DNA-Binding Proteins (DBPs) hold a strong affinity to interact with the major grooves of DNA for the purposes of transcription, translation, and repair. Although DBPs are found in nature, their specificity is difficult to predict and their production expends excessive resources. Therefore, our project’s goal is to efficiently generate DBPs that allow us to enable exact processes to occur. This is promising for future use in treating genetic diseases. In our research, we studied point mutations in the hexosaminidase subunit alpha (HEXA) gene and adenosine deaminase (ADA) gene, which can lead to Tay-Sachs disease (TSD) and Severe Combined Immunodeficiency (SCID), respectively. TSD is a rare genetic disease that affects infants and ultimately leads to brain damage, resulting in these children not making it to grade school age. Similarly, SCID is genetic, where children lack a strong immune system. This increases their susceptibility to infections, especially during their first year of life. Targeting the HEXA and ADA genes, we first developed designs utilizing computational software like RosettaFold, RFdiffusion, x3DNA, and LigandMPNN, followed by rigorous filtering via RosettaFold Nucleic Acid. Afterwards, we tested the final designs using yeast cultures and fluorescence-activated cell sorting (FACS) in the laboratory to determine which bind best to their generated DNA template sequences. Overall, we expect to find a few specific DBPs that bind effectively as predicted during the computational pipeline. These successful designs can be utilized as genome-editing proteins, correcting their target DNA sequences and restoring normal function.

Zooplankton are vital to the marine food web, supplying nutrients and energy from primary producers to secondary consumers. During Diel Vertical Migration (DVM), zooplankton travel between depth and the surface during day and night to capitalize on food and avoid predation. This study investigated diel differences in zooplankton community composition at two locations, one exposed and one protected, in the San Juan Channel, WA over four days in September 2023. Zooplankton were collected using net tows from surface waters at both sites during day and night times. Samples were analyzed using a stereoscope and different taxonomic groups were counted. Copepods were the most abundant zooplankton taxa at both locations, with mean abundances up to 1000 individuals per cubic meter. At the exposed site, there was a significantly higher (p<0.05) abundance of zooplankton at night versus during the day. The exposed site had significantly higher diversity than the protected site at night (p<0.05). At both locations, species richness was significantly higher (p<0.05) at night compared to day. The exposed location also had significantly higher richness (p<0.05) compared to the protected location during the day. Our results indicate that zooplankton abundance and diversity in surface waters of the San Juan Channel are controlled by DVM, and differences in locations perhaps due to exposure to different flow regimes. This study reinforces the flexibility of zooplankton community composition and emphasizes the importance of understanding factors that influence changes in the base of the marine food web.

Air pollution is a key component to understanding the Public Health of populations globally, with Diesel Exhaust Particles (DEP) being a significant contributor to traffic-related air pollution. Exposure to DEPs varies across populations and is therefore crucial to understanding the continual impacts of traffic-related air pollution on the public. Prior research has indicated that the formation of Amyloid-𝛽 (A-𝛽) plaques and activation of the  nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3 (NLRP3) inflammasome is linked with the development of Alzheimer’s disease (AD) later in life. AD is a form of progressive disease that impairs memory and other cognitive functions and impacts the lives of tens of millions of people globally. This study aims to confirm the linkage between exposure to DEP and memory impairment through NLRP3 inflammasome activation, utilizing an animal model to investigate a potential increase in AD later in life. We exposed male and female low-density lipoprotein receptor knockout (LDLR KO) mice chronically to inhaled DEP or filtered air as a control for 18 weeks. We then utilized the Object Location Memory (OLM) and Object Recognition Memory (ORM) behavioral tests to investigate the immediate impact of multi-week DEP exposure on short-term memory, another indicator in AD progression. Afterward, we sacrificed the mice and harvested a variety of tissues, including the brain. I conducted Immunohistochemistry (IHC) on cryosections of the exposed and non-exposed brain to assess DEP-induced AD-like brain architectural changes and to quantify the impact of DEP exposure in activating the NLRP3 inflammasome, ultimately leading to neurotoxicity, and to the development and progression of AD. Confirming the association between diesel exhaust and the NLRP3 pathway provides a potential therapeutic target in populations at an elevated risk for AD.

Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by Lewy bodies, pathogenic protein aggregates that include alpha-synuclein oligomers. The missense mutation p.G192R in the RAB39B gene was recently found to cause X-linked dominant PD. Loss of function mutations in RAB39B are associated with X-linked intellectual disability and autism spectrum disorder. RAB39B is a member of the human Rab GTPase family which plays a role in early autophagosome formation and is implicated in intracellular vesicular trafficking. This project investigates how defects in endolysosomal trafficking caused by the p.G192R mutation in RAB39B gene leads to parkinsonism and neurodegeneration. Because RAB39B is highly conserved, we developed a Drosophila model as human RAB39B and Drosophila RAB39 share 75% similarity in amino acid sequence, including 100% identity at p.G192 and flanking amino acids. Using CRISPR/Cas9 genome editing, we created a RAB39G196R Drosophila model that we are currently characterizing for possible neurodegenerative phenotypes. We are examining locomotor deficits and lifespan in RAB39G196R mutant flies compared to isogenic controls, as well as protein aggregation by Western blot. Complementary to the Drosophila model, we developed a human neuronal model by generating induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMC) of an affected male and similar age unaffected male family member kindred with X-linked PD due to the p.G192R mutation. We are investigating endolysosomal trafficking defects in neurons differentiated from iPSCs using antibodies specific for early and late endosomes and lysosomes. We are also examining whether insoluble ubiquitinated protein aggregates and oligomerizes alpha-synuclein are present in RAB39BG192R neurons compared to control neurons. Understanding mechanisms underlying the pathogenesis of X-linked Parkinson’s disease will elucidate the development of PD and potential novel therapeutic targets.

DNA-binding proteins play crucial roles in many biological processes. They can regulate gene expression by binding to specific DNA sequences and either promote or inhibit transcription, or they can serve as gene editing technology. This research focuses on the design of de-novo proteins with potential therapeutic applications in gene editing and transcriptional modulation. For our designs, we selected four short DNA sequences associated with disorders. Two of these sequences have point mutations that are associated with sickle cell anemia and hypertrophic cardiomyopathy. The other two sequences are promoter regions; one linked to the INS gene featuring a cc dinucleotide sequence that may contribute to hyperglycemia and one on the oncogene MDM2 in the MDM2p-52 pathway which has the potential to cause cancer. We then used the multi-step computational design pipeline developed at the Institute for Protein Design, which utilizes deep-learning models including RFdiffusion, LigandMPNN, and RosettaFold Nucleic Acid, to generate protein binders to the target DNA sequences. After filtering for the best theoretical designs, we would test them through yeast cell display, which visualizes the binding of the protein to the DNA. We then can analyze the binding pattern of these protein designs which vary in strength and specificity of binding to the targeted DNA sequences. Since the computational design pathway strictly filters thousands of designs to narrow our selection to the best protein designs, we expect that a few of these proteins will have significantly strong binding to their respective DNA sequences. For the designs that demonstrate strong and specific binding to the targeted sequences, we may continue to improve their structure. The success of this computational design pipeline could result in breakthroughs in protein design, and has the potential to drive medical advancement and produce treatment for genetic disorders in the future.

One of the most common types of vaccines used today are subunit vaccines. Subunit vaccines consist of an antigen that triggers the adaptive immune system to create antibodies but also require a separately added adjuvant, which is a substance that induces longer-term immunity by stimulating the immune system to pay attention to the antigen. Current adjuvants are non-specific - often things like oil-water emulsions that irritate the immune system and cause inflammation in unknown ways to draw attention to the antigen. This project aims to create a more specific adjuvant by directly stimulating B cells. In order for B cells to replicate antibodies, they need a primary signal from the antigen and a secondary signal that certain ligands on T-cells can initiate. We decided to investigate whether CD40Ligand (CD40L), an immune protein present on T cells that works to signal B cells to either replicate or create antibodies, could be used to achieve this goal. The idea behind the project is to co-display CD40L with antigen on the nanoparticle in hopes of creating a more specific adjuvant. We designed 10 different versions of this nanoparticle, where we tested two versions of CD40L, the placement of CD40L, and the linker length between CD40L and the nanoparticle surface. Our preliminary results also show that our cages retain their ability to bind both antibodies and CD40 as well as activate NFkB transcription - a proxy for B cell activation. We expect CD40L-displaying nanoparticles will promote B-cell proliferation to a greater extent than the nanoparticle vaccine displaying only hemagglutinin (flu) antigen accompanied with an adjuvant like Addavax. Ultimately, we hope to examine how co-display of CD40L with antigen will change the quality of immune response and memory in-vivo in comparison to currently used vaccine adjuvants, and begin testing in-vivo in the coming quarters.

Neurons, the fundamental cellular units of the human brain and nervous system, are crucial to transmitting signals throughout the human body. In human anatomy, anything that obstructs the communication between neurons could lead to neurodegeneration, decline in function, disorders, and diseases. Alzheimer’s disease (AD), which affects more than 6 million people across the nation as of 2023, is one such disorder. An extensive array of research has been done investigating the underlying cause of the neurodegeneration that occurs in AD. One such theory of neuronal dysfunction, the amyloid hypothesis, points to an accumulation of a protein called beta-amyloid that is present in the brain but in some people accumulates in excess and disrupts neuronal signaling. This ultimately leads to neurodegeneration and cognitive decline. I take part in conducting the AHEAD study, a Phase 2 clinical trial, which is currently underway investigating this theory and a new drug called lecanemab. The drug is designed to remove beta-amyloid from the brain and prevent further neurodegeneration in individuals who have accumulations of amyloid and are at risk of developing AD. This is one of the first preventive clinical trials for Alzheimer's. In this review, I have explored findings from Phase 1 of the AHEAD study and described the screening process for participants for the Phase 2 trial. I have also explored the science behind beta-amyloid, Alzheimer’s disease, and treatment with lecanemab. I expected a large population to pass the screening process, but the statistics show otherwise. I dive into why this is in this project.

Pediatric high-grade gliomas (pHGG) are highly aggressive brain tumors with limited therapies and extremely poor clinical outcomes. pHGG and many other cancers are often driven by genetic fusion events, which produce oncogenic fusion proteins such as TPM3-NTRK1. NTRK1 regulates signaling pathways promoting cell survival and differentiation. In cancer, NTRK fusions lead to constitutive activation of these pathways, resulting in uncontrolled cell growth. The purpose of my project is to validate TPM3-NTRK1 as a potential drug target for a novel therapeutic strategy known as targeted protein degradation and to clarify the role of TPM3-NTRK1 in cancer signaling. I hypothesize that degrading the TPM3-NTRK1 protein will decrease the activity of downstream signaling pathways that lead to pHGG. To model pharmacological degradation of TPM3-NTRK1, I applied the degradation tag (dTAG) system to rapidly and reversibly degrade a target protein fused with an FKBP12^F36V-tag. In this approach, dTAG molecules bind to the FKBP12^F36V-tag and recruit an E3 ubiquitin ligase to ubiquitinate the target protein, leading to its degradation by the proteasome. In my project, I used Gateway cloning to generate a lentiviral expression plasmid containing FKBP12^F36V-TPM3-NTRK1. I then produced lentiviruses, which I used to transduce NIH/3T3 cells and express FKBP12^F36V-TPM3-NTRK1. To test whether the FKBP12^F36V-TPM3-NTRK1 protein can be successfully degraded, I plan to treat the cells with dTAG molecules and use Western blotting at various time points to evaluate levels of FKBP12^F36V-TPM3-NTRK1 and known proteins involved in downstream signaling pathways, including pERK and pAKT. I expect that samples treated with dTAG molecules will show significantly decreased FKBP12^F36V-TPM3-NTRK1, pERK and pAKT. Importantly, these results will justify the development of small-molecule degraders to target TPM3-NTRK1, a promising therapeutic approach for pHGG and other hard-to-treat cancers. Additionally, a cell line expressing degradable TPM3-NTRK1 will serve as a powerful model system to further explore its role in cancer.

