Pembrolizumab (brand name KEYTRUDA), is a monoclonal antibody therapy classified as an immune checkpoint inhibitor, a type of immunotherapy that works by facilitating the detection of cancer cells by T cells. Specifically, it blocks the PD-1 pathway that cancer cells use to suppress and evade the immune system. With neoadjuvant-adjuvant treatment, patients receive systemic therapy both before and after the surgical resection of their tumor(s). There is limited research evaluating the association between tumor responses (changes in tumor measurements) and survival time with neoadjuvant anti-PD-1 treatment. In this study, I analyzed the association between tumor measurements and event-free and overall survival in a clinical trial of patients receiving neoadjuvant-adjuvant anti-PD1-therapy. Event-free and overall survival were estimated using the Kaplan-Meier method, and associations with RECIST tumor size were evaluated using martingale residual plots and Cox proportional hazards regression models. In this study, I found significant associations between event-free survival and baseline tumor burden and changes (quantitative and categorized) in tumor size between baseline and the end of neoadjuvant therapy. Increases in tumor size were associated with a lower 2-year event-free survival. Larger values of baseline tumor burden were significantly associated with event-free survival but were not significantly associated with overall survival. The findings from this study may serve to better inform clinicians on making prognoses for patients on anti-PD-1 therapy and indicate a need for more research to be done on associations between tumor size and overall survival outcomes.

The Disability Inclusive Development Initiative (DIDI) is a project that brings together students and faculty from various fields to promote the human rights of persons with disabilities. Our current project builds upon the findings of a 2020 Memorandum of Understanding (MoU) between the International Disability Alliance (IDA) and the United Nations High Commissioner for Refugees (UNHCR). We are conducting a comparative analysis of asylum procedures in Uganda and Colombia, specifically examining how these procedures consider the rights of persons with disabilities. Our goal is to ensure that refugees and asylum-seekers with disabilities have access to fair and accommodating processes. We are also looking into the challenges faced by persons with disabilities during the asylum process and how governments are addressing these challenges. To achieve these aims, we are conducting a comprehensive literature review, including primary legal research, using resources from the University of Washington libraries. Additionally, we are consulting with experts working in this field. Our team is dedicated to producing a report for the United Nations High Commissioner for Refugees, which will outline our findings, highlight effective practices, and offer recommendations for future actions.

People living with untreated HIV have a compromised immune system, which increases the risk for enhanced inflammation and disease severity in those co-infected with SARS-CoV-2. This emphasizes the importance of understanding the underlying mechanisms during immunosuppression that impact SARS-CoV-2 pathogenesis and disease outcomes. Since the microbiome plays an important role in immunity, microbial dysbiosis during HIV infection could contribute to prolonged SARS-CoV-2 pathogenesis. Microbial dysbiosis can be determined through the loss of diversity and changes to the composition of the microbiome. There is an established link between the increase in HIV disease progression and gastrointestinal microbial dysbiosis, however, the understanding of HIV-induced microbial dysbiosis during COVID-19 progression is unknown. In this project, we will investigate the gastrointestinal microbiome diversity and composition during SIV infection, to serve as a basis for understanding this undefined association. Utilizing the SIV macaque model for AIDS, we will test the hypothesis that the microbiome diversity and composition during SIV infection will be dissimilar between different gastrointestinal areas (stool, rectal swabs). Seven female rhesus macaques were intravaginally infected with SIVmac251. Rectal swabs and stool samples from the macaques were collected at baseline: 17~34 weeks post-SIV infection, and 7 days pre-SARS-CoV-2 infection. We extracted genomic DNA using a QIAgen PowerFecal Pro DNA kit and sequenced the ribosomal RNA after 16s amplification. We use the bioinformatics platform, QIIME2, to analyze the sequencing data. I am probing the data for relative microbial abundance, and diversity of microbial communities through metrics of richness, evenness, and specific indexes. Preliminary findings report that during SIV infection, the overall diversity of the gut microbiome is similar in stool and rectal swabs, and the microbiota composition is different between them. The results from these studies will then be used to understand the role of SIV-induced microbial dysbiosis on SARS-CoV-2 virological and disease outcomes.
 

