, in-depth knowledge about the mutations that arise in group B HIV-2 isolates and the roles that these mutations play in antiretroviral drug resistance is lacking. The aims of my project are (1) to develop a novel approach for creating full-length, infectious HIV-2 group B clones that encode patient-derived integrase sequences, and (2) to demonstrate that this approach can be used to characterize resistance to Integrase Inhibitors (INI), which are an important class of antiretroviral drugs. Specifically, the parental plasmid for cloning and virus expression is 7312A-JK; this plasmid contains a full-length HIV-2 genome with group B gag and pol gene sequences. To facilitate downstream steps, I have replaced the integrase-encoding portion of p7312A-JK pol with a short, synthetic linker. This cassette clone will serve as the acceptor for longer, synthetic DNA fragments that encode patient-derived group B HIV-2 integrases. My ultimate goal is to generate a panel of 10 replication-competent group B clones that can be given to members of the Gottlieb lab for culture-based drug susceptibility testing. This work is an essential step toward a more comprehensive knowledge of drug resistance in HIV-2 which, in turn, is important for evidence-based treatment of all HIV-2–infected individuals, including those infected with group B strains.

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.

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.

T cells are a crucial part of the human immune system that play a role in the response to diseases from type 1 diabetes to COVID-19. All T cells undergo genomic recombination to create distinct T cell receptor complex (TCR) cell surface molecules that recognize antigens presented by major histocompatibility complex (MHC) molecules, thereby triggering a series of signaling pathways that culminate in T cell activation and cell division. Afterwards, daughter progeny share the same unique TCR molecules. In my project, I will be examining the role TCRs shared within a single individual (private) and between individuals (public) during COVID-19 infection. While the existence of these classes of TCRs is well documented, their functional roles remain unclear. My hypothesis is that the presence of public TCRs is linked to the strength of an immune response and, hence, to different disease severity in humans. We reason that common evolution may have led to the rise of these public TCRs because of their major role in the human body’s antiviral immune response. This hypothesis predicts reduced levels of public TCRs results in an inability to fight disease, whereas high levels of public TCRs will be associated with less severe disease. To test this, I will use a mixture of single cell RNA analysis and statistical tests on a dataset of TCRs acquired from blood samples of 254 SARS-CoV-2 patients and 16 healthy patients. Using a multiomic approach will allow me to also find genetic links between cells of interest. The data includes baseline and acute readings for each patient, and can be subsetted to cells with expanded TRA and TRB junctions individually. This experiment will help advance our knowledge about the function of public TCR chains and their role in fighting disease. Furthermore, our data could have translational applications for disease biomarkers.

Cannabis use has dramatically increased in response to legalization in the U.S., with total sales in the U.S. jumping 46% from 2019 to 2020. ᐃ9-tetrahydrocannabinol (THC) is the primary psychotomimetic compound in Cannabis and has been shown to modify memory and motivation, processes mediated by the prefrontal cortex (PFC) brain region. I sought to test the effects of THC on PFC activity during appetitive Pavlovian conditioning in mice- a behavior in which a subject learns to associate a non-rewarding stimuli to a reward. THC acts on the endocannabinoid (eCB) CB1 receptor (CB1R), a presynaptic signaling protein responsible for modulating neural activity throughout the brain, with robust expression in the PFC. To monitor neural activity during behavioral trials, we implanted optic fibers into the PFC and virally expressed biological sensors: GCaMP6f to track Calcium activity, and the novel GRABeCB2.0 to measure eCB activity. VGAT-Cre and VGLUT1-Cre animals were presented with a house light prior to a sucrose reward to observe the neuronal GABAergic and glutamatergic activity during the conditioning, respectively. After 5 days of conditioning, I administered vehicle or THC (i.p., 5 mg/kg) to observe behavioral and neural effects of THC. We observed neural activity that transferred from the sucrose reward to the house light cue suggesting these neurons encode for this learning. Endocannabinoid activity also transitioned from sucrose reward to the house light cue suggesting cannabinoid involvement in regulating this association. THC pre-treatment reduced licking and motivation for sucrose while modifying neural activity without eliminating it. This provided much needed insight into the formation of memory during learning and reward motivation under the effect of THC.

Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive compound found in Cannabis sativa and acts on the cannabinoid-1 receptor (CB₁R). Given its well-documented analgesic effects, THC’s therapeutic value in treating pain such as those associated with motor neuron disease states, muscle spasticity-related pain, chronic pain, and muscular sclerosis has gained traction. THC’s psychotomimetic locomotor impairing effects causes patients to cease treatment. However, this relationship between THC and locomotor control is poorly understood. To address this, we are investigating THC’s effects on Pre-Frontal Cortex (PFC) neural activity during natural, unprompted movement behavior in mice. The PFC historically is known for its role in executive function but is also a target for THC’s psychotomimetic effects. We expressed GRAB_eCB2.0, an endocannabinoid biosensor, or GCaMP6f, a Ca²⁺ biosensor, in the PFC and recorded neural activity through fiber photometry during uninhibited movement behavior. We found a novel, THC- and locomotion-dependent transient of Ca²⁺ and endocannabinoid activity in the PFC at the initiation of movement. I investigated the activity of glutamatergic and GABAergic neuron activity in the PFC by utilizing genetic mouse lines and found the Ca²⁺ activity transients were primarily driven by the GABAergic interneurons that constitute 20% of the anatomical population. I hypothesized that this is due to THC-dependent activation of the CB1R on distinct GABAergic interneuron subpopulations in the PFC, which would disinhibit glutamatergic activity and in turn promote spontaneous movement. I utilized in situ hybridization to examine colocalization of CB₁R with distinct GABAergic interneuron subpopulations. We found that while CB₁R does, in fact, colocalize with GABAergic interneurons, there was no differential localization between subpopulations. Overall, this project furthers our understanding of the ways in which THC modulates neuronal activity and locomotive behaviors.

Lead perovskites have attracted the interest of the industry for optoelectronic devices applications due to their strong and tunable absorptions. However, their stability and environmental toxicity impose a challenge in its use for commercial application. Bismuth perovskites are a promising alternative due to their similar ionic radius to lead with long-term stability and lower toxicity. To replace lead-based perovskites with bismuth-based ones, it is necessary to increase the photoluminescent quantum yields and extend the emission wavelength range. The crystalline structure of bismuth perovskites can be altered with a dopant to redshift the usually blue light emission. We doped bismuth-based perovskites with rare-earth metals via sonication. The produced materials were analyzed by ultraviolet-visible spectroscopy and photoluminescence spectroscopy. The collected measurements determine if a redshift was produced on the emission spectra. It is expected to see a redshift from the current 400 nm wavelength on the doped perovskites. However, the current results do not show a redshift, instead they show a change in intensity. The comprehension of how to efficiently produce redshifted bismuth perovskites can propel the industrial level use of this less toxic alternative.