Over the past few years, patients have been identified with debilitating phenotypes due to mutations in MSTO1, a nuclear gene. These patients often have distal muscle loss and weakness leaving patients incapable of walking but to date there is no known treatment. One barrier to progress is that virtually nothing is known about MSTO1 function, making the development of therapeutics for these patients extremely challenging. The goal of this project is to use an unbiased approach to discover functions of MSTO1. To do this, I will find genetic interactors utilizing yeast to perform an unbiased screen. Yeast DML1 is the homolog to MSTO1 and is required to keep the yeast cells alive. This screen will identify genes in the yeast genome that support survival of cells lacking DML1 when the gene is overexpressed. We utilize an auxin-degron system that targets DML1 for degradation when the yeast are grown with auxin. To find genes from the yeast genome that keep the cells alive when DML1 is degraded, I express random fragments of genomic DNA. Those genes must, therefore, be linked to DML1 function in some way, thus providing insight into what MSTO1 does, how it works, and how to help MSTO1 defective patients. I have obtained hundreds of yeast colonies that survive without DML1 when other genes are overexpressed. Currently, I am extracting these overexpressed DNAs to determine the gene(s). This work is an essential step toward fully understanding MSTO1 function in cells and we plan to characterize these connections in yeast and human cells.

Lewy Bodies (LB) are É‘-synuclein protein aggregates that form when the protein is falsely over-expressed. This inappropriate cell behavior is a symptom of neurodegenerative ailments including Parkinson’s Disease. GATA-1 is a transcription factor that binds to a conserved region in the SNCA gene and promotes transcription of É‘-synuclein mRNA, which could be a contributing factor to disease presentation. DNA binding proteins serve a host of functions, primarily pertaining to regulation of gene expression. Through the Institute of Protein Design and their computational design pipeline, I designed proteins that bind to the GATA-1 binding site in the SNCA gene with the intention of inhibiting GATA-1 and hyper-expression observed in Parkinson’s patients. First I modeled the DNA target sequence using Chemnet software to best approximate the B form DNA structure that the final optimized proteins bind to. RoseTTAFold Nucleic Acid Diffusion generates protein backbones that have strong shape complementarity for the grooves of the target DNA. I then used LigandMPNN software to produce amino acid sequences for each of the backbone structures generated in the previous step. Designs are refined and diversified before the most plausible binders are selected via statistical metrics, AlphaFold2, and RoseTTAFold. I yielded roughly 5000 DNA binder models that were ordered as DNA libraries for high-throughput testing. Proteins are then expressed using yeast surface display and sorted via flow cytometry, where fluorescence indicates a binding event between the protein and DNA target. I had a robust sample of design outputs and intend on analyzing the successful protein models to quantify the importance of different structural features in DNA binding. Successful binders not only concurrently optimize and evaluate the efficacy of these deep learning models, but demonstrate the impact de-novo protein design can have on disease intervention via therapeutics research and development.

Alzheimer’s Disease (AD) is a progressive brain disorder that debilitates memory, learning, and decision-making. Early-stage AD represents the initial phase where individuals are still able to function independently, but with increasing age, their condition steadily progresses to dementia and loss of independence. Because a significant number of the aging population is affected by AD, understanding the neuroinflammatory processes would help develop more effective strategies for treatment. Examining markers such as MCP-1 and TNF-alpha, known to be associated with inflammatory response, will help identify the modulatory processes that lead to mild cognitive impairment associated with early-stage AD. Subsequently, higher levels of inflammation markers within the brain leads to mild cognitive impairment. This research study involved 40 C57BL/6 mice, 20 males and 20 females (21 months old), retro-orbitally infected with 80 µL of neurotrophic AAV-AD vector or AAV-Sham for a duration of 2 months before humane euthanasia. Brains were collected, and specific regions were examined by immunohistochemistry (IHC) and digital imaging to assess the expression levels and distribution of the inflammation markers. Preliminary observations showed that hippocampal regions of the brain from mice with early-stage AD had higher staining intensity for MCP-1and TNF-alpha compared to respective areas in Sham mice, suggesting increased inflammation is a very early lesion that develops in the presence of AD pathogenic components that might be controlled by anti-inflammatory drugs. The preliminary data suggests that the characteristics of AD manifest in part due to the neuroinflammatory response of brain factors that change with onset AD.

Myelodysplastic Syndrome/Myeloproliferative Neoplasm Unclassifiable (MDS/MPN-U) is a group of blood cancers that exhibit features of both MDS and MPN. Myeloproliferative Neoplasms (MPN) are cancers characterized by the proliferation of abnormal blood cells in the bone marrow, while Myelodysplastic Syndromes (MDS) are a group of cancers where immature blood cells in the bone marrow do not mature into healthy blood cells. Due to its overlapping features and the evolving morphology not being able to provide an accurate diagnosis, the molecular and cytogenetic abnormalities aimed at understanding the structure and properties of abnormal chromosomes could provide crucial insights for diagnosing and comprehending MDS/MPN-U. We hypothesized that the patients with MDS/MPN exhibit specific, unexplored molecular features that were not found in previous studies. We studied fourteen patients diagnosed with MDS/MPN-U overlaps and performed genetic analyses using proximity ligation sequencing (Hi-C sequencing) on Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens. Hi-C sequencing reconstructed all aberrations and rearrangements of the MDS/MPN-U genome. Cytogenetics and molecular data were collected and analyzed. New biomarkers and recurrent traits were identified. We expected Hi-C Sequencing to identify additional cytogenetics aberrations that were not detected using routine clinical cytogenetics techniques. Moreover, new insights into MDS/MPN overlap syndromes will help fill the knowledge gap in blood cancer, contributing to enhanced diagnostic accuracy, the development of treatment strategies, and improved patient care.

The kidney is composed of thousands of filtration units called nephrons. Within each nephron lies a tuft of capillaries, the glomerulus, that filters from the blood through a filtration barrier. Over time this filtration barrier thickens, ultimately causing decreased blood filtration. A main marker of this age-related degradation are telomeres which comprise the ends of chromosomes and protect the coding DNA from degradation. If telomeres become too short, the coding region of DNA will begin to degrade. To combat this, telomere shortening signals for cells to enter a state of permanent cell cycle arrest, senescence, which prevents replication of cells with degraded DNA. Accurately quantifying telomere length will enable the development of correlations between cell lineage and structural changes within the kidney. I hypothesize that Expansion Microscopy (ExM) and quantitative-Fluorescent In-Situ Hybridization (Q-FISH) will allow me to determine the relationship between physiological changes in the filtration barrier and single-cell telomere length. ExM enables a superresolution cellular view by embedding a tissue sample in a swellable hydrogel, achieving four-fold isotropic expansion. This technique confers greater resolution of Q-FISH signal versus traditional confocal microscopy. To determine telomere length, I developed custom analysis scripts to quantify Q-FISH signal brightness. Preliminary results indicate an increased brightness of younger mice compared to their aged counterparts. Additionally, to receive a base-pair output I compared the Q-FISH signal to the signal of a DNA region of known length, Major Satellites, determining young telomeres to have an average base-pair length of 30 kb. I am validating these results in collaboration with the Miller Lab using next-generation sequencing techniques. Future work includes concurrent application of general physiology stains to identify and measure the glomerular filtration barrier physiology. Results from this method will allow for a wealth of information regarding the relationship of single-cell telomere length and glomerular structural health.

Alzheimer's Disease (AD) is incredibly complex such that development of neuropathology and cognitive impairement is driven by multiple pathways. Therefore, targeting these pathways simultaneously, could provide a more effective treatment for AD compared to any single drug. Rapamycin, acarbose, and phenylbutyrate each have independent but overlapping effects on multiple pathways involved in cellular respones to pathogenic beta amyloid such as inflammation, glucose homeostasis, synaptic integrity, autophagy, and DNA damage. To test the safety and effectiveness of a cocktail of these three drugs, a proof of concept experiment was undertaken in transgenic mice carrying mutations for genes associated with early onset AD (5xFAD). These mice express neuronal amyloid plaques, a major feature of AD neuropathlogy. Transgenic and wild type mice were given either a control feed or feed containing the drug cocktail starting at 4 months of age and continued until 12 months of age. Medicated transgenic mice showed significantly less cognitive impairement in a spatial navigation learning task and reduced amyloid plaque levels in the hippocampal brain region compared to untreated transgenic mice. Immunohistochemistry will be used to identify specific biomarkers for inflammation, synaptic integrity, glucose homeostasis, autophagy, and DNA damage in the hippocampus of treated and untreated transgenic mice. Observation from this study will suggest the need to conduct additional preclincial experiments testing this specfic drug combination for a successful approach to treat Alzheimer's Disease. 

Epigenetic factors, including histone marks, change the patterns of gene expression without altering the DNA sequence. Variations in such marks are known to account for the ability of stem cells to differentiate into various cell types, but a preliminary experiment conducted by a former member of the Vaughan Group, Dr. Marcus Woodworth, has revealed that even in a phenotypically homogeneous, terminally differentiated cell population, the presence of H3K27me3, a repressive histone mark, varies on the HOXC gene of human retinal pigment epithelium (RPE1) cells at single-cell, single-loci level. My role is to evaluate the two possible origins of such heterogeneity: inheritance (histone mark varies due to events that happened during differentiation, or the random drift after differentiation, and the variations are kept within each lineage of cells), and multiple mark co-repression (one histone mark varies, but summing its effect with another histone mark that perform a similar function lead to the observed functional homogeneity), and to validate that such pattern exists among a broader range of genes. To achieve these ends, I profile selected histone marks (H3K27me3 and H3K9me3) on genes that experience different types of regulations during differentiation (HOXC, GAPDH and SIX6), using imaging-based methods, including the time-lapse imaging of live cells to map out lineages, and expansion microscopy (ExM) to capture fluorescently labeled histone marks at single-loci level. If the hypothesized origins are true, a significant difference in the number of histone mark clusters around the genes of interest would be observed between cells of different lineages, and complementary variation patterns would be observed between H3K27me3 and H3K9me3. The study reveals the nuances of histone mark dynamics on the single-cell, single-loci level, and optimizes an imaging-based method that has the potential for multiplexing at high spatial resolution, thereby providing a powerful tool for further studies on epigenetics.

Mitochondria are the main oxygen consumers in eukaryotic cells and as such are the primary organelles affected by oxygen deprivation, hypoxia. Hypoxia alters the size and shape of mitochondria, called the mitochondrial dynamics, but their role in hypoxic cell death is unknown. The Crowder lab has recently discovered that a mutation in the Mechanistic Target of Rapamycin Complex One (mTORC1) protein Raptor confers hypoxia resistance in the nematode C. elegans. mTORC1 is a master regulator of metabolism and is known to affect certain aspects of mitochondrial biology. Given these two facts, we tested the hypothesis that the hypoxia resistance of the C. elegans Raptor mutant is from alterations of mitochondrial dynamics. First, I showed that hypoxia induces small, rounded mitochondria in C. elegans caused from mitochondrial fission. Second consistent with the hypothesis, I showed that the mitochondria appear to have more normal morphology before and after hypoxia in the Raptor mutant. However, not consistent with the hypothesis, a C. elegans mutant with excess mitochondrial fission was not hypersensitive to hypoxia. Then combining the hyper fission mutant with the Raptor mutant did not diminish the hypoxia resistance produced by reduced Raptor function. Thus, our data demonstrates abrogating mitochondrial fission is not necessary for the hypoxia resistance produced by the Raptor mutant and leads us to reject our hypothesis. By exploring the interaction of mitochondrial fusion and fission with Raptor, we are beginning to understand how these important organelle and metabolic regulators combine to control hypoxic cell death.