Coccidioidomycosis, also known as Valley Fever (VF) is caused by the fungus Coccidioides. Pigtail macaques (PTMs) bred at the Washington National Primate Research Center (WaNPRC) in Mesa, AZ are naturally infected with Coccidioides and are similar to humans in their physiology, symptoms, and immune responses. Populations with a weakened immune system, notably older individuals, are at risk for severe complications from infection. Additionally, there is evidence that males have a higher incidence of VF than females in endemic areas. I characterized the immune responses in a PTM model across age and sex to better understand how VF affects the immune response of these populations. Forty-two PTMs (2.25-19.24 years, 3.66-18.29 kg, 37 female, 5 male) at the WaNPRC were sampled for blood. The frequencies of immune cell subsets in whole blood were characterized by flow cytometry and compared for significant differences based on age and sex. I analyzed sex-based differences with Brown-Forsythe and Welch ANOVA t-tests and found no statistically significant differences. For age-based differences, we used a simple linear regression to analyze differences by age in immune cell subsets. We found that old PTMs (10.07-19.24 years) have higher activation of CD8+ T cells, myeloid dendritic cells, intermediate monocytes, and higher frequency of γΔ T cells and CD4+ γΔ T cells than young PTMs (2.25-9.69 years). Young PTMs have a higher frequency of CD45+ granulocytes, PD-1 High CD8+ T cells, plasmacytoid dendritic cells, and NK cells. By correlating older PTMs with higher immune cell activation, and younger PTMs with higher immune cell frequency, we have a better understanding of how a vaccine or treatment could be developed to support older individuals, who are at greater risk of severe infection.

Convection in porous media within an inclined layer is relevant to a wide range of geophysical and engineering applications, e.g., in understanding large-scale convection in a geothermal reservoir. Previous work found that stable stationary localized convective structures are present at moderate Rayleigh numbers and a sufficiently large inclination angle when the boundary conditions are symmetric with respect to the layer midplane. In this project, I study the dynamics of traveling localized structures in inclined porous medium convection in the presence of asymmetric temperature boundary conditions. I conducted direct numerical simulations (DNS) of the fluid equations and found that one- and two-pulse structures exhibit a quadratic relationship between the travel speed of the structure and the symmetry breaking control parameter in the boundary conditions, while three- to five-pulse domain-filling structures display a linear relationship. With further simulations and increasing domain size, we discovered that, for sufficiently strong symmetry breaking, adjacent pulses repel each other while traveling and so tend to spread out, eventually becoming equidistant in the finite domain. The repulsion is sensitive to the travel speed (and thus to the asymmetric boundary conditions) and the domain size. I show that these interactions are associated with the spatial eigenvalues of the base flow that are responsible for the leading and trailing tails of the 1D along-slope temperature profile of the localized structures. These eigenvalues are complex implying that the tails oscillate while decaying exponentially. We employ the computed spatial eigenvalues to predict the tail profiles of traveling pulses and show that these successfully match observations from DNS. This comprehensive analysis enhances our understanding of the stability and bifurcations in the dynamics of traveling localized structures in inclined porous medium convection, offering valuable insights for geophysical and engineering applications.

Subtherapeutic drug levels can lead to the failure of antiretroviral therapy (ART) regimens used in Human Immunodeficiency Virus (HIV) treatment and prevention. However, gold-standard HIV drug level monitoring techniques—such as mass spectrometry—require bulky and expensive instruments that are not widely accessible at the point-of-need. Our group developed the REverSe TRanscrIptase Chain Termination (RESTRICT) enzymatic assay to rapidly (30 min) and inexpensively measure tenofovir diphosphate (TFV-DP), a nucleotide analog used in >90% of oral ART regimens and in all approved prevention regimens. However, RESTRICT currently requires trained operators to perform multiple time-sensitive liquid-handling steps. To reduce user intervention and minimize the need for laboratory equipment, we harnessed 3D-printed capillaric microfluidics to self-propel liquids using only surface tension effects encoded in microchannel geometry and surface chemistry. Specifically, we translated the manual tube-based RESTRICT to an automated microfluidic protocol by using autonomous trigger valves to pre-load multiple RESTRICT assay reagents and serpentine channels to control assay timing. Currently, RESTRICT reactions are incubated for 30 minutes at 37˚C, but we decreased the reaction time to 15 minutes and removed the need for an external heating source by incubating at room temperature (25˚C). There was only a 15% decrease in overall signal intensity in the faster, room temperature assays, and measured readout was distinguishable between clinically relevant concentrations of TFV-DP. Our results represent a first step towards integrating RESTRICT reactions and fluorescence readout onto a rapidly fabricated microfluidic chip. We hope to achieve a device that increases the accessibility of HIV drug level monitoring at the point of need without specialized equipment or highly trained operators.