The trapping of individual ions has allowed physicists to control and observe otherwise inaccessible phenomena. Ion traps have enabled the most precise measurements of fundamental physical constants, mass spectrometry for chemical characterization, atomic clocks that would only lose a fraction of a second over the entire age of the universe, and the direct observation of many core concepts in quantum mechanics. Many crucial developments in ion traps occurred here at the University of Washington in the group of Hans Dehmelt, who shared the 1989 Nobel Prize in physics for that work. Today, techniques in ion trapping continue to be developed because trapped ions are one platform for creating qubits in quantum computers. With the growth of quantum information science in academia and industry, there is a need for inexpensive, scalable educational labs to introduce students to concepts in quantum computing. To fill this need, we developed a reproducible lab, which demonstrates key concepts in ion trapping. Our process utilized, first, a comparative approach with reference to literature and, second, iterative improvement on built components. The lab consists of two, independent quadrupole traps: a four-rod trap and a planar five-rail trap. To reduce cost and complexity, we trap charged particles with 25 µm and 50 µm diameter, rather than atomic ions. The particles are trapped in air, at atmospheric pressure. Due to the damping forces provided by this background gas, the trapped particles are easy to control. The result of our project is a lab capable of several experiments, including controlling the number of particles trapped through voltage modulation at a constant frequency, studying the phase transition between one- and two-dimensional Coulomb crystals, exploring micromotion compensation, observing two- and three-particle secular modes, and demonstrating particle shuttling along the trapping axis of the planar trap.

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.

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.

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.

Phytoliths, silica bodies formed within and around plant cells, are a key part of a plant's physiological structure that can vary in shape between species. These phytolith shapes (so-called morphotypes) can be found abundantly within grasses and vary between taxa within a subfamily either in shape or in their relative abundances and could therefore provide important evolutionary data on how specific grass subfamilies may be related. Previous work has been done on certain grass subfamilies, including Bambusoideae, to identify similarities and differences in shape within a grass subfamily. This study aims to investigate the distribution of phytolith morphotypes among three closely related grass subfamilies (Arundinoideae, Danthonioideae, and Micrairoideae). To collect this data, we conducted a morphological study on over 300 phytoliths in samples from many species within our three subfamilies. The samples were taken from leaf clearings wherein the phytoliths were isolated through chemical treatment and centrifuging to remove other organic material before staining. Samples are imaged using a confocal microscope and then patched together with computer processing to form three-dimensional phytolith images. These sample objects were compared based on phytolith morphotype three-dimensional shape, their relative abundance, location in plant tissues, and size. In addition to the morphological study of individual phytoliths, we studied cleared leaves to obtain a greater sense of the composition of morphotypes within the tissue of the grasses. The results are expected to show an overlap in similar phytolith morphotypes across clades that have similar ecological niches such as photosynthetic systems. Overall, this research aims to find a link between these three close modern subfamilies that could be compared to fossil phytoliths in order to document their evolutionary history and past distribution.

In the 400 million years since they first emerged on land, plants have acquired numerous adaptations to the terrestrial environment. One of these adaptations is modification of the photosynthetic pathway, from C3 to C4. The C3 pathway, the ancestral form of photosynthesis found in most plants, has limited efficiency under high temperatures and light intensities, whereas the C4 pathway offers improved productivity. C4 photosynthesis has evolved numerous times in flowering plants in the last 66 million years, with C4 grasses being the most diverse and ecologically dominant. Despite their current importance, we still do not understand exactly when and where the evolution of C4 photosynthesis in grasses occurred because the fossil record of grasses is sparse. Phytoliths (hardened silica structures precipitated within plant cells) offer a novel tool for tracking C4 evolution. We will use phytoliths as a comparative tool to examine morphological changes associated with C4 evolution. All C4 grasses are contained within the PACMAD (Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonioideae) clade of grasses. Panicoideae, one of the largest and most diverse subfamilies within PACMAD, containing both C3 and C4 species, is an ideal group to study the ecological and evolutionary factors that drive the distribution of C4 photosynthesis. Through analysis of phytolith morphology, as well as overall density and distribution of phytoliths within leaf tissue using leaf clearings, we will examine a broad sampling of Panicoideae, looking for common trends amongst C4 photosynthesizing groups as compared to their C3 counterparts. To extend the scope of these conclusions beyond Panicoideae, we will seek to confirm these trends by comparison to Aristidoideae, another clade that evolved C4 photosynthesis. Preliminary data indicate that the majority of phytolith morphotypes will be bilobates, crenates, and rondels. We expect these results to correspond between observed leaf clearings and the 3D models.

My project involves making new tools for the genetic manipulation of Staphylococcus aureus. S. aureus is an important and pervasive human pathogen, however, much is still not known about its pathogenesis pathway and virulence genes. To better understand the genes and mutations that make S. aureus successful at causing disease, it is necessary to induce specific genetic changes and assess their impact on the organism’s virulence. However, currently available tools for bacterial genome engineering have yet to be optimized for S. aureus. Building on a successful recombineering system, previously developed by the Salipante lab, that is able to edit the S. aureus genome, we aim to address this need by using a dominant negative mutant protein of the mismatch repair (MMR) system to achieve suppression of DNA repair. Recombineering is the process of incorporating mutagenic DNA molecules into a host genome through the recombinase enzymes. Dominant negative mutations in the highly conserved MutL protein have been shown to disrupt the DNA base MMR pathway in several other microorganisms but have not yet been evaluated in S. aureus. My project aims to construct a temperature sensitive vector that highly expresses a ssDNA recombinase that is active in S. aureus along with a dominant negative mutL mutant able to bypass the MMR pathway. We anticipate that WT mutL should be able to repair a single base mutation since it has a functional MMR, reducing the number of successful recombinants relative to experiments performed with the multiple base pair mutations. The dominant negative mutL strains should not be able to correct a single base pair mutation, resulting in a similar number of drug-resistant transformats when recombineering with either a single base pair or many base pair mutations. This will allow us to better generate custom mutant strains and subsequently test the functions of various S. aureus genes towards a variety of clinically relevant phenotypes.

When managing our natural resources and assessing human impacts on ecosystems, it is important to understand how plant communities respond to disturbance events. The geologic record has the potential to provide an important source of information for scientists to observe how plant communities of the past have responded to disturbances. Currently, there is a limited ability to recognize disturbance as the primary driver of change because there is limited evidence of how functional traits - plant traits that relate directly with plant function and ecological strategy that are measurable in fossil leaves - vary across succession. To improve this ability, I am measuring the carbon stable isotopic composition (δ¹³C) of bulk organic matter in leaves sampled across a successional gradient. This functional trait is often preserved during leaf fossilization and is representative of a plant's water use efficiency (WUE), an important ecological strategy representing the carbon assimilated per water lost in a plant during photosynthesis. The extent to which carbon isotopes measured at the community scale reflect the successional stage of a plant community is not currently known. To improve this knowledge, I am testing the hypothesis that the WUE of plant species within a community will become more conservative in later successional stages. In support of this hypothesis, I predict that the abundance-weighted community average of leaf δ¹³C will increase through succession. In addition, I hypothesize that δ¹³C as a proxy for WUE will be most confounded in early succession, before a tree canopy forms, due to seedling utilizing water resources more rapidly without having established root systems and thus predict a higher variance of δ¹³C values in this earliest stage of succession. This research is helping develop a method of identifying disturbances within geologic records which can give guidance on management decisions regarding modern ecosystems