We use genes to survive and reproduce, but this means that genes can hold our survival hostage to ensure their own propagation, without providing any benefit to us. Selfish genes are a brutal, and poorly-characterized, demonstration of this concept. Instead of producing beneficial proteins, they produce nonessential proteins that prevent individuals who carry the selfish gene from successfully reproducing with non-carriers. One such example, the PEEL-1/ZEEL-1 system, is natively found in C. elegans. In this system, PEEL-1 is a toxin protein that kills cells when it is expressed without the antitoxin protein ZEEL-1. My aim is to determine the mechanism of toxicity employed by PEEL-1. AlphaFold predictions suggest that PEEL-1 contains an amphipathic helix. The amphipathic property of this region is hypothesized to play a critical role in PEEL-1 toxicity. To test this hypothesis, I am conducting a Deep Mutational Scanning (DMS) on the amphipathic helix of PEEL-1, with the goal of identifying key polar or nonpolar residues in this region that are essential to PEEL-1 toxicity. First, I generate a library of single-residue PEEL-1 mutants. Second, I transfect these constructs into HEK293T cells. I sample this initial pool of cells for sequencing, to identify which PEEL-1 mutants the pool carries, and in what proportions. Third, I induce expression, exposing each cell to the effects of the PEEL-1 mutant they carry. Only the cells expressing loss-of-function mutants survive. Now, I sequence this final surviving pool of cells, similarly to the initial pool. Mutations that drastically alter the polarity of the residue and thus break the overall amphipathic structure of the region are expected to be overrepresented in the surviving pool. This result would provide a broader understanding of the various methods of cell death in nature and provide novel insight into how animal-derived selfish genetic systems function.

Glomeruli are the basic filtration unit of the kidney. The current understanding of its physiology is limited by the partial or 2D analysis of its structural components. The Vaughan Group uses optical super-resolution microscopy in combination with advanced chemical labeling techniques and powerful data analysis approaches to perform high-resolution 3D reconstruction of the whole mouse glomeruli. Overall, the work has the potential to provide a novel understanding of the glomerular structures and how they are altered in aged and diseased conditions. The labeling of the overall morphology of the glomeruli is achieved by chemically labeling the distribution of abundant macromolecules (carbohydrates, amine, and DNA) using Fluorescence Labeling of Abundant Reactive Entities (FLARE). Though we could visualize the general physiology of the sample with FLARE, incorporating specific targeting of molecules with FLARE is still challenging. My role is to optimize the FLARE protocol to add the capability of labeling the distribution of specific molecules using immunolabeling. The most challenging part is that all the fluorophores labeled prior to FLARE will be bleached out by the strong oxidation step while labeling carbohydrates. I am focusing on exploring possible workarounds to incorporate immunostaining with FLARE. The only way to bypass the bleaching fluorophores is to label dyes after the FLARE. However, the FLARE involves the gelation part, and the gel makes antibodies which are linked to fluorophores hard to get into the sample. So, instead of using regular secondary antibodies, I use biotin and then link to the streptavidin dye, which is smaller in size and easier to enter the sample. With this optimization working, we could incorporate whatever target of interest with high resolution on top of three general stains provided by FLARE, giving us an extra degree of information for our 3D reconstructions of glomeruli.

Evaluating women in Ancient History could be difficult as patriarchal societies limited the representation of women, which resulted in little or biased documentation. Another obstacle was evaluating the accuracy of evaluating the authenticity and accuracy of reported events when there is such limited evidence of certain figures and events in Ancient History . However, regarding the feminist martyr narrative, I was able to evaluate the effects of the representation in martyrdom and how they could be applied to modern feminist narratives. To investigate the significance of the texts of women and martyrdom to values in the Patristic Era of Christianity, I analyzed women in the context of the Roman Empire compared to the Early Christian Church using primary sources. Introducing the consequences of martyrdom, I addressed the etymology of the word “martyr” and the connotations associated with the term. As my primary sources, I used the accounts of Perpetua and Blandina because their narratives were among the most well-known. Studying the divergence of feminine virtues of the Roman Empire and Early Christianity, I explained how the differences influenced the divisions and spread of the Early Christian Church. I also studied the retelling of their stories throughout history, which illustrated society’s expectations of women as opposed to the possible motivations of the martyred women. I discovered that the stories of martyred women were altered throughout history to perpetuate different ideals of femininity but at a closer look, the original narratives depicted a progressive depiction of women. This research exposed how the representation of women could be altered to fit an agenda and revealed the values of the authors of the narratives as well as the surrounding culture. This could be used to evaluate the representation of influential women figures today as well as the consequences of altering feminist narratives.

Pulling apart DNA during replication induces DNA strands to wrap around each other, producing “positive supercoils” ahead of the replication fork. These positive supercoils present a significant obstacle for further DNA replication. Type II topoisomerases (Top2s), a group of essential DNA replication enzymes, cleave these positive supercoils and relax DNA to a state that is easy to separate. Errors in the resolution of supercoils are implicated in many health conditions including autoimmune diseases and cancer. However, the mechanism by which Top2s localize to positive supercoils is unknown. We recently discovered that GapR, an essential DNA binding protein conserved in alphaproteobacteria, binds positive supercoils and stimulates the activity of bacterial Top2s DNA Gyrase and Topoisomerase IV. Although GapR stimulates Top2s in vitro, we do not know the mechanism by which GapR may recruit Top2s. We hypothesize that GapR recruits Top2s to positive supercoils by direct interaction. We investigated this mechanism by using a Bacterial Two-Hybrid assay to screen for GapR interaction with Top2 subunits, and I formed GapR truncations to probe for interaction in biochemical and genetic assays. We identified an interaction between GapR and the homologous Top2 subunits GyrA of DNA Gyrase and ParC of Topoisomerase IV. I discovered that this interaction terminates with the truncation of the last 13 amino acids of GapR. In the future, we aim to identify the surface that mediates direct interaction between GapR and Top2s aided by structure prediction. This work will reveal a previously unknown mechanism of Top2 recruitment. Because GapR is broadly conserved by alphaproteobacteria, our research could reveal a novel mechanism to inhibit with antibiotics. As GapR is the first identified Top2 recruiter, our work could reveal a novel pathway to target with anticancer therapeutics if conserved, as human Top2 inhibitors are important chemotherapy drugs.

Although the relationship between the structure, function, and physiology of organs is well documented, the mechanisms by which cells collectively coordinate into three-dimensional tissues and organ components remains unknown.The countless factors that inform the morphogenesis of mammalian organs poses a challenge to understand organogenesis from first principles. However, the Malpighian tubules of the fruit fly Drosophila offer an excellent model system for investigating this question due to their rapid development, relative simplicity, and the degree to which scientists can manipulate variables that affect their development. These tubules are the renal equivalent of the fruit fly excretory system; further, many of the genes involved in sculpting these tubules are conserved from flies to humans. One conserved gene is the fly homolog of β-catenin, which is known to play an essential role in cell-cell adhesion. The goal of my research is to define how β-catenin impacts organ morphogenesis. To do this, I use fluorescence microscopy and live imaging to compare wildtype Drosophila to those with decreased β-catenin expression. Using tissue-specific fluorescent protein tagging, I can differentiate Malpighian tubule cells from other embryonic cells under the microscope so that their shapes can be analyzed, and I control the level of β-catenin expression specifically in Malpighian tubule cells using RNAi. Due to β-catenin’s integral role in cell-cell adhesion, I expect to find localization of β-catenin to the cell membranes of the tubules, with high concentration along membranes undergoing the greatest adhesion or motion, and interrupted tubule morphogenesis in reduced-expression lines. I also suspect that cells may completely fail to adhere and will be unable to transmit tension effectively along the tissue. The results of this experiment will contribute to our understanding not only of Malpighian tubule morphogenesis, but of one of the components of morphogenesis in general.

Tuberculosis remains a global public health threat due to the rising number of multi- and extensively drug resistant strains of the causative pathogen Mycobacterium tuberculosis. Development of novel drugs and an understanding of their resistance mechanisms is urgently needed. Aminothiazoles (AmT) are potent molecules with killing activity against M. tuberculosis; these compounds act as copper ionophores and target a key enzyme (enolase) by displacing its Mg²⁺ co-factor, a substance required for its activity, with Cu²⁺ imported by the compounds. Spontaneous mutations in an essential protein export system (the Esx 3 Type VII secretion system) confers resistance to AmTs. My research focuses on understanding how mutations in the secretion system cause AmT resistance. We hypothesize that copper imported by AmTs could disrupt other metallo-proteins including EccA3, a key ATPase of the of the Esx-3 secretion system that hydrolyzes ATP into ADP and inorganic phosphate, and that resistance mutations (e.g. E237K) reduce Mg²⁺ co-factor displacement by Cu²⁺. To test this hypothesis, I expressed wild-type (WT) EccA3 and mutant EccA3 [E237K] proteins in Escherichia coli BL21(DE3) expression strain and purified the proteins via Ni-NTA His-tag chromatography. Subsequently, I measured the activity of the purified EccA3 (WT) and EccA3 [E237K] proteins via an ATPase assay based on colorimetric detection of free inorganic phosphate released by ATP hydrolysis. I aim to understand whether copper inhibits EccA3 activity through this assay, anticipating that copper reduces EccA3 (WT) ATPase activity while EccA3 [E237K] ATPase activity is unaffected. Thus, my work will provide an avenue for understanding AmT resistance in M. tuberculosis.

How do organs have such consistent and reproducible shape, form, and volume? One factor of this complex phenomena is cell-cell adhesion. Cell-cell adhesion plays a vital role in organ formation, as it is an essential driver of cell shape, cell arrangements, and tissue structure. To determine the role of adhesion in organ formation, I define the role of E-Cadherin, a cell-cell junction projection that adheres neighboring cells. The developing renal system of Drosophila, Malpighian tubules, are an excellent system because I can selectively manipulate expression of E-Cadherin in the organ and can utilize fluorescence microscopy to observe how these changes affect tubule morphogenesis. I observe where the adhesion protein is located during organ growth, and what happens to organ growth when expression of the adhesion protein is reduced. To track the dynamic localization of E-Cadherin, I take measurements of specific location of E-Cadherin between cells and concentration of E-Cadherin throughout organ development. I expect the concentration of E-Cadherin to increase during elongation, and that it will be enriched in more looped parts of the organ. To define the requirement of E-cadherin during organ formation, I use RNA interference to reduce E-Cadherin expression. Because of how vital E-Cadherin is in other developmental morphogenetic processes, I expect a decrease of expression to have profound impacts, leading to severe organ developmental defects. I measure these defects by comparing cell shape change and organ shape in control and E-Cadherin reduced organs. The results of this study will not only help us understand Malpighian tubule morphogenesis, but it will also help us understand organogenesis more generally. Elucidating the precise mechanisms behind cell behavior, shape, and cell-cell interaction has important human health implications and will enable work in many other fields such as cancer, regenerative treatments, tissue growth, and organ synthesis.

