Microsporidia are a group of obligate intracellular fungi that infect nearly all animals. Two microsporidia species, Nematocida ausubeli and Nematocida parisii, infect and cause structural changes in the free-living nematode Caenorhabditis elegans, an important model system for response to microsporidia infection. N. parisii and N. ausubeli invade the intestinal cells of their host after ingestion where they reproduce and emerge as spores. Previous studies have shown that mechanisms of microsporidia infection are highly conserved and C. elegans responses to N. parisii infection vary across wild isolates. To build on these previous studies, we plan to identify genetic differences in the C. elegans population that influence microsporidia responses. We first needed to remove the bacterial contamination from natural C. elegans strains that were previously infected with N. parisii or N. ausubeli. Once these strains were free of bacterial contamination, we isolated and quantified microsporidia spores from the infected worms. We are currently performing dose response assays to determine the spore concentration that maximizes broad-sense heritability for a large-scale fitness assay. Next, we will quantify the fitness of 330 natural C. elegans isolates after exposure to five genetically distinct N. parisii and N. ausubeli strains. We anticipate that the large-scale fitness assay will help identify novel mechanisms of microsporidia resistance, which will further our understanding of the genetic basis of microsporidia infection across species.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world. Bile acids (BAs) are cholesterol-derived molecules synthesized in the liver through the classical and alternative BA synthesis pathways that promote intestinal lipid absorption. We hypothesize that manipulating the BA pool size or its composition can alter lipid absorption. Specific control of absorption by specific BAs provides new treatment avenues for NAFLD and diet-induced obesity. Our lab uses AAV-CRISPR/Cas9 (adeno associated viral - clustered regularly interspaced short palindromic repeats-associated cas9) to facilitate liver-directed gene disruption in adult mice to genes involved in BA synthesis and regulation. We sought to disrupt steroid 5β-reductase (Akr1d1), aldo-keto reductase family 1 member c14 (Akr1c14), and bile acid-CoA:amino acid N-acyltransferase (Baat) to assess editing efficiency and changes in lipid metabolism. We designed CRISPR guide RNAs specific to our genes of interest, and validated them for specificity, efficiency, and possible off-targets using CRISPOR and COSMID web-tools. Mice were injected with AAV-CRISPR, followed for 10 days, and liver tissue was harvested for analysis. We performed polymerase chain reaction (PCR), Sanger sequencing analysis, quantitative PCR (qPCR), and western blot to assess the efficiency of CRISPR-mediated disruption. My data show successful gene disruption of Akr1d1, Akr1c14, and Baat at the DNA level, and thus far efficient loss of BAAT protein. After validating gene disruption, I will analyze BA composition and lipid-related phenotypes where we predict to observe decreased lipid absorption and protection from NAFLD and diet-induced obesity due to disruption of key BA synthesis genes.

Cholesterol is a vital lipid necessary for the proper regulation of T cell function due to its role in cell structure and signaling. Oxysterols, naturally produced oxygenated metabolites of cholesterol, have been found to accumulate in tumor microenvironments (TME) and atherosclerotic plaques. Some oxysterols have been shown to activate the LXR pathway for cholesterol efflux and suppress the Sterol Regulatory Element-Binding Protein (SREBP) pathway for sterol synthesis. We hypothesize that oxysterols present in the TME dysregulate T cell cholesterol homeostasis and hinder their antitumor response. However, the extent to which oxysterols influence the T cell’s ability to activate, proliferate, and function is not well understood. To investigate this, we activated isolated T cells from murine splenocytes and cultured them in vitro. We utilized flow cytometry and qPCR to evaluate T cell count, activation, SREBP gene targets, and LXR gene targets. Results showed downregulated gene expression of SREBP gene targets and upregulation of LXR gene targets in response to increasing concentrations of 25-hydroxycholesterol (25-HC), 22R-hydroxycholesterol (22R-HC), and 7α-hydroxycholesterol (7α-HC). Overall, the extent to which cholesterol metabolism genes were dysregulated correlated with inhibition of T cell activation and proliferation. Thus, providing a better understanding of how T cells are affected by oxysterol accumulation in tissue environments. Additionally, we found that genetic ablation of LXR rendered T cells less susceptible to oxysterol-mediated inhibition of proliferation. These results also suggest that manipulation of oxysterol metabolism could be used to target cholesterol homeostasis in T cells and, by extension control T cell immune responses.

The model yeast (Saccharomyces cerevisiae) is adapted to a wide variety of natural and domesticated environments including Beer, Dairy products, Trees, and the Ocean. Large-scale genomics of wild populations of yeast have revealed substantial genetic diversity, but our understanding of how these genetic differences impact fitness remains poor. To assess the effects of a large number of variants on yeast fitness, we will perform genetic mapping in a cross between the highly divergent Taiwanese strain (CEI) and the reference strain (BY). CEI harbors 160,000 single nucleotide differences from the reference strain, whereas the average number of changes between strains varies from between 40,000 to 60,000. We crossed CEI with BY and sporulated the diploid hybrid to create a population of 200 haploid segregants, that through recombination inherited unique combinations of the two parental genomes. We will perform whole genome sequencing to determine for every segregant the combination of alleles that were inherited from each parent. I will collect the fitness profiles of these segregants in different environments and perform genetic mapping to identify the genetic changes that are responsible for any observed growth differences. I will perform population genetics analysis to discover which causal variants are maintained by natural selection. Our results will improve our understanding of the role that genetic variation plays in the ability of yeast to survive and reproduce in a huge range of natural and domesticated environments.

Glaucoma is a significant eye disease that results in blindness to millions worldwide. Glaucoma is caused by optic nerve damage resulting in permanent vision loss and dysfunction in the trabecular meshwork, a tissue near the base of the cornea by the ciliary body. Dexamethasone alters the structure of the trabecular meshwork and hinders its function, suggesting that it could serve as a therapeutic for glaucoma. A bulk collagen contraction assay and a novel single-cell contraction assay was used to measure the impact of dexamethasone exposure on contractions at the trabecular meshwork. Findings to date view expression changes of contraction related proteins after dexamethasone treatment through RNAseq. Findings up to show that dexamethasone treated for trabecular meshwork exhibits increased cellular contraction in both aggregate and single cell assays, and decreased expression of cadherins. Findings also show that integrins’ expression changes after treatment appear to be donor dependent. The integrins’ expression changes align with the literature trends in heterogeneity of dexamethasone response to patients. What is proposed for research is that Dexamethasone may lead to increased ocular pressure by disrupting contractility through dysregulated mechanotransduction in trabecular meshwork.