Early-onset Alzheimer’s Disease (EOAD) patients have a greater risk of developing seizures. Consequently, EOAD patients who develop seizures have worsened comorbidities, including mortality and behavioral outcomes. The causal relationship between chronic seizures and AD is still unknown. Neuroinflammation has been postulated as one of the links between these two disorders. Our lab has previously demonstrated that young mice with a presenilin 1 (PS1) genetic variant and amyloid precursor protein (APP) overexpression, mimicking an EOAD phenotype and genotype, subjected to chronic seizure are at high risk of mortality. This behavior was not observed in young mice with an EOAD-associated presenilin 2 (PS2) genetic variation. We hypothesize that chronic kindled seizures evoked in 2 months-old APP/PS1 mice worsen neuroinflammation and other neuropathological hallmarks of EOAD, including β-amyloid (Aβ) accumulation in the brain. To address this, 2-month-old male and female APP/PS1 and PS2 mice were subjected to seizures through a corneal kindling model of chronic seizures. The right brain hemisphere was collected before and after mice achieve the kindled criterion (five consecutive 5 Racine scale scores). Brains were harvested and cryopreserved for immunohistochemistry. My role in the project was to process tissues for immunohistochemistry. 20-µm thick sections of hippocampus from each mouse were sectioned on a cryostat and slide-mounted before processing with commercially available antibodies for molecular markers of neuroinflammation and neuropathology. Photomicrographs were collected and images were analyzed as the number of immunoreactive cells for each molecular and protein marker. We anticipate increased neuroinflammation and Aβ accumulation in kindled APP/PS1 mice versus their respective wild types and non-kindled littermates. We do not expect differences in PS2 variant animals. These results add to a larger research study in the laboratory to suggest that targeting seizures, and its inflammatory response, may be a potential therapeutic strategy to mitigate the behavioral and neuropathological burden of AD.

Trapped ions are one of the promising candidates for an operating quantum computer. Ions trapped in an electromagnetic trapp serve as a physical qubit, where the qubit states are manipulated by applying lasers to the system. As quantum computers use quantum gates with a given precise angle of rotation of the qubit state within the Bloch sphere, applying a laser with very narrow bandwidth is essential for minimizing errors, and thus stabilization of laser frequency is a required process for trapped ion qubit control. In our project, we stabilize the 1762 nm InfraRed fiber laser by using an optical cavity lock, where we obtain the resonant frequency of the cavity by measuring the intensity of the laser across the Fabry-Perot cavity, while varying the laser frequency. However, this model cannot distinguish between the laser intensity noise and the laser frequency noise. To address this, we eliminate the intensity noise by analyzing the signal reflected back from the cavity, where we observed a frequency dependent signal which reaches zero at resonance, allowing us to stabilize the laser to the desired frequency. A deeper understanding of the laser stabilization techniques may help us to minimize the trapped ion qubit control errors.

The increasing availability of both prescription and illicit opioids has caused substance use disorders to skyrocket. Treatment options such as therapeutics that can inactivate Kappa opioid receptors (KOR) have been shown to reduce drug-seeking behavior through the modulation of intracellular signaling pathways. Downstream KOR activation, G-protein coupled receptor kinase 3 (GRK3)/arrestin-dependent pathway leads to activation of p38 mitogen-activated protein kinase (p38 MAPK) and feelings of dysphoria. In another pathway downstream of KOR receptor activation, a G-protein mediated response and activation of cJun kinase (JNK) leads to the generation of reactive oxygen species (ROS). Selective activation by biased ligands of the JNK mediated pathway result in the release of ROS, which leads to the eventual depalmitoylation of the G-αi/o subunit of the KOR. This results in the long-term inactivation of KOR, which is predicted to improve stress resilience and to prevent drug-seeking behavior. Drugs such as Nalfurafine and Nalmefene can selectively activate KOR such that ROS is produced. Using 2-photon microscopy to detect fluorescence that indicates the release of ROS by Nalmefene and Nalfurafine into the ventral tegmental area of transgenic mice, I can better understand the potential of these drugs for long-term inactivation of KOR. I have observed under a light microscope that when Nalfurafine was washed onto slice, an increase in ROS was observed. Nalmefene showed a similar trend to Nalfurafine but increased ROS to a lesser extent. Additionally, when either Nalfurafine or Nalmefene were added to a solution of naloxone, there was no significant increase in ROS. Using the data collected from slice and behavioral assays such as the tail-flick test, we can illustrate the positive therapeutic effects that KOR inactivators can have on substance use disorders in the long term.

Have you ever watched a true crime show? I’d be surprised if you said no. Stories of violent crime inundate the entertainment offered to individuals whether you’re looking for a TV show or a podcast. People’s worst moments are retold for TV audiences under the guise of investigation. While crime media has always been popular, true crime media, television in particular, is having a moment of unprecedented popularity. Drawing from my own experiences as a white middle-class viewer of true crime I wonder what the dangers of this genre's popularity are. This project investigates this by asking how the consumption of others' trauma through true crime television impacts how individuals who have no contact with the criminal justice system understand violence and crime. It asks whether or not this creates additional distance between individuals who are not system impacted and those who are. And finally, it wonders if true crime's popularity can be used to put its viewers in conversation with abolition. Using Dateline NBC episodes as its archive, this project hopes to navigate the complicated nature of true crime viewership and its harms. The archive of Dateline episodes provides examples of key narratives within true crime as well as facilitates a true crime viewing experience informed by abolitoinist politic. The patterns observed in Dateline are put in conversation with my academic research to both attempt to answer my research questions as well as articulate critiques of true crime television. These intentions are creatively rendered into a zine that synthesizes my research as well as my relationship to the genre and Dateline specifically. I hope that this project inspires individuals to engage critically with their entertainment and understand that entertainment is a means of perpetuating hegemony.

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.

Urolithin A (UA) is a metabolite produced by gut bacteria from ellagitannin and ellagic acid, which is present in pomegranates, berries, and walnuts. Previous studies have shown that UA supplementation improves skeletal muscle health in both mice and humans. UA supplementation also appears to be beneficial to the heart, resulting in both systolic and diastolic functional improvement in old mice. This study aims to determine a mechanism for the improvements in the aging heart. We hypothesize that the mechanisms behind improved heart function from UA supplementation are related to the pathways that directly improves mitochondria, contractile properties and mitophagy. To investigate the mechanism behind improved heart function with UA supplementation, we split the treatments into two staggered cohorts with 20, 24-month-old mice per cohort (10 control/10 UA treatment). Both the control and the treatment group were kept on the control diet during week 1 for acclimation. In the following 8 weeks, the UA treatment group were moved to a UA 50 mpk supplemented diet. Echocardiography measurements were done pre and post supplementation. At the end of the study, all mice were euthanized under anesthesia after an overnight fast and sample collection was performed. Heart samples were collected from each mouse. Western blots of heart samples on mitophagy, mitochondrial and cardiac contractile protein markers indicate that those pathways remain unaffected by UA supplementation. In future work, we will explore other informatory pathways in order to determine the mechanism behind UA supplementation improvement of heart function.