Toll-like receptor 4 (TLR4) is an immune protein which binds lipopolysaccharide (LPS) present on the outer membrane of Gram-negative bacteria and activates the innate immune response. In mice, an mRNA splice variant composed of only the extracellular domain of TLR4 was shown to encode a soluble product (sTLR4) capable of inhibiting inflammatory response to LPS. sTLR4 has been recovered from human saliva and demonstrated to dampen the production of pro-inflammatory cytokines by macrophages. Prior work showed that TLR4 was also present in endometrial glands, uterine tube epithelia and endocervical glands. However, there are no published studies exploring the presence or role of sTLR4 in lower genital tract secretions. We tested primary female genital epithelial cells’ supernatants as well as human endocervical cytobrush and vaginal swab samples for the presence of sTLR4 by using a chemiluminescent immunoassay. We found sTLR4 in cervicovaginal secretions, with increased concentration of sTLR4 present in participants with endocervical ectopy and in those sampled during the proliferative phase of the menstrual cycle. Supernatants from endocervical cell lines possessed higher levels of sTLR4 than those derived from ectocervical or vaginal cells. sTLR4 concentration was not correlated with the presence of bacterial vaginosis, age, the concentration of common vaginal Gram-negative bacteria or with genetic variation in the TLR4 locus. By western blotting, we demonstrated that sTLR4 is composed of a ~100 kDa polypeptide, corresponding to the entire TLR4 ectodomain. In a reporter monocytic cell line, we showed dose-dependent inhibition of the LPS/Interferon-regulatory factor (IRF) pathway when LPS was preincubated with endocervical cells’ supernatants. These results point to an unappreciated form of innate immune regulation in the cervicovaginal niche, which could potentially open new avenues for understanding inflammatory disorders such as cervicitis and pelvic inflammatory disease.

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.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an opportunistic pathogen of wounds, cornea, and the Cystic Fibrosis lung. P. aeruginosa is also a model organism for the study of bacterial biofilm formation. Biofilms are multicellular communities that form from bacterial growth concomitant with the production of extracellular polymeric substances (EPS). EPS includes polymers such as polysaccharides, DNA, and proteins; these polymers provide structure and protection to the biofilm cells. Proteomics experiments by the Parsek Lab and others have demonstrated that a notable component of the biofilm matrix are the secreted proteases. Secreted proteases have defined roles in virulence and nutrient acquisition, but their role in the biofilm matrix of P. aeruginosa has not been explored. I hypothesize that these secreted proteases recycle nutrients, remove cell waste, and protect cells from host immunity. To test my hypothesis, I generated a mutant strain of P. aeruginosa that lacks the six major secreted proteases. While we see that loss of the proteases does not impact planktonic growth, preliminary data suggests that loss of proteolytic activity results in moderately increased biofilm formation. Using a general proteolysis assay relying on casein hydrolysis, I have determined the relative contribution of each of the six proteases to the total proteolytic capacity of P. aeruginosa in planktonic growth. I will further test the impact of the proteases on biofilm growth in different growth environments, including under flow conditions and in artificial sputum medium. I will also assess which proteases contribute the most to proteolysis during biofilm growth. My work fits into a growing body of literature that suggests that the biofilm matrix is not an inert scaffold, but is instead a dynamic and active network.

Damage to the spinal cord causes one of the most debilitating injuries to the human body. The challenge of promoting the regeneration of this dense network of neurons and glia after spinal cord injury has been seen as insurmountable. However, new techniques emerging from the field of regenerative medicine have illustrated the possibility of encouraging the body to repair these injuries on its own. In the Wills Lab, we study the model organism Xenopus tropicalis, or the Western clawed frog, which has the ability to regenerate its spinal cord and associated tissue following amputation. My project focuses on how X. tropicalis uses the developmental morphogen Sonic Hedgehog (Shh) to re-establish the dorsal-ventral (DV) patterning of the spinal cord during regeneration. I have used cyclopamine, a Shh inhibitor, and SAG, an agonist, in order to perturb Shh signaling during regeneration. I then monitored the effect on DV patterning via immunohistochemical labeling of dorsal and ventral markers. Work so far has shown that Shh signaling is in fact necessary to the establishment of proper DV domains in the regenerate spinal cord. However, my research has also hinted that this specification is complex. Shh appears to have a more proliferative role early on, with patterning effects coming later. In addition, there appears to be an interaction between Shh and other signals that specify anterior-posterior polarity. Overall, my research so far has generated new evidence for how developmental signals are repurposed in the context of regeneration. 

In the face of a global crisis, the poaching of pangolins (Manis spp) has emerged as a dire threat, with Africa at the epicenter of this illicit trade; these enigmatic creatures, sought after for their scales and meat, face exploitation driven by illegal activities that endanger their populations and disrupt the balance of the African ecosystems. Studying the lineage distributions of pangolins could prove crucial for their protection as it provides valuable insights into their evolutionary history, genetic diversity, and ecological adaptations. This information may be essential for formulating effective conservation strategies, understanding their vulnerabilities to diseases, and identifying key habitats for preservation. We aim to address these gaps, by exploring whether pangolin (Manis temminckii) lineages from Mozambique can be genetically distinguished from those from Southern Africa and by investigating the region of origin for two scale samples seized by law enforcement in Singapore in 2019. We conducted genetic and phylogenetic species delineation by sequencing two mitochondrial genes, Cytochrome b oxidase (cytb) and the Control Region (D-loop), from pangolin skin tissues from 10 villages in 3 provinces in Mozambique to complement existing data from Southern Africa. We will then use RAxML (Randomized Axelerated Maximum Likelihood) for phylogenetic analysis and to estimate phylogenetic trees based on our collected data. This multi-locus approach ensures robust species delineation and provides essential support for species recognition. Our focus on mitochondrial DNA (mtDNA) in Mozambique aims to make a reference map of the distributions of genotypes throughout Southern Africa, and our research contributes valuable genetic insights, offering a nuanced perspective on the spatial dynamics of pangolin populations. Ultimately, these findings play a pivotal role in addressing the complex issue of illegal wildlife poaching in Africa, providing essential resources for conservation efforts and informed management strategies as well as contributing to the field of forensic science for counter wildlife trafficking.

In response to acute genotoxic stress, such as chemoradiation therapy, stem cells undergo temporary cell cycle arrest at the G1/S phase transition. This state, called quiescence, is reversible once stress-free conditions allow reentry into the cell cycle. We have previously identified the underlying mechanism behind quiescence in Drosophila Germline Stem Cells (GSCs) and human-induced pluripotent stem cells (hiPSCs). Mitophagy, or autophagy of the mitochondria, is required to enter quiescence. Surprisingly, we have observed a reserve of cyclin E (CycE) associated with the outer mitochondrial membrane that’s present in normal GSCs and hiPSCs but is reduced in quiescent stem cells. The role of CycE in quiescence remains unclear. Previously we have shown that reduced levels of CycE via inhibition of mTOR have driven cells toward mitophagy-dependent quiescence. This reveals that mitophagy serves as an alternative mechanism of CycE inhibition in contrast to the typical p21-mediated inhibition. Additionally, Parkin, a ubiquitin ligase activated by a serine/threonine kinase PINK1, is a key protein involved in mitophagy required for quiescence, and it has been found that CycE is a degradation target of this protein complex. Our hypothesis is that CycE degradation is necessary for entry into quiescence. To test this we upregulated CycE with a deleted portion of its PEST domain, which is a target for ubiquitination, under UAS-GAL4 control and used the GSC spectrosome morphology to observe quiescence. We observed a six-fold reduction of quiescent GSCs with overexpressed CycE, and hence concluded that CycE degradation is necessary for entry into quiescence. Determining the mechanism of CycE in stem cell quiescence is critical to understanding how cancer stem cells can avoid chemoradiation therapy. This project allows us to characterize the role of CycE within mitophagy and strengthen our understanding of the mechanisms that govern the cell cycle and quiescence.

Repetitive transcranial magnetic stimulation (rTMS) is a psychiatric treatment which has shown promise for experimental treatment of memory loss in Alzheimer’s Disease. rTMS uses a coil and electric current which is able to create a magnetic field that can depolarize neurons noninvasively and induce synchronized activity of large populations of neurons, ultimately inducing, lasting changes through synaptic plasticity. Alzheimer’s disease patients show disruptions in the Default Mode Network (DMN), a network of brain regions which is typically active at rest. The DMN has an important role in memory consolidation and is disrupted in Alzheimer's Disease. We hypothesize that strengthening the default mode network through rTMS applied to area left 8AV of the frontal cortex will create improvements in patient memory. To answer this question, we are performing a single-blind, single-arm, randomized cross-over trial of rTMS on early-stage Alzheimer's disease patients. Region 8AV is located by using MRI scans obtained before patients receive either the sham or experimental procedure. This region was chosen due to its connection to the default mode network and previous promising TMS research. Our primary outcome measure is the speed of forgetting, a new, reliable index of memory function obtained by fitting a computational model of episodic memory to behavioral data from an adaptive memory test. Due to the frequent use of rTMS in mood disorder treatment, we are using depression and anxiety scales to track possible mood improvements as a secondary outcome measure. MRI scans will also be analyzed to see if the experimental treatment caused any structural differences in patient brains. Should our hypothesis be correct, we expect to see improvements in memory or cessation of memory decline in patients. Successful treatment would provide a novel target for Alzheimer’s Disease treatment using rTMS, and additional evidence for the continued investigation of rTMS for Alzheimer’s Disease.

Infants perceive speech and acquire language amidst noisy and complex auditory environments. Thus, elucidation of the cognitive mechanisms governing speech perception under noisy conditions is crucial. Cortical encoding of the speech envelope has been one approach used to study speech-in-noise perception in adults. For infants, research shows that Infant Directed Speech (IDS) facilitates cortical encoding of the speech envelope in quiet conditions more than adult direct speech. However, it is unclear whether infants are able to track the IDS speech envelope amidst competing speech. To investigate this, we recorded the neural responses from 40 typically-hearing infants (20 seven-month-olds, 20 eleven-month-olds) to continuous IDS using electroencephalography (EEG) in three conditions: Quiet, Co-located Noise, and Separated Noise. The target stimuli consisted of naturally recorded IDS produced by two female English speakers. The noise stimuli consisted of a four-person babble constructed from audiobooks read by 2 male and 2 female English speakers. We presented stimuli at an overall level of 70 dB SPL via speakers placed at 0°, +90°, and -90° azimuth to infants sitting on a caregiver’s lap in a sound-attenuated booth. Our team analyzed EEG signals using the Multivariate Temporal Response Function (mTRF) toolbox in MATLAB. This backward modeling approach assesses whether the stimulus envelope can be reconstructed based on the recorded neural responses. Reconstruction accuracies greater than chance were observed in all three conditions for the majority of infants, suggesting that we were able to decode the speech envelope in both quiet and noise. Participants demonstrated the capacity to process speech, even amidst competing auditory stimuli, emphasizing speech perception competencies from an early developmental stage. These results support using the envelope model and mTRF method as a feasible method for investigating the development of speech-in-noise perception in infants and young children.

It is increasingly recognized that exposures during sensitive developmental time windows may lead to delayed onset of diseases later in life. Polybrominated diphenyl ethers (PBDEs) are legacy flame retardants that bioaccumulate in the environment as well as human breast milk. In both animal models and humans, PBDE exposure is linked to thyroid toxicity, neurodevelopmental disorders, and hepatotoxicity. Our previous work showed that neonatal oral exposure to BDE-99, a human breast milk-enriched PBDE congener, produced dysbiosis of the gut microbiome associated with a pro-inflammatory transcriptomic signature with the gut-liver axis. However, very little is known whether the BDE-99 mediated host effect is caused by the changes in the gut microbiome. To address this knowledge gap, large intestinal contents from adult male pups that were neonatally exposed to BDE-99 (57 mg/kg p.o. once daily from postnatal days 2-4) or corn oil (10 ml/kg) were transplanted to adult germ-free mice (i.e., mice without microbiome). After one month colonization period, total RNA was isolated from the colon and subjected to RT-qPCR. Ex-germ-free mice receiving the microbiome from BDE-99 exposed pups had decreased expression of genes involved in gut barrier integrity (tight junction protein 2 and claudin 7 [Cldn7]), indicating increased gut permeability. These mice also had increased expression of several pro-inflammatory cytokines including interferon gamma (Ifng) and interleukin 17 (Il17), but decreased expression of the major drug-metabolizing enzyme cytochrome P450 (Cyp) 3a11. In summary, our results suggest that early life BDE-99 exposure mediated persistent dysbiosis in the gut microbiome mechanistically contributes to a proinflammatory and leaky gut state with reduced xenobiotic metabolism capacities.

