Using a large NOAA Storm Prediction Center database covering United States and Canadian tornadoes, our group analyses the traits and tendencies of tornadic activity across North America. With recent improvements in tornadic data collection, there is a surplus of topics to thoroughly explore. Some of our work includes investigating relationships between two parameters such as CAPE (convective available potential energy), and CIN (convective inhibition) or precipitable water and temperature. Furthermore, we created histograms with Gaussian curves and probability plots for the most critical parameters for tornadic development to better understand the distribution of data and how much it deviates from a centered mean. Currently, we are in the process of creating filled contours to visualize the geographic distribution of parameters across the United States. Our goal is to create a more nuanced tornado climatology. In doing so, we hope to further understand tornadoes and their tendencies with hopes of making our interactions with these events much safer.

IMPDH1 catalyzes the rate limiting step of de novo guanine synthesis. cGMP is a critical signaling molecule involved in phototransduction in rod and cone photoreceptors. In humans, nine mutations in IMPDH1 lead to Retinitis Pigmentosa and Leber's Congenital Amaurosis; however, the cause of retinal degeneration is unknown. In zebrafish, IMPDH1a is the major variant in the retina, exclusively expressed in rod and cone photoreceptors. To understand the function of IMPDH1, we utilized an IMPDH1a knock-out (KO) zebrafish line. Loss of IMPDH1a does not lead to retinal degeneration, and cGMP levels remain unchanged. However, retinas lacking IMPDH1a show a 61.6% reduction in guanine. Since photoreceptors do not undergo cell division, mitochondrial DNA (mtDNA) synthesis would be a major use of guanine during mitochondrial biogenesis and turnover. Under normal conditions, the amount of mtDNA between the KO and WT zebrafish was the same. To eliminate the potential that KO fish could supplement guanine from their diet, fish were starved for 24 hours. Starved IMPDH1a KO showed a trend of increased mtDNA compared to WT fish. Interestingly, starved fish had about four times the amount of mtDNA for both WT and KO compared to fed fish. Starvation may increase mitochondrial DNA copy number to increase efficiency of ATP production by increased utilization of oxidative phosphorylation. Reducing guanine levels by knocking out IMPDH1a in zebrafish retina does not affect mtDNA or nDNA levels, and the photoreceptor cells appear healthy. Although more research is needed, silencing the gene encoding for IMPDH1 could be a possible therapy for those suffering from the effects of its mutation.

Cancerous cells have a modified metabolism that supports their demands for increased proliferation. One of the essential molecules in cancer cell metabolism and proliferation is the amino acid aspartate. Aspartate is not only incorporated into proteins, but is also a substrate for nucleotides and other amino acids, including asparagine. Aspartate availability can constrain tumor growth rate, and the consumption of aspartate to generate downstream products can alter aspartate levels. One gene that draws from the aspartate pool is asparagine synthetase (ASNS). ASNS converts aspartate into asparagine, which is used in the production of proteins, but does not increase cell proliferation. Thus we hypothesized that ASNS expression and activity can affect aspartate levels. With this, we aimed to determine if ASNS expression could alter aspartate availability and change sensitivity to aspartate suppressing therapies. Since cancer cells express ASNS to varying degrees, my project sought to determine if ASNS expression could be used to identify those cancers that are most amenable to aspartate suppression therapies. To test this I generated cell lines that express ASNS in different ways and treated them with multiple electron transport chain inhibitors. Preliminary results suggest that cells that express ASNS to a higher degree are more susceptible to mitochondrial inhibitors. More broadly, this research sought to better understand the conditions that determine aspartate levels, and how to exploit those conditions to inhibit tumor growth in association with asparagine synthetase.

Hydrogen sulfide (H₂S) is a toxic gas in the environment, but it is also an important cellular signaling molecule. Our goal is to understand the genes and pathways that mediate the physiological effects of H₂S. Previous work from the Miller lab has shown RHY-1 to be a component of a pathway that mitigates H₂S toxicity. RHY-1 is an integral membrane protein with predicted acyltransferase activity that localizes to the endoplasmic reticulum. We are attempting to identify proteins that work with RHY-1 to promote survival in H₂S. In this project, we have optimized biotinylation by antibody recognition (BAR), a proximity-labeling approach, to identify proteins that may physically interact with RHY-1. In these experiments, we introduced an antibody conjugated to horseradish peroxidase (HRP) into C. elegans that expresses the epitope-tagged RHY 1::FLAG::GFP protein, so that upon addition of biotin peroxide, biotin radicals were formed only in proximity to RHY-1. These biotin radicals react with other proteins that are localized near RHY-1. We visualized biotinylation using a fluorescent label and showed that the biotinylated proteins colocalize with RHY-1, indicating a successful BAR reaction. To our knowledge, this is the first use of BAR in C. elegans. Going forward, we will use mass spectrometry to identify the proteins that are biotinylated, and then test the functional role of these proteins in mitigating H₂S toxicity. Identifying the genes and pathways downstream of RHY-1 that promote survival in H₂S will reveal new factors that can modulate H₂S signaling in cells and may lead to treatments for H₂S poisoning.