Iron centers which feature metal ligand multiple bonds can be powerful group transfer agents, for example, terminal Fe-oxo intermediates in soluble methane monooxygenase can perform oxo-atom transfer for the selective oxidation of methane to methanol. Abiologically, ligand constructs which enforce desirable electronic and structural configurations have been shown to enhance group transfer to a range of organic substrates. We developed and studied an iron (Fe) molecular complex with two aminophosphine selenide ligands (Se=PPh₂NTol; Ph=Phenyl, Tol=4-Tolyl) that chelate the metal center via the selenium and nitrogen. The iron complex (FeL₂) was synthesized by a reaction between Fe(HMDS)₂ (HMDS = bis(trimethylsilyl)amide) and the aminophosphine selenide. Characterization shows a tetrahedral, high spin, symmetric compound. We hypothesized that FeL₂ can activate and transfer heteroatoms and explored the reactivity of FeL₂ with oxidants, oxo atom donors, and organic azides. Treatment with iodine (I₂) results in oxidation of the iron center (Fe(II) to Fe(III)) and coordination of the iodide counterion results in structural reorganization to a five-coordinate square pyramidal complex. Reactivity with oxo atom donors shows that either the ligand or Fe center are oxidized, and we identified a µ₂-oxo dimer, which is the first Fe-O-Fe dimer to have selenium in its first coordination sphere. We found that FeL₂ forms Fe-nitrenoid intermediates and can perform nitrene transfer to form diazos or do C-H amination, when treated with aromatic and aliphatic azides, respectively. The presented complexes are characterized by single crystal X-ray diffraction (XRD), Evan’s method, nuclear magnetic resonance (NMR), and Ultraviolet-Visible Spectroscopy (UV-Vis). This research builds upon the knowledge of transition metal complexes for heteroatom transformations.

Benzalkonium Chlorides (BACs) are widely used antimicrobial disinfectants in a variety of settings, including large scale food processing and consumer environments. Persistent usage of BACs raises concerns about the potential disruption of the gastrointestinal microbiota, an increasingly recognized regulator of an individual’s health. Furthermore, the gut microbiota has been shown to regulate drug metabolizing enzymes (DMEs) and the Gut-Liver Axis is a known prominent crosstalk pathway. Previous work in our lab has found BACs are capable of altering gut microbiome composition in BAC exposed C57BL/6 male and female mice, with notable differences between the male and female sexes. Therefore, we hypothesize that exposure to the BACs can alter the composition of gut microbiota, leading to sex specific changes in bile acid homeostasis as well as the metabolic phenotype and DME expression of the liver. In this study, we exposed male and female mice to C12- and C16-BACs at 120 ug/g/day for one week via oral dosing. Additionally, through a targeted bile acid quantitation analysis, we found sex specific decreases in secondary bile acids in BAC-treated mice. This finding is supported by decreases in bacteria known to metabolize primary bile acids into secondary bile acids, such as the families of Ruminococcaceae and Lachnospiraceae. We also aim to elucidate both transcriptomic (RNA sequencing) and functional (enzyme activity assays) analyses of the harvested livers from both male and female cohorts. Upstream pathway analysis from the results of these analyses is expected to yield sex specific differences in the downregulation of genes responsible for a variety of pathways such as protein digestion and absorption and transcriptional regulation in cancer. This study is expected to provide novel insights into the sex specific alterations in the relationship between the gut microbiome and liver caused by BAC exposure and the mechanisms underlying BAC toxicity.

Urban trees are an increasingly essential part of the city environment, as lining streets with greenery has been shown to benefit residents’ health and quality of life. Trees especially impact air quality, which has implications for chronic illnesses like asthma, and existing literature suggests a relationship between air quality and children's health. This paper examines the impact of increasing urban tree canopies in a large, evolving city like Chicago on children’s asthma rates over a decade. I use tree census data from the Morton Arboretum to evaluate change in tree cover from 2010 to 2017, and use Chicago health records to track rates of emergency department visits due to pediatric asthma. Testing the strength of correlation through a regression model can determine the existence and strength of a correlation and relationship between tree canopies and health. The results suggest a need for increased tree planting policies to improve local health outcomes, especially in low-income neighborhoods with sparse canopy cover. As the climate gets warmer and air quality decreases, infrastructure that addresses both climate change and public health will become increasingly essential. By continuing to study the impacts of urban forests, we can develop strategies for mitigating these global issues in a local context.

Hypoxia ischemia encephalopathy (HIE) is characterized as a lack of oxygen and blood flow to the brain, and is a leading cause of neonatal mortality and morbidity within the United States. HIE causes immediate cell death and oxidative stress resulting in inflammation, energy failure, and ongoing injury. Though there is a lack of effective therapies for HIE, extracellular vesicles (EVs) have shown incredible potential in attenuating oxidative stress and inflammation. EVs are biological nanoparticles with a lipid membrane containing essential biomolecules. EVs participate in cell-to-cell communication as they travel between membranes of cells within the central nervous system (CNS). Previous studies on adult brain injury models show the potential for EVs to drive neuroprotective and anti-inflammatory processes in the brain. The aim of my project is to evaluate neonatal injury responses to brain-derived EVs (BEVs) following HI injury on ex vivo brain tissues. To mimic an ischemic brain environment, I used an oxygen glucose deprivation (OGD) model to induce hypoxia in neonatal rat brain tissues. I quantified time-dependent changes in the gene expression profiles of brain tissues after BEV treatment by performing RNA extractions and reverse transcription-quantitative polymerase chain reactions (RT-qPCR). This allowed me to compare the expression levels of pro-inflammatory and anti-inflammatory markers to determine the therapeutic efficacy of BEVs on an ischemic model. My results suggested that BEV exposure in OGD-injured models decreased cytotoxicity by encouraging microglia (the brain’s immune cells) to transition from inflammatory to anti-inflammatory phenotypes. Results from the RT-qPCR analysis further suggested that BEVs reduced inflammation through the upregulation of anti-inflammatory cytokines observed in the study. This demonstrates that BEVs play a role in reducing cell death and activating anti-inflammatory pathways in the neonatal brain, providing insight into their potential as a therapeutic tool for future interventions aimed at treating HIE.

The geologic record provides opportunity to provide actual examples of how plant communities have responded to climatic changes, providing important perspective for modern anthropogenic-driven climate change. Two important climatic events in the Miocene offer such an opportunity, including a global warming event, the Miocene Climatic Optimum (MCO; 17-14 million years ago), and a global cooling event, the Middle Miocene Climatic Transition (MMCT; 14-12 million years ago). This study is assessing how the diversity and prevalence of ecological strategies within Pacific Northwest (PNW) plant communities changed in response to these events, by analyzing ~6 PNW fossil plant sites that span these events in time. At each site I characterize ecological strategies of taxa comprising these ancient communities by measuring leaf vein density (LVD) of fossil angiosperm leaves, which relates strongly to the maximum photosynthetic rates of the plant. Photosynthetic rates influence ecological strategy by placing plants along a spectrum with fast growth but low tolerance to resource scarcity at one end, and slow growth and high tolerance at the other. I am digitally measuring leaf vein density using microscope images of fossil leaves previously taken at several museums where these fossils are housed. I expect that during the MCO, evergreen plants with slower growth rates become more dominant and the diversity of ecological strategies increased (lower mean and higher variance of LVD). Across the MMCT, I expect that deciduous plants with high growth rates became more dominant and stronger abiotic filtering caused a decrease in the diversity of ecological strategies present (higher mean and lower variance of LVD). This study provides a real-life example of how climatic events reshaped the assembly of plant communities and provide an important perspective for present and future climate change.

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.