Mycoplasma genitalium (MG) is a sexually transmitted bacterial pathogen commonly associated with urethritis in men and cervicitis, endometritis, pelvic inflammatory disease, infertility, and preterm birth among women as it invades the upper reproductive tract. Infections can persist for months to years without effective treatment due to antimicrobial resistance. Current first-line drug choices are only successful in less than half of all patients. Preliminary in vitro data suggests that MG is susceptible to tinidazole (Tdz) and may fill the need for additional treatment options for drug resistant infections as it is already FDA-approved for other indications. As strains can vary in their susceptibility to particular drugs, we aim to identify the minimum inhibitory concentration (MIC), the concentration that inhibits growth by 99%, of Tdz against 10 MG clinical isolates. This data will determine if these strains are susceptible to Tdz, define the range of MICs, and reveal whether current strains have already developed resistance. Twofold dilutions of Tdz and doxycycline (DOX) antibiotics are added to MG clinical strains in 48-well plates and incubated at 37 C/ 5% CO2 for 21-28 days. DOX is one of the first-line drug choices with a known MIC; it is used to confirm that assays are performed correctly and to compare the effectiveness of Tdz. Four of the wells have no drugs to serve as a control to compare the number of genomes against those in the Tdz wells to determine MG inhibition. MG growth in each Tdz dilution is quantified with qPCR by isolating DNA from the wells. Calculations of the percent inhibition will dictate which antibiotic concentration is useful for treating infected patients. As physicians are already beginning to treat MG patients with Tdz, data regarding susceptibility of multiple isolates is crucial in informing these treatment regimens.

Body shape provides useful insight into the diversity and evolution of vertebrate body plans. Previous research revealed that body shape scales with body size in carnivoran mammals, but whether this trend occurs in other mammals remains unknown. Our goal is to examine the relationship between body shape and body size in Leporidae, which consists of rabbits and hares. Leporids exhibit a unique gradient of locomotion types between the smallest rabbits that are primarily saltatorial and the larger hares that are primarily cursorial. We quantified body shape using the head-body elongation ratio and body size using the geometric mean of all measured traits from osteological specimens held at the Burke Museum and other natural history museums. We tested the allometric relationship between body shape and size using a phylogenetic regression and also tested if these allometric relationships differed between saltatorial rabbits and cursorial hares using phylogenetic ANCOVAs. We predict that the differences in locomotory modes within the family could influence the correlation between body shape and allometry, possibly leading to different amounts of correlation in rabbits and hares. Specifically, we predict that larger body size corresponds with more elongate body shape. Elongate body shape would facilitate cursorial locomotion more common in larger leporids. More elongate and flexible bodies would allow the forelimbs to reach farther forward, enabling longer strides while running. This study informs allometric relationships of body shape and size of saltatorial and cursorial members of leporids and can lead to future research into the relationship between locomotion and body shape in other mammalian clades.

Saccades are rapid eye movements that are essential for tasks like reading, and their accuracy is maintained through motor adaptation across life stages and in response to neural injuries of diseases. Our long-range goal is to identify the neural mechanisms of such saccade adaptation. While saccade adaptation has been intensively studied before, our understanding of the neurophysiological basis of this phenomenon is based largely on amplitude-decrease adaptation. However, the clinical relevance of amplitude-decrease adaptation is marginal at best, as overshooting saccades rarely occur in real life. Most erroneous saccades fall short of their target, necessitating an amplitude-increase adaptation. This is what we propose to investigate in this application. The challenge in studying amplitude-increase adaptation lies in the requirement of numerous saccades over extended periods to observe significant amplitude alterations. Our preliminary studies, conducted over daily sessions spanning more than a month, discovered a bimodal distribution of adapted saccades characterized by low and high gains, with gain defined as the ratio of saccade amplitude to the target step. Low-gain saccades surface initially, reaching peak velocity saturation leading to a gain increase saturation. Conversely, high-gain saccades emerged after extensive trials, characterized by their reduced velocities and prolonged durations, suggesting a novel adaptation mechanism through the fusion of consecutive saccade pairs. Our project aims to (1) characterize this newly found adaptation mechanism and (2) investigate the superior colliculus's (SC) role, which is pivotal in commanding saccade size, in generating both low and high-gain adapted saccades and the integration of saccade pairs. The overarching impact of this research lies in its potential to enhance our understanding of motor adaptation's role in recovering from motor deficits caused by neural damage. Understanding these changes may help refine the strategy of rehabilitation for patients with saccade dysmetrias, and perhaps motor hypometrias in general.

Signaling of fibroblast growth factor receptors (FGFR) is critical for the development of vascular cell types. FGFR exists as two alternative splice variants: the b and c isoforms. Previous experiments have shown that activation of the c isoform leads to arterial endothelial cell development and inhibition of the c isoform is critical to perivascular development. These results were found using a c isoform-specific computationally designed protein. The goal of my project is to replicate these isoform specific results in an endogenous context. Our hypothesis is that induced pluripotent stem cells (IPSCs) overexpressing the b isoform will develop into pericytes and IPSCs overexpressing the c isoform will develop into arterial endothelial cells. I used the Gibson assembly method to create b/c isoform overexpression plasmids that can be inserted into the AAVS safe harbor site and used bacterial transformation to increase the amount of DNA. I am using stable transfection to create IPSC overexpression cell lines and adapting a previously verified 14-day protocol for creating endothelial cells from IPSCs to monitor each cell line’s differentiation. I am performing assays such as qPCRs, Western Blots, and immunofluorescence to quantify perivascular and endothelial markers in the cell lineages. Our findings should agree with our isoform specific hypothesis. In future experiments, we plan to engraft the overexpression cell lines into immunodeficient mice and assay how varying ratios of the two cell types affect their regenerative potential in vivo.

Glutamine synthetase (GS) is a highly regulated enzyme critical for converting glutamate to glutamine and associated with ammonia assimilation. Dysregulation in the GS interconversion process can lead to hyperammonemia, potentially resulting in death or brain damage. GS is conserved across prokaryotes and eukaryotes. Among enzymes, glutamine synthetase has the ability to polymerize but the functional characteristics of its self-assembling filaments remain unknown. This study aims to elucidate the occurrence of filament formation in GS and its effects on enzyme activity. We hypothesized that filaments might influence the association of GS substrates or allosterically regulate the enzyme. I purified GS from Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Helicobacter pylori using Ni-column and size exclusion chromatography (SEC). The focus was primarily on Pseudomonas GS, examining it under various buffer conditions (Mg²⁺, Co²⁺) through negative staining. Under magnesium conditions (10 mM), dodecamer strcture of GS was observed and filaments was induced under cobalt conditions (10 mM). To investigate the structural mechanism of filament formation further, we utilized cryogenic electron microscopy (Cryo-EM) to create a model of the GS filament interface and identifying involved residues. Additionally, I am conducting mutagenesis on key residues of Pseudomonas GS to disrupt filament formation. This research holds significant implications for metabolic engineering, as understanding the structure and role of filament formation in GS could lead to new therapeutic targets in metabolism.

Adoptive Cell Therapy (ACT) is a novel modality of cancer therapy, in which immune cells can be engineered with T cell receptors (TCRs) to aid in targeting specific antigens presented on the surface of cancer cells. ACT has already brought curative therapy to many previously treatment-refractory blood cancers. However, TCR-T cells often have limited persistence after transfer into patients, which has hampered the effectiveness of this therapy for solid tumors, which require a sustained anti-tumor response. I evaluated if functional improvements could be induced by a small molecule inhibitor of LSD1 (LSD1i), a histone lysine demethylase that modifies chromosome accessibility, as a strategy to improve long-term activity against tumor cells after infusion into patients. Previous research has also found that “helper” CD4+T cells enhance the functionality of cytotoxic CD8+ cells, and I am now seeking to assess broader activity and benefits of LSD1i on a combined population of CD4+ and CD8+ cells. I first treated CD8+ T cells with a small molecule drug, Bomedemstat (an inhibitor of LSD1) during the in vitro cell expansion process, and demonstrated enhanced function in a co-culture system that models chronic stimulation as would occur in a solid tumor. To assess activity of Bomedemstat on CD4+ T cells, I used lentiviral transduction to insert functional TCR constructs into both CD4+ and CD8+ T cells, and plan to expand these cells with the optimized concentration of Bomedemstat in a second coculture assay, this time with combined CD4+ and CD8+ populations. I expect Bomedemstat will prevent dysfunction similar to our previous results with a LSD1i in CD8+ T cells, as illustrated by improved tumor killing and changes in T cell phenotype and function. Demonstrating applicability of LSD1i pretreatment on combined T cell populations would establish a stronger foundation to advance this epigenetic perturbation to clinical applications.

Chemical communication is the oldest and most widespread form of communication across the tree of life, and markedly present among lizards. However, the drivers of chemical profile variations in this group remain for the most part uninvestigated. In South American lizards of the Tropiduridae family, semiochemicals are produced by epidermal gland organs called α-glands, exclusively found on the ventral side of male individuals of at least 40 species from four genera. The chemicals produced by these glands are hypothesized to interact with their environments in different ways since chemical species are naturally reactive and tend towards their lowest energetic state. Thus, the intrinsic properties of a semiochemical impact its survival and efficacy for communication. Given the diverse ecology and broad geographical distribution of tropidurids, we investigated whether variation in the chemical composition of α-gland secretions correlates with temperature, humidity, and habitat openness. We performed liquid chromatography-mass spectrometry (LCMS) to obtain the metabolomes of three different sample types. We sampled male skin containing the α-glands, undifferentiated male skin, and female skin. Environmental and chemical property data were extracted from online databases, literature, and field observations. Preliminary tests were done by making Venn diagrams comparing the metabolomes of each sample type. These revealed differences in metabolite compositions, notably between males and females as well as between glandular and undifferentiated skin. From the metabolomes of α-glands, we expect to see chemical species with properties that confer greater survival given the specificities of the environment. For example, given a lizard from a hot and humid environment, we expect the metabolome of the α-glands to contain higher molecular weight species with less functional group complexity. Understanding how environmental parameters drive the chemical composition of α-glands is expected to provide a deeper understanding of the evolutionary history of chemical signaling in terrestrial vertebrates.

Patients with Human Immunodeficiency Virus (HIV) are known to have increased risk of cardiovascular complications. High Density Lipoprotein (HDL) is a circulating lipoprotein responsible for removing lipids, such as cholesterol, from the blood and returning them to the liver, and is known to have a large impact on cardiovascular health. HDL is also known to have a protective effect on endothelial cells, which line the blood vessel walls, and it normally stimulates nitric oxide to cause an anti-inflammatory response. However, little is known about whether HDL from HIV patients has unique effects on the function of endothelial cells. I hypothesize that HIV-positive patients have increased inflammation due to impairment of HDL’s protective anti-inflammatory function. To test this, I am determining whether there is an increase in pro-inflammatory cytokines in plasma from HIV-positive patients compared to control patients, using enzyme-linked immunosorbent assays. I am also testing the hypothesis that HDL from HIV patients has a more pro-inflammatory effect on endothelial cells. I am culturing human microvascular endothelial cells (HMEC) and treating them with HDL from HIV-positive and non-HIV patients, along with appropriate control stimuli, followed by in-cell Western assays to measure activation of NFkB protein, a master pro-inflammatory regulator. I am using the same methods to measure activation of Akt, an intracellular signaling protein that can activate the production of nitric oxide via the enzymatic activity of endothelial nitric oxide synthase. I anticipate that HDL from HIV patients will cause increased activation of NFkB and decreased activation of Akt, which could explain, at least in part, the increased inflammation and cardiovascular issues in HIV patients. This research will begin to reveal possible mechanisms by which dysfunctional HDL may contribute to cardiovascular risks in HIV patients, and such findings could ultimately identify novel targets for therapeutic intervention.