There is a strong need for rapid and inexpensive tests for monitoring human immunodeficiency virus (HIV) drugs to prevent treatment failure. The REverse TranscrIptase Chain Termination (RESTRICT) Assay provides fast and inexpensive measurement of HIV reverse transcriptase (RT) inhibitors by measuring the extent of DNA synthesis with the HIV RT enzyme in the presence of a patient’s blood. RESTRICT uses an intercalating dye, PicoGreen, to provide fluorescence as a function of the amount of DNA synthesized. However, PicoGreen interrupts DNA synthesis and binds to any double-stranded DNA (dsDNA) present in the sample including background DNA in blood samples, precluding real-time measurements and potentially decreasing the accuracy of results. This project uses RESTRICT in conjunction with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) complexes and reporters to accurately measure RESTRICT results in real-time. Fluorescence is produced by reporters which are made up of a quencher and a fluorophore. When the CRISPR complex detects a specific complementary sequence formed from the single-stranded DNA (ssDNA) template, the Cas enzyme is activated and cleaves the fluorescent reporter. Once cleaved, the fluorophore is released from the quencher thus generating fluorescence. Because the CRISPR complex binds only to specific segments of DNA produced by RESTRICT, we can obtain more specific results without interference from background DNA. In addition, CRISPR complexes and reporters do not interfere with DNA synthesis and can be added at the same time as other RESTRICT reagents, enabling real-time monitoring of RESTRICT reactions. Using a portable fluorescence reader (T8-ISO, Axxin Pty Ltd.), positive control reactions containing RT enzyme provided at least 5X more fluorescence than negative controls without RT. Future work will focus on measuring clinically relevant levels of HIV drugs in aqueous buffers using RESTRICT with CRISPR readout.

Heschl’s gyrus (HG) is a region of the brain containing the primary auditory cortex. The extent of folding within the HG shows high morphological variability across individuals. Interestingly, increased HG folding is more likely to be found in expert than amateur musicians, suggesting a possible role of auditory experience in shaping HG gyrification. In my research, I examined HG folding in blind individuals—another population with extensive auditory experience. I hypothesized that, if experience alters HG structure, then individuals with early-onset blindness might have increased HG gyrification compared to those with late-onset blindness or those who are sighted. I analyzed T1-weighted images collected from previous MRI studies at the University of Washington, University of Pennsylvania, and Oxford University. The combined dataset included 6 anophthalmia (individuals born without eyes), 48 early blind, 18 late blind, and 28 sighted control participants. I created hand-drawn HG regions of interest for each participant in both hemispheres and measured HG gyrification in two ways: 1) by visually categorizing the extent of HG folding (single, partial, or complete duplication), and 2) by obtaining continuous metrics (gyrification index and curvedness index) using FreeSurfer. A chi-squared test revealed that the degree of HG folding was not different across the four groups. A linear mixed-effects model (controlling for the effects of age, hemisphere, and scan location), similarly showed no effects of group on the gyrification index or the curvedness index. To conclude, my findings show that blindness does not affect HG gyrification. The results challenge the idea that auditory experience alters HG structure and offer important insights into previous findings in professional musicians. Our results suggest that the prevalence of duplicated HG in musicians may be the result of individuals with larger processing capacity within the auditory cortex being more likely to take up music as a profession.

Most eukaryotes use histones to package the genome. However, many animals package their sperm genomes using specialized DNA-binding proteins called protamines, which package DNA in sperm more tightly to fit inside the sperm head. Based on the transcriptional silencing role of protamines, we hypothesize that protamines can suppress meiotic drivers, which kill other sperm to bias their own transmission. Previously, we found that one protamine gene, Mst77F, is required to suppress meiotic drivers on the Y-chromosome in Drosophila melanogaster. Since drive is generally deleterious for the transmission of autosomal alleles (due to lower male fertility for example) theory predicts that multiple suppressors of drive will arise in populations; Mst77F may represent just one such suppressor. We hypothesized that multiple natural variants in distinct genetic loci interact with and impact meiotic drive in Drosophila melanogaster. To identify these natural variants, I crossed wildtype flies to knock out flies and generate hemizygous Mst77F flies carrying genetic backgrounds from four different populations. I measured the fertility and drive strength by crossing a single hemizygous male from each cross to five wild type females. In all cases, I found that the sex ratio was skewed to favor male offspring, indicating they all carry X-linked targets. However, I did not identify any dominant genetic variation associated with the drive strength, indicating Mst77F might be the major suppressor of this drive. I am conducting reciprocal crosses to determine whether Y chromosomes from different populations carry the same strength of drive. In the future, I will extend my analyses to other genetic backgrounds. My study contributes to a better understanding of the pervasive effects of meiotic drive in natural populations and unexpected functions of protamines.