 Patients with end-stage renal disease (ESRD) are 25 times more likely to be admitted to an intensive care unit (ICU) compared to those without ESRD. About 70% of ICU patients are aged 55 and older. Older ICU survivors may report new impairments in cognitive, physical, and psychological health, referred to as post-intensive care syndrome (PICS). It is unknown whether older ICU survivors with ESRD experience greater severity of PICS symptoms than those without ESRD. We conducted a secondary analysis of pooled data from 2 studies of 51 older ICU survivors. Diagnosis of ESRD was obtained from electronic health records. Assessments from the National Institutes of Health Toolbox (attention, cognitive flexibility, grip strength, dexterity) and Patient-Reported Outcomes Measurement Information System (depression, anxiety) measured PICS symptoms. Wrist actigraphy measured activity and sleep. We conducted independent samples t-tests to compare fully corrected T scores of each PICS symptom between older ICU survivors with ESRD and those without ESRD. Results indicated older ICU survivors with ESRD scored worse on attention (25 ± 10.3) compared to those without ESRD (33.8 ± 6.9), t(48) = 2.76, p = 0.008. Older ICU survivors with ESRD also scored worse on grip strength (24.8 ± 14.9) compared to those without ESRD (39.1 ± 12.6), t(48) = 2.54, p = 0.014. Older ICU survivors with ESRD were less active during the daytime (21.2 ± 10.3 activity counts/minute) compared to those without ESRD (51.9 ± 33.1), t(48) = 2.25, p = 0.03. Older ICU survivors with ESRD slept more during daytime hours (70.5%) compared to those without ESRD (51.2%), t(48) = -2.53, p = 0.015. Older ICU survivors with ESRD may be at risk for PICS throughout recovery from critical illness. Further research with targeted interventions focusing on screening and treating PICS symptoms is needed to improve health in this population.

Neonatal hypoxic-ischemic encephalopathy (HIE), caused by a lack of blood flow and oxygen to the brain, is a major cause of infant mortality. Primary and secondary energy failure caused by HIE activates microglia, resulting in morphological changes and inflammatory cascades that mediate ongoing pathology. Proinflammatory microglia release cytokines and reactive oxygen species that damage oligodendrocytes, the myelinating cells in the brain that supports neuronal function, thereby causing demyelination of neurons. Previous studies in term-equivalent in vivo ferret models showed that microglia respond to injury and treatments with region-dependent cell morphology changes. However, the effect of combinatorial therapy on microglia and oligodendrocyte in a preterm model is unknown. This project aims to quantify image-based morphological features of microglia and oligodendrocyte in response to neuroinflammation and separate and combinatorial treatments in different brain regions of an in vivo preterm ferret model. Using machine learning supported image processing, I quantified microglia and oligodendrocyte morphology in the healthy control group, injury group of two hours of oxygen-glucose deprivation, and treatment groups of azithromycin (AZ), erythropoietin (Epo), and combined AZ+Epo treatment followed by injury. The machine learning algorithm clusters microglia and oligodendrocytes into distinct shape modes with different morphological parameters, such as perimeter, circularity, and aspect ratio. Perimeter and circularity of both microglia and oligodendrocytes show regional heterogeneity within each shape mode while aspect ratio is homogeneous. Microglia perimeter decreases upon injury in crescent and rod-like shape modes. Epo treatment reverses the decrease to the level of nontreated control, but AZ+Epo treatment only partially reversed the decrease. By quantifying microglia and oligodendrocyte morphological response to neuroinflammation and treatments across regions, I non-destructively assessed therapeutic performance of separate and combinatorial treatments in the preterm ferret model. The assessed performance informs therapeutic choices for preterm populations and have the potential for translating to larger animal models.

The grass family, Poaceae, dominates over 40% of land ecosystems and is found in every biome except areas covered by ice sheets. Within Poaceae are two major clades, one being the PACMAD clade, named for the six subfamilies: Panicoideae, Arundinoideae, Chloridoideae, Micrairoideae, Aristidoideae, and Danthonoideae. The PACMAD clade is the only lineage of grasses that evolved C₄ photosynthesis. This derived trait allows plants to efficiently photosynthesize under low CO₂ concentrations and in hot, arid climates. Chloridoideae is the largest subfamily within the PACMAD clade with over 1,500 species across five tribes. Most Chloridoideae species use C₄ photosynthesis and it is likely that some of the first transitions from C₃ to C₄ occurred in this subfamily; however, fossil evidence for this deep history is currently lacking. Phytoliths, which are silica bodies that form in living grass tissues and can be preserved in soils for millions of years, have great potential for filling this gap. We are studying the three-dimensional shape of phytoliths from modern Chloridoideae grasses to better recognize them in the fossil record. By linking modern Chloridoid phytolith shapes to their respective climatic conditions, we will be able to create a robust reference database to be used for future research to identify past Chloridoids and their past growing environments. To do this, we are processing 3D Chloridoid phytolith models from 2D confocal microscope images to analyze and characterize the morphology, abundance, distribution, and diversity of Chloridoid phytoliths. Thus far, preliminary data suggests our findings will be especially useful in making comparisons between past and present bilobate or saddle-shaped phytoliths, though we expect to conduct further analysis on current phytolith shapes. Future studies will be able to compare our 3D modern renderings to fossil phytoliths to infer periods of climatic warming through deep time.

Metabolites are small organic compounds that are the products of cellular metabolism and the building blocks of macromolecules. The analysis of a multitude of metabolites in a sample simultaneously is known as metabolomics and is a powerful tool for understanding microbial interactions in the ocean. In particular, metabolomics provides a way to investigate how marine communities vary in composition during shifts in environmental conditions. The North Pacific Subtropical Gyre (NPSG) is a region where inorganic nitrogen availability limits phytoplankton productivity and microorganisms rely partially on diazotrophs for fixed nitrogen in the surface ocean. Because N2 fixation is often iron-limited, bioavailable iron should control fixed nitrogen levels in the gyre. Here, we tested this hypothesis by collecting metabolomic samples during a large-volume incubation in which tanks were amended with various nutrient combinations of iron, nitrogen, and phosphate during a month-long incubation. It was expected to stimulate a diazotroph bloom by limiting the incubation for nitrogen. In these nitrogen-limited tanks, we expect to see a strong metabolic response to the absence of fixed nitrogen, followed by the ingrowth of nitrogen fixers with their own metabolite fingerprints as the experiment progresses. I will compare metabolomes of incubations to those of phytoplankton cultures, including the nitrogen-fixing cyanobacteria UCYN-A and Trichodesmium. I will also use the incubation's nutrient concentration and microbial community metabolomics to test the hypothesis that altering nutrient supply ratios (Fe: N: P) in the NPSG microbial population will result in metabolite shifts. As the critical link between inorganic matter and the formation of the organic material that powers the ocean’s food chains and biological carbon pump, metabolomics provides a way to better understand the critical role that nitrogen fixation plays in regulating the taxonomy and biochemistry of the world’s largest biomes.