The four letters in DNA (ATGC) construct the basis of life as we know it. Unnatural base pairing xenonucleic acids (ubp XNAs) are synthetic nucleic acids that can be used orthogonally to the 4-letter code. XNAs have the potential to revolutionize a myriad of biotechnologies, but commercial sources of XNA nucleotides are limited and expensive. Here, we fill one step of an enzymatic cascade required to sustainably produce XNA nucleotides. Nucleoside phosphorylases (NPs) are enzymes that catalyze the reversible phosphorolysis of nucleosides to their base and sugar components. We purified and assayed promiscuous NPs from two thermophiles, Geobacillus thermoglucosidasius (GtNP) and Thermus thermophilus (TtNP). Using a combination of mass spectrometry and fluorescence assays, we show that these phosphorylases have activity on a subset of three XNA substrates (B, Sⁿ, and P). This enzymatic pathway allows us to synthesize non-standard nucleotides in a cost-efficient manner and provides a crucial tool for the biosynthesis of XNAs.

When a cell undergoes stress conditions, such as oxidation or aging, an increase in protein instability can occur and prevent proper cell functions. Small Heat Shock Proteins (sHSPs) are molecular chaperones that work to maintain a healthy proteome by associating with misfolded “client” proteins to delay aggregation under such conditions. HSPB5, a human sHSP, is ubiquitously expressed throughout the body. HSPB5’s disease mutant, R120G, is a defective chaperone associated with cataracts and desmin-related myopathy. It is still unknown how this mutation is detrimental despite many years of research. My research aims to understand how this mutation retunes the electrostatic properties of HSPB5, affecting its chaperone activity. Residue R120 is part of an electrostatic network that helps create an important structural feature in the folded region of HSPB5, the alpha-crystallin domain (ACD). In the unmutated (WT) protein, the ACD surface is overall positively charged. Substitution of the positive R120 to glycine alters both ACD’s structure and electrostatics. I generated two mutants, R120K (retaining positive charge) and R120D (switching to negative charge) to investigate how R120 plays a role in ACD’s conformation. Using a negatively-charged molecule, ATP, as an “electrostatic” probe in 2D NMR, I observed differences between its binding affinity to my R120 variants. I found that only R120K ACD behaves similar to WT ACD, suggesting a possible correlation between charge potential and ACD’s interactions with ATP. Currently, I am investigating if charge potential affects chaperone activity through aggregation assays with a client protein, human γD-crystallin, found in the lens and implicated in cataracts. I predict that WT and R120K, with similar electrostatic properties, will have similar chaperone activity. R120G and R120D, prevalently in an “active” state, will have higher chaperone activity. Understanding how such mutations affect HSPB5’s conformations and chaperone activity is a step forward in understanding sHSPs’ chaperone mechanism.

Traumatic spinal cord injury (tSCI) is a devastating condition that causes sensory and motor dysfunction and permanently impairs normal life. Spasticity is one of the most common complications associated with tSCI that limits independent functional abilities. Spasticity is defined as a velocity-dependent increase in muscle tone, in response to passive movement and it is accompanied by pain and stiffness. Unfortunately, current treatments provide only transient and often incomplete relief of spasticity and individuals often experience long-term adverse effects. Through a collaborative project between three labs, we aim to develop a durable non-invasive electrical stimulation treatment to alleviate spasticity. I participated in preparing the model of spasticity by performing spinal surgeries on the cervical spine of rats. To evaluate spasticity, we studied the loss of Rate-Dependent Depression (RDD) of the H-reflex which is considered the electrophysiological hallmark of spasticity. To do so, I fabricated an electrode nerve cuff that was implanted on the median nerve of the rodent’s forearm to study the H-reflex of the affected muscle in the rat’s forelimb. I then recorded and analyzed the temporal development and change of spasticity. H-reflex results validated the spasticity model by showing RDD reduction in injured rats compared to the uninjured rats. The developed treatment shows promising modulation of the H-reflex and recovery of RDD in injured animals. Additionally, to measure velocity-dependent muscle tone, we developed a robotic device that passively moves the rodent’s forearm at different velocities. Employing this robotic behavioral assessment allows me to objectively assess the effect of stimulation on spasticity in the rodent forelimb. Obtained data reveals the muscle resistance to be three times higher in the injured rodent. This novel therapeutic stimulation protocol could potentially be used for clinical use as a non-invasive therapy, to help patients with spasticity in the hand after suffering from cervical tSCI.

Kathak is a classical dance form which originated in Uttar Pradesh, North India. Kathak loosely translates to “story-teller,” and has become a symbol of Indian culture and national identity. Additionally, this dance form is very physically demanding, with skills that challenge the biomechanics of human anatomy. Therefore, the purpose of this research is to explore the cultural context of Kathak, as well as examine Kathak from a medical perspective. Specifically, I sought to examine the injuries which are common to Kathak dancers, particularly focusing on the risk factors, prevalence, and mechanisms of these injuries. The methods to investigate this research question were primarily through literature review. Kathak dancers experience injuries in the lower extremities due to high rates of twisting, jumping, and stomping in the dance form. These repetitive and compressive motions may lead to hyperpronated feet, flattened arches, and extreme dorsiflexion and plantarflexion. These foot injuries overall produce a lack of alignment in the dancer’s anatomy and can decrease proprioceptive orientation and neuromuscular function and control of the lower extremities. The outcome of this research has the potential to bridge the gap between dance and medicine. Education is powerful for dancers so that they can be aware of high-risk injuries and perform exercises as preventative efforts. Preventative efforts may include reducing static stretches and instead incorporating more dynamic stretches into their daily warm-up routine. Additionally, dancers should be empowered to take initiative of their health and seek professional guidance when necessary, and dance schools should play an active role in encouraging this.

Atherosclerosis is characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue in arterial walls, forming plaques. Plaque accumulation can lead to stenosis and potentially severe outcomes such as myocardial infarction or stroke. The gut microbiome, including Collinsella aerofaciens, is believed to play a role in the prevention or development of atherosclerosis. Gut bacteria can directly influence systemic inflammation, a factor correlated with the pathogenesis of atherosclerosis, and produce metabolites that alter the disease course. This study explores the potential link between C. aerofaciens and atherosclerosis by investigating the abundance of C. aerofaciens in the gut microbiome of individuals with and without atherosclerosis. We collected 179 stool samples from participants at the Kisumu District Hospital HIV Clinic in Kenya and conducted a comprehensive analysis of their gut microbiomes. 100 participants had carotid ultrasonography, categorized as showing atherosclerosis with visible plaque or intima medial thickness ≥ 0.7 mm. We employed bacterial 16S ribosomal RNA gene sequencing to characterize the stool microbial composition and noted that the relative abundance of C. aerofaciens was 2.6-fold less in participants with atherosclerosis (p=0.006). To validate these findings, I employed a Quantitative Polymerase Chain Reaction with a cloned plasmid control for targeted quantification of C. aerofaciens. We found 6.9-fold more C. aerofaciens copies per total 16S in Kenyan adults without atherosclerosis versus with (p=0.020). This suggests a potential protective or mitigating role for this bacterium in cardiovascular health. Future work could include assessing changes in C. aerofaciens abundance over time and its association with cardiovascular disease progression. Additionally, in vitro or preclinical studies could reveal the specific mechanisms by which C. aerofaciens influences atherosclerosis development and progression. This research contributes to our understanding of the intricate interplay between the gut microbiome and atherosclerosis, offering insights that may inform future therapeutic strategies and personalized interventions for cardiovascular diseases.

Small heat shock proteins (sHSP) are a family of molecular chaperones whose function is to delay the harmful aggregation of other proteins. Protein aggregation is associated with neurological disorders such as Alzheimer's disease and Parkinson's disease. In many tissues, multiple sHSPs are coexpressed and tend to assemble into hetero-oligomers. Hetero-oligomers are complexes of two or more different protein species. The extent and mechanism by which these hetero-oligomeric complexes form is yet to be fully understood. The goal of my discovery-driven research is to assess how the properties of sHSP hetero-oligomers differ from the properties of homo-oligomers. In my project, I focus on three sHSPs that are highly expressed in muscle: HSPB1, HSPB5, and HSPB6. Each of these proteins exhibit different behavior when on their own. HSPB1 and HSPB5 form a distribution of large homo-oligomers, whereas HSPB6 forms a small homo-dimer. One of the most characteristic properties of the small heat shock proteins is formation of oligomers that span different sizes. Thus I am primarily determining the sizes and composition of the sHSP hetero-oligomers. I performed a comprehensive study to characterize the sizes of the hetero-oligomers using three complementary methods: analytical size exclusion chromatography, mass photometry, and native gel electrophoresis. I have found that HSPB6 is able to readily incorporate into hetero-oligomers as the concentration of the other sHSP is increased, and that the complexes are formed in a distribution of intermediate sizes. I am currently working on assessing the ability of the hetero-oligomers to act as molecular chaperones by aggregation assays. I predict the hetero-oligomers will delay protein aggregation more efficiently than HSPB6 on its own. The findings of my project give insight into why sHSPs are coexpressed and form hetero-oligomers in cells. Understanding these hetero-oligomers sheds light into the complex pathways of sHSP function. 

The effective treatment of individuals with HIV relies on maintaining therapeutic drug concentrations, necessitating accurate measurement of antiretroviral (ARV) drug levels. Current methods, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), are limited by cost and accessibility. Our research addresses this gap by developing the INTEGRase activITY (INTEGRITY) assay for measuring integrase strand transfer inhibitors (INSTIs), a leading class of ARV drugs. This 2-step assay quantifies INSTIs using a DNA strand transfer reaction and quantitative polymerase chain reaction (qPCR). The presence of INSTI drugs disrupts the strand transfer reaction, inhibiting full-length target DNA formation, which is then measured through real-time qPCR. My work focused on optimizing the limit of detection of INTEGRITY by altering the strand transfer reaction conditions and protocol. Specifically, I conducted experiments altering INSTI drug concentrations and optimizing pre-incubation times of integrase with the drug to enhance the LOD. I observed that preliminary incubation of integrase and INSTI drugs for 5 minutes at 37 degrees Celsius improved the LOD of INTEGRITY by an order of magnitude. The simplicity of the INTEGRITY assay, utilizing standard laboratory equipment, holds immense promise for broadening access to routine clinic-based ARV drug level monitoring. This advancement has the potential to significantly enhance HIV care on a global scale by offering a cost-effective and accessible solution for monitoring therapeutic drug concentrations.

Treponema pallidum subspecies pallidum (T. pallidum) is growing in incidence in high-income countries like the United States and remains endemic and highly prevalent in low-income countries, primarily sub-Saharan Africa, and South America. Despite being treatable, syphilis is associated with significant fetal and perinatal mortality in low-income settings, due to congenital transmission of the infection. Improving our understanding of syphilis pathogenesis, immunology, and T. pallidum biology, could result in novel measures to curtail syphilis spread, including improved diagnostics, novel therapeutics, and a preventive vaccine. We are exploring the use of a chimeric antigen, composed of a scaffolding/carrier protein of T. pallidum and a series of protective epitopes from other T. pallidum antigens previously described and patented by our laboratory, to be used as a recombinant vaccine. The first step to using this protein as a vaccine scaffolding is determining the most immunogenic sequences of the protein to be used for replacement. Specifically, we used sera from 63 patients with syphilis at different stages, and sera longitudinally collected from rabbits infected with either the Nichols or SS14 isolates of T. pallidum, which represent the model strains for the two known clades of this pathogen. Recognized amino acid sequences were then mapped to the experimentally determined Tp17 structure. Reactive epitopes in both serum groups mapped predominantly to the α-helix preceding the Tp17 soluble β-barrel and to the loops of the barrel. We are currently using the same Enzyme-Linked Immunosorbent Assay (ELISA) to assess reactivity levels in control sera of naive human patients. These results identify sequences of the Tp17 antigen that could be replaced by protective epitopes. Our work provides the basis for future research on the use of this scaffolding to develop a syphilis vaccine able to confer protection from this this serious infection.