DNA damage occurs regularly, and although the cell has numerous repair mechanisms to counteract it, some DNA lesions may persist. Many of these lesions can be bypassed by DNA and RNA polymerase, with replicational mutagenesis leading to permanent mutations in the genomic sequence, and transcriptional mutagenesis leading to mutant transcripts that may direct the production of mutant proteins. We hypothesized that transcriptional mutagenesis is a mechanism for initiating adaptive mutagenesis, which occurs in nondividing cells in direct response to a selective pressure, allowing cells to overcome the selection and resume cell division. Our lab has previously demonstrated that DNA damage leading to adaptive mutagenesis in yeast is biased to the template strand relative to transcription, supporting this hypothesis. Unexpectedly, DNA damage leading to replicational mutations in our system is similarly skewed to this strand, although the sequences surrounding these two classes of mutations seems to differ. We hypothesize that sequence context is important for determining whether certain mutation sites are more likely to contribute to replicational or adaptive mutagenesis. To address this hypothesis, our aim was to create Trp5 mutant yeast strains using CRISPR/Cas9 in which the mutation occurred in a sequence context-specific manner, and the reversion of which could be scored for both replicational and adaptive mutagenesis. The CRISPR/Cas9 system required generation of a plasmid containing the target sgRNA (via molecular cloning techniques) as well as a linear repair template with the mutation of interest (via a two-step PCR protocol). Creating both of these components proved challenging, and optimization of the protocols will be required. Once we have all the components for CRISPR/Cas9-mediated mutagenesis, future research involves creating the mutant strains and assessing whether the sequence context affects the mutation frequency with respect to either replicational or adaptive mutagenesis.

Herpes Simplex Virus 1 (HSV1) is a prevalent lifelong virus transmitted through oral-to-oral contact resulting in painful oral sores. HSV1 may also give rise to encephalitis, or inflammation of the brain, and has even been linked to sporadic Alzheimer’s Disease (AD). Typically, HSV1 enters through the oral cavity, replicates, invades nearby clusters of sensory neurons that innervate mucosal membranes called the trigeminal ganglia, and can ultimately lead to infection of the brain. HSV1 can establish latency in these trigeminal ganglia and other neuronal nuclei, and can reactivate in the brain in response to external stresses at any point in time. Previous studies have even shown presence of HSV1 in the brain. However, this process of reactivation is still not understood. Our goal is to comprehend the mechanisms of HSV1 reactivation in the brain by generating two recombinant HSV1 through the CRISPR-Cas9 gene editing system. One strain will contain an insertion into the HSV1 genome of luciferase and mEmerald, a green fluorescent protein, for detection of cells with reactivating HSV1 through in vivo imaging of bioluminescence. The second strain will have an insertion of Cre recombinase and mEmerald. When this strain is injected into a mutant mice line with a Cre-dependent reporter gene, the Cre recombinase can lift genetic repression of a red fluorescence protein. This prompts all cells with an HSV1 infection, including those with past reactivation, to be detected. Through these novel recombinant HSV1 strains, we can monitor viral reactivation in brain cells in response to different external stresses in vivo, examine the time course of the infection, and distinguish areas of the brain with high viral load, especially cell types vulnerable to HSV1 infection. These experiments will be the stepping stone for improved investigation of viral latency and reactivation and understanding the connection between HSV1 and AD.

Environmental stressors trigger physiological adaptations in organisms that allow them to dynamically remodel tissues. As many of these processes in the gastrointestinal tract of Drosophila melanogaster are studied in the context of stem cell proliferation, we instead chose the novel approach of investigating visceral muscle adaptations in response to stressors to the gut. The three stress conditions we employed were starvation, damage, and aging. For starvation, we treated adult flies with only water following nutrient-enriched recovery. For epithelial damage, we fed them with the chemical damaging agent, bleomycin. Finally, for aging, the flies were subjected to different prolonged time periods on normal media. The flies were then dissected and processed for confocal imaging and phenotypic analysis, allowing us to categorically define and quantify the phenotypes in order to measure the degree to which the visceral muscle has changed. We discovered that various responses of the visceral muscle are induced by the types of stressors, and will further look into whether it is adaptive remodeling, muscle repair, or other undescribed mechanisms. We will investigate the molecular mechanisms that contribute to the remodeling of the visceral muscle using RNAi approach. This research would provide valuable resources as a model system for studying complex tissue responding to environmental challenges and understanding of its mechanisms utilizing the vast genetic tools of Drosophila.