Syphilis is a sexually transmitted infection (STI) caused by the bacterium Treponema pallidum subspecies pallidum (T. pallidum). Syphilis is still a global health concern, as its incidence is rising in high-income countries like the United States and it is still endemic in low-income countries, where it causes significant mortality due to congenital transmission. Our chances at syphilis control would improve if a vaccine against syphilis was available. The past century of research has only yielded experimental vaccines able to produce partial protection, but the use of new techniques could improve this outcome. One possibility would be to use an array of known protective epitopes from other T. pallidum antigens crafted onto a protein carrier to induce a protective immune response. Currently, we are investigating the use of the Tp17 protein of T. pallidum as a scaffolding for a future vaccine. The Tp17 protein has a β-barrel structure with loops around the edges of the barrel. To use this protein as a scaffolding for a vaccine, we will be performing epitope mapping to determine the hierarchy of immunodominant epitopes in Tp17 to find the ideal candidate regions that will be substituted with other epitopes. We are using Enzyme-Linked Immunosorbent Assay (ELISA) to attain these data in combination with human sera to define the reactivity of the different peptides. The results will determine the most immunogenic peptides, thus indicating the ideal areas to be replaced by protective epitopes in a potential vaccine. We anticipate that several of the most reactive peptides will correspond to the protein loops outside of the β-barrel, which should be easily replaceable without affecting Tp17 structure.

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.

The Miocene Climatic Optimum (MCO) was a period of global warming 17-14 million years ago, where temperatures increased 2-4°C and CO₂ levels increased to ~400-600 ppm. Overlapping with the MCO were the Columbia River Basalt eruptions (CRB: 6.6-15.9 Ma), where extensive lava flows spread across the Pacific Northwest, resulting in primary succession. My study is focused on reconstructing the vegetation across the MCO and during CRB eruptions using epidermal phytoliths (i.e., Microscopic Biosilica) to understand how these conditions impacted plant communities. Epidermal phytoliths are formed within living plant matter reflecting the current environmental conditions in their size and undulation. The plant matter then falls to the forest floor and decays leaving behind the resilient Microscopic Biosilica, which is preserved within that sediment. Leaves formed in ecosystems with an abundance of sunlight reflect open-canopy vegetation, with small circular phytoliths; while large-undulated phytoliths come from closed-canopy, shady environments. Previous work has shown a correlation between the average size and undulation of epidermal phytoliths with leaf area index (LAI; i.e., a measure of canopy openness). I am using this process with sediment samples collected across four sites in Central Oregon to calculate ancient reconstructed LAI (rLAI), and thus reconstruct the canopy cover. Each site was chosen due to the time period it represents, with different exposure to increasing variations of CO₂ and CRB impacts. I hypothesize increased temperature and atmospheric CO₂ concentrations during the MCO created favorable conditions for plant communities, which promoted a productive closed-canopy forest structure. Additionally I hypothesize, the primary succession induced by CRB volcanism prevented the re-establishment of forests, leading to open-canopy vegetation structure. As modern day anthropogenic-driven climate change invokes alterations in our planet's ecosystems, we need to better predict and anticipate future responses of plant communities to these environmental perturbations. 

Morphology of leaves informs plant functions from structure to growth rate, all summing to diverse strategies that plant communities employ to thrive. By analyzing strategy changes over time, morphology can describe the strategic response to disturbance events - particularly those precipitated by human activity. My study aims to develop a framework for characterizing those strategies on a temporal scale during ecological succession based on leaf vein density (LVD), which is the length of vein tissue within a given leaf area. I am accomplishing this through the chemical isolation of vein tissues, analytic microscopy, and image analysis, examining temperate deciduous leaves from 5 sites in North Carolina with varying amounts of time since the plot was clear-cut for timber harvest. Once I prepared slides of 4 cm2 of leaf matter, I began imaging the leaves under 8x magnification and following this, I plan on using image analysis software to measure LVD over 2-3 mm2. Based on correlations between early successional species - species populating a cleared area before slow-growth taxa regenerate - and high vein density organisms demonstrating faster growth, I hypothesize that taxa prioritize growth via resource allocation during initial phases of recovery post-disruption due to the increased availability of sunlight due to altered canopy openness, increasing photosynthetic rate. This would be characterized by higher LVD observed in early successional species. Should this prediction bear out, it indicates that LVD can be used to better understand varying ecological strategies in a community over time. Specifically, we will be able to use it to analyze how that spectrum changes based on environmental changes and determine which strategies are prioritized in which stages of community change. By garnering a clearer grasp on the diversity in early versus late successional strategies, I plan on connecting the prevalence of certain functional traits and environmental changes across time.

Cell death and the processes surrounding it are essential parts of life. Ferroptosis is a distinct type of regulated cell death characterized by increased lipid peroxidation leading to cell membrane damage. The exact mechanism of ferroptotic death is currently unknown, so research is underway to discover pathways that can modulate ferroptosis. The goal of this project is to determine how different isoforms of the ACSL gene impact ferroptosis mediated by non-conjugated and conjugated polyunsaturated fatty acids (PUFAs). ACSL4 (long-chain acyl-CoA synthetase 4) is a gene of importance in ferroptosis because it incorporates PUFAs into the cell membrane. The membrane-incorporated PUFAs can be oxidized via lipid peroxidation in the cell, leading to membrane damage and eventual cell death. It has been found that knocking out or silencing the ACSL4 gene can make cells resistant to ferroptosis. I compare how ACSL4-knockout cells react to different PUFAs, and how these PUFAs sensitize wild-type and ACSL4-knockout cells differently when treated with ferroptosis-inducing drugs. Cell viability assays are a way to measure the amount of cell death in response to a treatment. I perform these assays to create dose-response curves for different lipid and drug treatments and use flow cytometry to quantify the amount of lipid peroxidation. These assays help establish a baseline comparing the response of wild-type and ACSL4-knockout cells to different PUFAs and ferroptotic drugs. Preliminary results demonstrate greater percent viability in two different knockout cell lines compared to wild-type cells when treated with the drug RSL3 and arachidonic acid. ACSL4-knockout cells are expected to have decreased cell death if they are protected from ferroptosis. Results will demonstrate the extent to which knocking out the ACSL4 gene affects cell survival. Ultimately, ferroptosis is a process of interest due to its therapeutic potential in treating tissue damage and as a targeted cancer therapy.

The Middle Miocene (23-5 Ma) represents a period of rapidly changing climate and active volcanism, particularly in the Pacific Northwest. Our understanding of the structure and composition of plant communities during this timeframe is complicated by a limited or degraded leaf fossil record. Plant fossil assemblages are also often time averaged, representing accumulation of plant material over an extended period. A plant community that was preserved because of a single, short-lived event can provide insight into the composition and structure of that community in life. The Watersnake locality of the Sucker Creek Formation in southwestern Idaho contains two thick (8-14 m) ignimbrite tuffs (volcanic ash layers) that preserve charcoal fragments, reflecting plants that were burned when the tuffs were deposited. In order to identify the woody taxa that made up this community, thin section microscopy is used to examine the preserved cellular detail of the fossil charcoal fragments from the lower tuff. Since ignimbrites are deposited as a part of a single event, the impacts of time-averaging are minimized. Although these deposits reflect a short time, they are likely to integrate across space, providing a view of the broader landscape. The results of this study will reveal the structure of this plant community immediately prior to the eruption that caused the ash flow. I hypothesize that the taxa identified within the ash will be very similar to those of the leaf fossil record found in the shale beds below. A lowland community consisting primarily of wetland Glyptostrobus oregonensis, and Quercus simulata found near flowing water will likely be represented. Upland vegetation consisting of conifers (Pinus, Tsuga) were also likely incorporated into the ash flow as it moved downhill. This study will provide insight into the dynamics of plant community change during the Miocene in relation to volcanic disturbance.

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.