Syphilis remains a serious global health concern, underscoring the need for better control strategies. If left untreated, the syphilis agent, Treponema pallidum subsp. pallidum (T. pallidum), can persist for decades due to its ability to evade the host immune response. Antigenic variation of the surface-exposed outer membrane protein TprK is believed to mediate persistence. TprK contains seven discrete variable (V) regions. TprK antigenic variation occurs via non-reciprocal gene conversion between the variable regions in the tprK expression site and 53 donor cassettes (DCs). We previously engineered a T. pallidum strain impaired of antigenic variation (SS14-DC^KO) by eliminating 51 of the 53 DCs. Rabbits infected with the DC^KO strain developed an attenuated infection phenotype with a reduced burden of T. pallidum cells compared to wild type (WT). Therefore, we hypothesized that if immunosuppressed rabbits are infected with SS14-DC^KO, the disease would undergo similar progression to rabbits infected with the WT strain. In this study, two rabbit groups (n=8) were either immunosuppressed with Depomedrol or untreated. Four rabbits in each group were infected intradermally with SS14-WT or SS14-DC^KO strain on clipped backs. Sera was isolated weekly to measure antibody titers using VDRL and TPPA. In addition, DNA was isolated from lesion biopsies to perform TprK profiling. Based on TprK profiling data, synthetic peptides for the most abundant V5, V6, and V7 variants were used to measure antibody reactivity by ELISA. Antibody titers measured by TPPA and VDRL were similar between immunosuppressed rabbits regardless of infecting strain. TprK profiling shows that the most abundant V sequence at the inoculum decreases over time in lesions as humoral reactivity to these peptides increases. Overall, this research demonstrates the role that TprK plays in the persistence of T. pallidum during syphilis infection and the need for novel control strategies.

Engineered heart tissues (EHTs) have emerged as a promising tool for cardiac disease modeling and drug screening, allowing for better study of cardiovascular diseases (CVDs). However, most current EHTs are composed of only a mixture of an extracellular matrix and heart muscle cells, called cardiomyocytes (CMs), without a vascular element. This prevents the study of the impacts of flow and the endothelium on cardiac function, and the role that endothelial cell (EC) dysfunction may play in cardiovascular disease. Endothelial function is closely related to cardiac homeostasis, as risk factors for CVD (smoking, obesity, diabetes, etc.) lead to an increase in pro-inflammatory cytokines, which can trigger EC dysfunction. Thus, this interaction is important to study further. The Zheng lab has developed a perfusable collagen-based EHT model, which incorporates a vascular element. The constructs form a lumen through utilization of needles and collagen, support CMs within the bulk collagen matrix, and the inner lumen of the tube can be endothelialized, serving as an effective in vitro model of cardiac vasculature. This project aims to identify healthy and unhealthy EC flow conditions within the EHTs, hypothesizing that physiologically relevant shear stress will lead to EC alignment and strong barrier properties. . We optimized the fabrication and culture process of the EHTs by fabricating a secondary dish for the EHT constructs while they are under perfusion, in order to avoid contamination risks. We then employed this model to look at EC retention and health at different flow rates, and examined the effects of altered shear stress on EC dysfunction. ECs perfused under physiological shear stress have shown markers of healthy barrier function and alignment. This project establishes a perfusable EHT model that allows us to interrogate EC function under perfusion and, in the future, assess the effect of endothelial dysfunction on cardiac dysfunction.

Phosphoribosyl Pyrophosphate Synthetase (PRPS1) is an enzyme in the nucleotide biosynthesis pathway that makes a molecule necessary for de novo nucleotide synthesis. It is known that PRPS1 protein hexamers can stack into linear filaments in the presence of ADP and phosphate. When these filaments are broken, catalytic activity is lost, and it is hypothesized that enzyme inhibition is lost as well. Mutations in PRPS1 lead to a wide spectrum of diseases in humans. In addition, changes in cell regulation of the enzyme have been linked to cancer. Motivated by research that connects PRPS1 phosphorylation to increased cancer proliferation, my project investigates the effects of phosphorylation on PRPS1 structure, enzyme activity, and inhibition properties. I have transformed plasmid DNA containing the PRPS1 phosphomimetic mutations S47E, S103D, and S308E into E. coli strains BL21 and pLysS. I then grew overnight bacterial cultures and induced protein expression using IPTG. After verifying protein expression with gel electrophoresis, I purified the protein from bacteria using nickel resin affinity and size exclusion chromatography. Having made and purified protein mutations that mimic phosphorylation, I conducted a negative stain screen to analyze filament formation trends. This has yielded preliminary findings that S47E and S103D phosphorylation mutations of PRPS1 break enzyme filament formation. Variation in filament formation between mutations points to the importance of phosphorylation location and its potential impact on enzyme activity and inhibition. To assess the catalysis of the phosphomimetic mutations in PRPS1, I will conduct biochemical assays which measure the activity and inhibition of the enzyme. Through these ongoing experiments we will learn how phosphorylation modifies PRPS assembly and activity and the implications of PRPS1 dysregulation in cancer proliferation.

Transportation between the Endoplasmic Reticulum (ER) and Golgi is the first step of the secretory pathway, essential for correctly localizing intracellular proteins. USO1 is a long tethering protein between the ER and Golgi. USO1 acts as the first contact between transport vesicles and the Golgi initiating the transport process. Deletion of USO1 is lethal. YPT1 is a GTPase that is needed to recruit USO1 to these membranes. It is believed that physical interaction between YPT1 and USO1 is required for this transport function. Using AlphaFold2 predictions, we identified potential binding sites of YPT1 on USO1. If those sites are mutated, how would it affect the cell? I mutated sites proposed to bind to YPT1 on USO1 by Alphafold2 in order to break this interaction. Using yeast, I plan to determine if these mutation sites break the physical interaction. The resulting mutants cannot grow at elevated temperature. Further experiments will test how important these sites are in the overall process of protein secretion.

The nucleolus is an essential subnuclear organelle that performs central regulatory roles in cellular metabolism, epigenetic programming, and stress signaling. In mammals, nucleoli are disassembled and rebuilt de novo with each cell division, through an elaborate assembly mechanism that has long eluded molecular characterization. This assembly process is spatiotemporally controlled by a long noncoding RNA termed the 47S pre-ribosomal RNA (47S pre-rRNA), which initiates nucleolar assembly at the site of its transcription, and for which continued expression is required to maintain nucleolar integrity. Yet, while the 47S’ roles in nucleating and scaffolding nucleolar architecture are well established cytologically (they were first observed nearly a century ago), the structural elements on the 47S that enable these architectural functions remain unknown. I hypothesize that an RNA domain within the 47S, termed the 5´–External Transcribed Spacer (5´–ETS), harbors the long-sought structural scaffolds of the nucleolus. To test this, I am implementing a live-cell reporter assay that will monitor, in real time, if transcripts derived from the 5´–ETS drive nucleolar localization and architecture. My approach leverages recent advancements in artificial gene synthesis and live-cell RNA imaging. A novel drug-inducible promoter will enable me to temporally control expression of 5´–ETS sequence variants in live cells. I will monitor the kinetics and efficiency with which these transcripts localize into the nucleolus by two-color live cell imaging, using the newly discovered fluorescent RNA aptamer RhoBAST, and a fluorescently tagged nucleolar marker protein. To design our negative controls, I implemented a bioinformatic pipeline that generates scrambles of long, low-complexity RNA sequences—ablating primary structure but preserving dinucleotide content. This allows us to investigate whether nucleotide composition or sequence affects nucleolar formation. We anticipate that this powerful system will set the stage for detailed molecular characterization studies, revealing the long-elusive molecular interactions that control nucleolar architecture in health and disease.

Kaposi’s Sarcoma (KS) is among the most common tumors in central Africa and is a prevalent AIDS-associated malignancy. Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of KS. While all herpesviruses are capable of both lytic and latent replication programs, KSHV is predominantly in the latent state in the main KS tumor cell, the spindle cell - a cell expressing markers of the endothelium. There is limited viral gene expression during latency so it is difficult to target the virus directly. Therefore, our approach is to target host cellular requirements for KSHV latent infection. Previously, the Lagunoff Lab performed a genome wide CRISPR-Cas9 screen targeting over 18,000 human genes to identify cellular genes essential only to cells latently infected with KSHV. ACADS and CHD1 are two genes identified as some of the top hits from the screen. ACADS encodes a tetrameric mitochondrial flavoprotein, which catalyzes the first step of mitochondrial beta-oxidation. CHD1, or chromodomain helicase DNA binding protein 1 alters gene expression by chromatin modification. I hypothesize that ACADS and CHD1 are required for survival of latently infected KSHV cells but not uninfected cells. To test this hypothesis, I created knockout tert-immortalized microvascular endothelial (TIME) cells of each gene with CRISPR-Cas9 and plasmids containing guide RNAs used in the original screen. Then, I infected control and knockout cells with either KSHV or mock, and conducted trypan blue assay at 72 hours post infection to measure percent of live cells. Preliminary data suggests an increase in cell death for KSHV infected ACADS knockout cells compared to the control cells. In future experiments, I expect a significant decrease in the percentage of live cells in the ACADs and CHD1 knockout cells compared to the control and uninfected cells.

A key neuromodulatory system involved in anxiety disorders is the locus coeruleus noradrenergic system (LC-NE), which projects broadly throughout the central nervous system. The LC is stress responsive and tonic activation of the LC and its projections to the BLA is anxiogenic. Previously, the Bruchas Lab has used two-photon calcium imaging to show that a powerful stressor (predator odor) increased synchronous activity of LC neurons. They also found that mimicking this predator odor evoked activity with optogenetics altered the activity of individual neurons downstream in the BLA in a β-adrenergic receptor (β-AR) dependent manner. Although these data support the LC's involvement in promoting aversion and increasing anxiety-like behavior, the specific neurotransmitter, neuronal cell types, and receptors responsible for these effects remain unidentified. Therefore in hopes of identifying these specific signaling molecules and neuronal cell types and receptors, I first used fiber photometry and a novel biosensor (GRABNE2m) to detect norepinephrine (NE) release in the BLA while mice were exposed to a predator odor. I found that predator odor produced robust increases in NE release in the BLA compared to control odor (n=5, 3 male, 2 female) Further, we found that optogenetic activation of terminals from the LC to the BLA produced very similar levels of NE release compared to what was evoked by predator odor. To determine the cell type and receptor that is sensing this stress-induced NE release, I used a CRISPR/SaCas9 virus, developed in collaboration with Dr. Larry Zweifel’s lab, to knock-down β2-adrenergic receptors (β2-ARs) in glutamatergic BLA neurons to test their causal role in stress-induced anxiety-like behavior. CRISPR knockdown of β2-ARs in the BLA blocked several stress-induced anxiety-like behaviors (n=4, 4 female). By understanding the circuit-based mechanisms of how stress-induced anxiety is regulated, researchers could identify potential targets for therapeutic treatments of anxiety disorders.