Fire is a fundamental disturbance that drives changes in biome structure. Knowledge of ancient fire regimes may help predict future fire regimes resulting from anthropogenic climate change. Charcoal morphometry (quantified shape of charcoal), particularly charcoal aspect ratio (length:width), is an emerging proxy of ancient fuel type wherein higher mean aspect ratios are associated with grassy fuels. This study aims to experimentally validate this proxy method. Thirty-four modern plant species were sampled from UW Herbarium collections, separating leaf, stem, and reproductive body tissues for each species. Each sample was burned at 500°C for 20 minutes, crushed in a water slurry, and imaged under a binocular microscope. Charcoal particles were enumerated and morphometrics were measured using ImageJ with charcoal particles 125-250 μm and >250 μm analyzed separately to account for differences due to differential particle breakage. Strong evidence was found in the 125-250 μm size fraction through an analysis of variance test (F = 2.66, p = 0.03), that aspect ratio varies as a function of taxonomic group. The strongest evidence for a difference in aspect ratio is found, through Tukey's Honestly Significant Differences to be between graminoid and conifer charcoal (p = 0.03). Evidence is even stronger for a taxonomic effect on aspect ratio in the >250 μm size fraction (F = 3.64, p= 0.007). This variation seems to also be driven by a difference between graminoid and conifer charcoal (p = 0.002), corroborating earlier findings. Future validation of this methodology will be focused on the potential effects of burn temperature and charcoal transport in charcoal morphometric records. Rigorous verification of charcoal morphometry as a proxy of fuel type will help increase confidence in paleo reconstructions of fuel type. 

Despite the abundance of research surrounding the influence of organized religion on emotional well-being and the sense of community, women in Spanish-speaking Christian churches have been overlooked. In most churches, women are treated differently than men and are less likely to hold leadership positions, yet, previous research findings imply high religiosity is positively associated with well-being among women. However, Spanish-speaking religious communities have notably different cultures than the largely white church communities that previous studies focused on. My aim with this research is to understand how gender impacts the relationship between religion, emotional well-being, and sense of community outside the previously studied, predominantly white populations. The cultural differences between white and Spanish-speaking religious communities are numerous, these differences may lead to different outcomes. This study investigated whether the results of the previous studies would be the same within Spanish-speaking churches. This study used qualitative semi-structured interviews with women (lasting 15-45 minutes), in private settings within their churches. These interviews were transcribed and patterns that emerged among the women were coded. I selected churches using non-probability sampling and snowball sampling to recruit women within the identified churches. My hypothesis suggests that women within Spanish-speaking Christian churches experience a better state of well-being and a strong sense of community despite being treated differently than men. Understanding how religious institutions can have positive effects on marginalized identities allows for those institutions to be used as safe spaces where one might do outreach, engagement, and design interventions in ways that might be more effective than through other social institutions such as schools or hospitals.

In face-to-face surveys containing sensitive questions, Social Desirability Bias (SDB), respondent’s tendency to provide socially acceptable responses rather than truthful ones, can compromise data accuracy. Randomized response techniques (RRT) are survey models that allow respondents to provide scrambled responses, thereby circumventing SDB. In this study, we introduce a mixture optional quantitative RRT model that combines the elements of both the Pollock and Bek (1976) additive RRT model and the Greenberg et al. (1971) unrelated question quantitative RRT model. We examine the utility of the proposed mixture model using a unified measure of efficiency and privacy introduced by Gupta et al. (2018) that provides a metric of both predictive accuracy and respondent privacy. We also account for the lack of trust in RRT models. Both empirical and theoretical results show that the mixture model outperforms the two component models. The proposed optional quantitative mixture RRT model provides a survey technique that can account for not only SDB but also respondent lack of trust, leading to more accurate and interpretable data used to inform decision making that does not compromise the privacy of respondents.

In the midst of World War I and the Mexican Revolution, a document known as the Plan de San Diego emerged in South Texas. Discovered by local police in January of 1915 in McAllen, Texas when they arrested a Mexican national, the Plan called for a total racial revolution against Anglo-Texan power. A few months later, raids in support of the Plan broke out. Historians have primarily disputed over whether the Plan was an authentic product of local Tejano radicalism, or part of a wartime master plan by German and Mexican conspirators. The mystery surrounding its authorship and the raids has contributed significantly to this lack of consensus. My research traces the roots of the Plan’s rhetoric and the movement around it through the history of Mexicanos in Texas. I argue that the racial logic of the Plan demonstrates a real grounding in local history, regardless of its authorship. Rather than focusing on the moment in itself, I follow the lineage of the “Mexican race” which it attempted to mobilize. To do so, I closely read the wording and differences across time and space in legal policy, court cases, proclamations, newspapers, and folk music. In particular, I read the operation and formation of social-political categories between the lines of these texts. This approach draws significantly on racial formation theory, which offers a means to understand how such a movement could come about. By incorporating this critical approach, I hope to make a step forward in critically analyzing history as an element in racial history and the development of the identity "la raza," which the Chicanx movement mobilized later in the 20th century.

Immunofluorescent images are a common way to analyze cell response in the presence of brain disease. Microglia - the brain's immune cells - have a range of functional states dependent on their local environment to keep the brain environment healthy. Microglia are typically stained and viewed with immunofluorescent imaging to study the brain's immune response. Microglial functionality and microglia morphology (shape) are highly correlated [5]. By taking and quantifying images of microglia in healthy and diseased brains, we can gain insights into their functional state and their local environment. In addition, most fundamental research about microglia involves the use of animal models, where many species are used to model brain disease. However, limited research directly compares microglia response in one species to another. Previously, research within the Nance Lab has focused on quantifying rat microglial features such as area, perimeter, or circularity [3]. Here, we developed a method to quantify features of microglia, with a focus on microglial branching – the arm-like protrusions from the cell body expanding upon previous work by adding additional branching features to the quantification pipeline to look at the number and length of branches around each cell, which gives us information on the functional state of the cell. We investigated the species-dependent effect on the microglial shape by analyzing images of cells obtained from the neonatal human-term equivalent rat (postnatal day 10, P10), ferret (P21), and mouse (P12). We see qualitative differences in morphology, such as more extensive branching in the rat compared to the ferret. Our ongoing work aims to quantify feature differences in microglia between the rat and ferret and expand to other species.

Hypoxic Ischemic Encephalopathy (HIE) is an injury to a newborn that can occur during pregnancy or the birthing process. HIE is caused by a restriction of blood flow to the brain which leads to inflammation and neuronal death. A myriad of symptoms and disorders including developmental delays and cerebral palsy can result from HIE. The widespread treatment for HIE among clinicians is therapeutic hypothermia; however, this treatment method can only reduce the severity of the injury, is not a curative procedure, and is only effective in a small percentage of babies with HIE. One possible solution to the lack of effective HIE therapies is a nanoparticle-based therapeutic used to decrease inflammatory responses in the brain following HIE. Prior work in the Nance Lab has shown a polymer nanoparticle can specifically target microglial cells in a pro-inflammatory state in an injured brain. In this study, I have successfully loaded the antioxidant N-Acetyl-Cysteine (NAC) into the polymer nanoparticle platform. Using an ex vivo model of neuroinflammation, I displayed that polymer nanoparticles loaded with NAC can be harnessed to change microglia in a proinflammatory state to a more anti-inflammatory phenotype. This nanoparticle treatment was also combined with a priming dose of azithromycin (AZ) prior to NAC-encapsulated nanoparticle administration to further reduce the rate of microglial inflammation in the injured brain. The reduction of microglial inflammation and increase in anti-inflammatory microglial phenotype presence caused by these two therapeutics provides a platform that can be tested in preclinical models of HIE, with the long-term goal to improve the quality of life for children and families affected by HIE.

Hypoxic-ischemic encephalopathy (HIE), resulting from a lack of blood and oxygen flow to the brain, is the leading cause of morbidity and mortality in newborns, and currently has no cure. Our lab is investigating curcumin for use as a neuroprotectant agent, as it has anti-inflammatory, antioxidant, and antiapoptotic effects. Our current studies have been focused on further improving the drug encapsulation of curcumin in a polymeric nanoparticle platform, as well as methods to increase long-term shelf-life stability of the nanoparticle therapeutics. Previous research from our lab has successfully loaded curcumin into poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) nanoparticles as a delivery vehicle. PEG-PLGA is an FDA approved, biodegradable polymer platform that allows for improved drug delivery efficiency, controlled and sustained drug release, and improved penetration and diffusion in the brain. We have shown that curcumin loaded PEG-PLGA nanoparticles have resulted in significant neuroprotection when used as a treatment for hypoxic-ischemic neonatal rats (term equivalent to human). In order to progress towards scale-up and clinical translation of the therapeutic, I have tested variations to the formulation method at every step of the formulation process, including changes in PEG-PLGA molecular weight ratios, surfactants, and organic solvents used. I have assessed the impacts of each formulation parameter on colloidal stability and drug loading, with the aim to create a scalable, stable platform that can retain drug delivery and drug activity properties during distribution and shelf-life storage. I have identified that nanoparticle drop size, surfactant type and concentration, and freezing protectant (cryoprotectant) have the biggest impact on drug loading and stability. Improving the stability is the first step in making the therapeutic more accessible, cheaper, and easier to transport for a larger impact.

The marine intertidal is subject to challenging hydrodynamic forces. Organisms that reside within these zones; such as purple (Strongylocentrotus purpuratus) and green (Strongylocentrotus droebachiensis) sea urchins, must have strong attachment to prevent dislodgement, ambulate, and feed in intense environmental conditions. Sea urchins attach to the substrate via their podia (tube feet). The disc found in the distal part of tube feet has a duo-gland that releases specialized adhesive compounds and de-adhesive enzymes to achieve adhesion. Adhesive performance was evaluated to determine if purple sea urchins, that are predominantly found in high wave exposure areas, have better adhesive performance than green sea urchins, which are abundantly found in areas protected from wave action. We evaluated the following adhesive performance variables: stem mechanical properties, tube foot disc tenacity, attachment area, speed of detachment and whole animal adhesive force. We found that for most adhesive performance metrics, purple urchins performed better than greens, except for detachment speed. These results suggest that, in the field, behavioral changes may play an important role in determining risk of detachment for sea urchins. Understanding how morphology, behavior and mechanical properties of tube feet work in conjunction to provide strong attachment, is critical to predict sea urchin fitness in challenging environmental conditions.

Hypoxic-ischemic encephalopathy (HIE), a brain injury that occurs when infants do not get enough blood flow or oxygen to the brain, is a leading cause of neonatal mortality and morbidity worldwide. Therapeutic hypothermia (TH) is the current standard of care for newborns with HIE, but TH is only available in high-resource settings and only provides partial neuroprotection. Thus, the search for additional neuroprotective treatments is critical. The ferret provides an excellent model for investigating novel treatments for HIE as, unlike rodents, it has a gyrified brain and gray-to-white matter ratio that is like humans. Previous research has shown that the ferret brain is resilient to brain injury, requiring additional hypoxia periods and increased pro-inflammatory stimuli to create the same injury as in rats. However, no previous studies have evaluated why the ferret brain is so resilient. This study will use live rat and ferret organotypic brain slices to investigate this resiliency. Whole hemisphere brain slices from postnatal day (P)10-12 rats and P21-23 ferret, equivalent to term gestation in humans, will be collected. The slices will be randomized to oxygen-glucose deprivation (OGD) injury, to mimic HIE, or control groups. OGD slices will be in 0% oxygen for 2 hours, resulting in partial, but not total, cell death. Subsequent analyses will assess transcriptomics using NanoString nCounter technology, which provides a neuropathology panel of 770 genes, as well as cell-specific regional death in brain regions affected by HIE: hippocampus, cortex, corpus callosum, subcortical white matter, basal ganglia, and thalamus. Preliminary results show that genes such as UCHL1 and TLR4, both associated with injury, are upregulated in ferret OGD slices, but rat data are currently incomplete. Identifying the pathways associated with the resiliency of the ferret brain to injury at the transcriptome level could inform future therapies to treat infants at risk for HIE.

An estimated 5-8% of the American population have cerebral aneurysms, showing higher rates of development in patients with common risk factors like hypertension, smoking, and family history of cerebral aneurysms (CA). This study focuses on understanding the causes of aneurysmal subarachnoid hemorrhage (aSAH), where a CA ruptures, resulting in bleeding in the brain. Endovascular coiling is a minimally invasive surgical treatment method for aSAH. Unfortunately, up to 30% of endovascular coiling treatments are unsuccessful, leading to aneurysm recurrence, growth, or rupture. The risk of these outcomes can be predicted using Computational Fluid Dynamics (CFD), a tool that quantifies the hemodynamic environment by solving the equations of motion for a fluid. The CFD simulations calculate factors significant in predicting the effectiveness of coiling treatment including flow rate, wall shear stress, and pulsatility. In this project we have studied the effect of using patient-specific blood viscosity values (the resistance of the blood to fluid flow), that have typically been standardized for all patients in CFD simulations. We have analyzed the effect of using patient-specific blood viscosity on pre-treatment patient-specific computational fluid dynamics simulations of endovascularly-coiled cerebral aneurysms. Preliminary results show that there is an expected improvement in CFD simulation predictive power of treatment effectiveness when patient-specific blood viscosity values are used. We hope to improve the predictive power of CFD simulations regarding the treatment outcome of aneurysm coiling, allowing us to better predict aneurysm recurrence, and eventually guide treatment outcomes.

As the field of astrophysics continues to grow, the quantity of data to analyze is constantly expanding. With projects like the James Webb Space Telescope each sending back hundreds of gigabytes of data every day, Artificial Intelligence (AI) technologies is needed to assist manual analytical techniques in processing these volumes of information. One of the most apparent tasks for AI in astrophysics is image categorization – identifying what sort of astronomical object a certain body is. If a machine could categorize these bodie in significantly less time than a person, it would free tens of thousands of human hours every year. I created a Machine Learning program using a Deep Neural Network (DNN) implemented in Keras and TensorFlow capable of classifying astronomical images based on photometric data. Built from scratch, it utilizes existing labeled images to “learn” how astronomical bodies differ in appearance and assign them a category. The value of automated classification of astronomical phenomena cannot be understated. DNN allows the model to find unique identifiers in images humans often cannot spot, leading to often-more reliable predictions, recognizing possible discoveries in far less time, and freeing astronomers to undertake higher-cognition tasks only humans can accomplish. As the model is continuouly improved, it will be able to make increasingly accurate classifications and be of ever-growing value.

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

 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.

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

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.

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.

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

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

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.

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

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.

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

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

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

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

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

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

The 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.

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