Cannabidiol (CBD), a non-psychoactive cannabinoid compound found in cannabis, has been reported to attenuate morphine tolerance and can potentially be used as an alternative to opioids in treating chronic pain. Previous work has established connections between morphine tolerance and Reactive Oxygen Species (ROS) production through JNK-mediated Peroxiredoxin 6 (PRDX6) activation. Excess ROS production promotes desensitization of opioid receptors, which in turn leads to opioid tolerance. CBD administration is associated with decreasing pain-related Reactive Oxygen Species (ROS) production, and it was hypothesized that CBD directly interacts with JNK, blocking JNK’s activities. This project aims to investigate the connections between CBD administration and ROS production to determine CBD’s effects on JNK-mediated ROS production. To quantify ROS production through fluorescence imaging, I will transfect wild-type HEK 293 cells with oROS, a genetically encoded sensor, which fluoresces proportionally to ROS production. Coverslips of HEK 293 cells expressing oROS are treated with buffer (control) and CBD before administration of Tumor Necrosis Factor alpha (TNFα), a known activator for JNK released during pain states. After imaging with oROS, I will quantify ROS production and compare this between groups with and without CBD pretreatment to determine CBD’s activity on inhibiting JNK-mediated pro-inflammatory pathways. I predict that relative to the control, cells treated with CBD will have significantly less ROS production. If the results are consistent with this prediction, CBD could be a potentially promising co-treatment with opioids in managing chronic pain as it can potentially attenuate opioids' side effects like tolerance. 

Eukaryotic DNA is wrapped around nucleosomes to be packaged into a cell’s nucleus. Nucleosomes are made up of a combination of canonical histones (H2A, H2B, H3, and H4) and histone variants. Histone variants are evolutionarily derived from their canonical counterparts and can replace canonical histones to perform specialized chromatin functions. Given their crucial and widespread functions, mutations of histone genes are correlated with poor prognosis in cancers. 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. Here, we study two such changes in the H2A repertoires of budding yeast and fruit flies. Most eukaryotes including humans have an H2A repertoire of canonical H2A and two variants– H2A.X important for DNA damage response (DDR) and H2A.Z essential for gene regulation. However, in yeast, H2A.X entirely replaced H2A, and in flies, H2A.X and H2A.Z are fused into a single variant H2Av. We take two approaches to discover the adaptive advantages of these changes. First, we are studying the evolutionary origins and diversification of H2A repertoires in yeast. We find that many basally branching fungi have a canonical H2A, suggesting that only some yeast have lost canonical H2A. Second, we are recreating the fly and human H2A repertoire in S. cerevisiae. We find that while yeast with a fly-like H2A repertoire have a DDR, it is dramatically reduced compared to wild-type yeast. This raises the intriguing possibility that a fly-like repertoire might lead to a trade-off of compromised DDR in fly genomes. We are now analyzing how DDR-dependent processes like meiosis are altered in fly-like yeast. By leveraging the power of evolution and yeast genetics our work will reveal the biological consequences of unexpected histone innovation.

Cryo-electron microscopy (Cryo-EM) plays a crucial role in macromolecular complex structure analysis, which helps to understand the cellular mechanism and pathologies to facilitate drug discovery and delivery. However, due to factors such as the target conformational change, damage during imaging, or improper sample preparation, low-resolution maps could be reconstructed and pose challenges to the structural analysis. Prior studies have employed different generative deep learning models to enhance the resolution or readability, performing well most on medium-resolution maps but failing to operate with even lower ones. Here, we proposed another modified diffusion model to enhance the cryo-EM density map resolution, expanding the input range of up to 10 Å. Based on the latent diffusion models, we enable it to work directly in 3D density maps with the conditioning factor to utilize the local resolution estimation rather than a fixed global resolution value for the attention mechanism. Compared with the previous work, the conditioning factor further improves the model's stability and robustness, especially in cases where the structure undergoes conformational changes. Our ultimate objective is to provide an improved and efficient solution for cryo-EM density map analysis, enhancing the process's accuracy and efficiency.

Traditionally known for its toxicity, hydrogen sulfide (H₂S) also possesses physiological roles as an endogenous gaseous signaling molecule in multiple biological processes. Previous research has demonstrated changes in levels of H₂S-producing enzymes during oxidative stress, hypoxia, and inflammation in various tissues including the liver and heart. H₂S protects cells from cytotoxicity in part by promoting the synthesis of glutathione, neutralizing reactive oxygen species, and inhibiting apoptosis signaling pathways. Thiol methyltransferases TMT1A and TMT1B can methylate endogenous H₂S to methanethiol. TMT1B has been shown to have a potential role in mediating the toxic effects of methanethiol. Gene silencing of TMT1B was found to significantly alleviate the observed cytotoxicity induced by methanethiol in human bronchial epithelial cells (16HBE). Methanethiol may induce harm to human respiratory tract cells, and understanding the mechanisms involved, including the role of TMT1B, could potentially lead to insights for mitigating these harmful effects. Doxorubicin (Dox) is a widely used chemotherapy drug for the treatment of various cancers but can induce oxidative stress in cells. In my preliminary experiments, I assessed the cell viability of HepG2 liver cells that were supplemented with various concentrations of NaSH (H₂S donor) and sodium methanethiolate (NaSMe, MeSH donor), followed by Dox treatment. Supplementing the cells with H₂S significantly increased cell viability in the presence of doxorubicin, while the methanethiol had no effect. The goal of my project is to identify H₂S-dependent protective pathways during cellular stress in HepG2 versus cardiomyocytes. My preliminary data indicates that both H₂S and its metabolite, methanethiol, may alter cellular responses following treatment of exogenous compounds that induce cellular stress such as CoCl₂, hydrogen peroxide and Dox. My goal is to pinpoint genes altered during the stress response. This understanding of H₂S-dependent pathways may pave the way for designing novel therapeutics that maintain or enhance H₂S levels.

Human Immunodeficiency Virus (HIV) is a rapidly evolving pathogen with no effective vaccine for eliciting broad protection against HIV infection. The HIV Envelope protein (Env) is a trimeric glycoprotein that is responsible for host-cell membrane fusion and infection initiation. As the only protein on the HIV virion surface, Env is the sole target for neutralizing antibodies. Characterizing the local structural dynamics of Env provides valuable insight into HIV host-virus interaction mechanisms. HDX-MS is an excellent tool for determining structural dynamics by measuring local backbone amide solvent accessibility. Generally, less structured protein regions uptake deuterium more rapidly compared to buried regions or those that are stabilized by secondary structure. We can use mass spectrometry to measure the kinetics of deuterium uptake for peptides throughout the Env protein. HDX-MS provides a detailed portrait of local structural dynamics and order, effectively identifying switching between completely closed prefusion and more open conformational states. A particular HIV Env isolate, A4, is of interest due to its unusually dynamic nature compared to other well-studied Env isolates, such as BG505. Dynamic Env exhibit more conformational flexibility, allowing them to sample various intermediary conformations between open and closed. We hypothesize that this attribute could increase HIV resistance to broadly neutralizing antibodies (bnAbs) that selectively target the closed Env conformation to prevent virus entry in immune cells. We may be able to correlate antibody binding to local dynamics measured in A4 versus BG505 Env trimers to verify this hypothesis. Biolayer interferometry will be applied to quantify antibody association and dissociation rates, as well as binding affinities. These studies will advance existing knowledge in Env-based vaccine therapeutics to improve immune responses to HIV.

Oxygen deficient zones (ODZs) are large, naturally-occurring regions of the world's oceans where dissolved oxygen concentrations drop to low levels—less than 10 nM. These regions are important to the biogeochemical cycling of carbon, nitrogen, methane, and sulfur and are expected to expand as ocean temperatures rise due to anthropogenic climate change. Located above and below the anoxic ODZ core are oxyclines where dissolved oxygen concentrations change rapidly with depth. The deep oxycline extends into the deep ocean supporting an understudied microbial community adapted to these low-oxygen conditions. In this study, I examine a metagenomic library previously generated from a water sample collected at 1000 meters on the RR1804 POMZ cruise to determine both the diversity and genetic potential of microbes in the deep oxycline. I found that three groups of prokaryotes dominate in the deep oxycline: the cosmopolitan alphaproteobacteria, Pelagibacter ubique (20%); the uncultured candidate phylum SAR324 (16%); and archaea of the phylum Thaumarchaea (12%). I generated metagenome-assembled genomes (MAGs) from this sample to determine the genetic potential of this microbial assemblage. By examining these MAGs, I expect to find genes encoding for processes such as low-oxygen stress responses, alternate terminal electron acceptors, and carbon-fixation pathways. By better understanding the contributions of the deep oxycline microbial community to biogeochemical cycles, we can more accurately predict how nutrients will be consumed and regenerated in ODZs as they expand.

Benzalkonium Chlorides (BACs) are widely used as an antimicrobial disinfectant in a variety of food and consumer goods processing. Exposure to BACs has increased significantly due to the COVID-19 pandemic. BACs have been reported in common foods like fruits, milk, and other dairy products, raising concerns about the impact of BACs on human health via oral exposure. Recent work in our lab has reported that BACs are metabolized by cytochrome P450 (CYPs) 4Fs and 2D6 in the liver. However, there is a gap in knowledge regarding how BACs and BAC metabolites are distributed throughout the body, post-oral exposure. We hypothesize that insight into BAC disposition and distribution following an oral exposure route could lead to valuable knowledge of BAC accumulation and subsequent toxicity. In this study, we exposed male and female C57BL/6 mice to deuterated C12- and C16-BACs at 120 μg/g/day for one week via a gel food diet. We harvested liver, lung, heart, spleen, and intestinal section tissues at the end of the study, as well as fecal samples at two time points, and a singular urine time point. Through a targeted BAC and BAC metabolite quantitation analysis using liquid chromatography-mass spectrometry, we found omega-oxidation of the alkyl chain to carboxylic acid followed by beta-oxidation to be a major route of metabolism. Additionally, we found that the liver and big intestine had a higher metabolizing capacity than other tissues and the C16 BACs were preferentially metabolized compared to the C12 BACs. This work provided a deeper look into the disposition and metabolism of BACs and revealed organs that are susceptible to BAC exposure for future studies

An important and universal aspect of cancer cells is the ability to proliferate rapidly. Rapid proliferation imposes specific metabolic demands which are often targeted for cancer therapies, and yet these demands are not well understood. A crucial aspect of cell metabolism is from the Tricarboxylic Acid (TCA) cycle. The TCA cycle is amphibolic, both catabolic and anabolic, and disruptions in the cycle are implicated in the onset and progression of various human cancers. Succinate Dehydrogenase (SDH) and Fumarate Hydratase (FH) are two TCA cycle enzymes that are tumor suppressors, proteins that when lost contribute to the malignant phenotype. In the TCA cycle, SDH catalyzes the conversion of succinate to fumarate, and FH catalyzes the subsequent step of fumarate to malate. Due to this proximity, one may predict SDH and FH mutations would have similar metabolic effects. However, this prediction, surprisingly, does not hold true. Paradoxically, loss of SDH generally impairs cell proliferation by disrupting synthesis of the amino acid Aspartate, a crucial output of mitochondrial respiration. Our lab recently discovered that SDH-deficient cancer cells adapt to overcome this metabolic deficiency by downregulating Complex I of the Electron Transport Chain (ETC). By downregulating Complex I, SDH-null cells increase the capacity of alternative aspartate synthesis pathways aside from the usual TCA cycle dependent pathway to enable faster proliferation. For SDH-null cells, treatment of a Complex I inhibitor improves proliferation for reasons discussed above. However, when FH-null cells are treated with the same Complex I inhibitor, there is a decrease in proliferation rate. It is not well understood why this difference exists, but characterizing it can provide insights on the roles of these enzymes and could inform better treatments for SDH and FH linked cancers.

Asthma exacerbations often begin and increase in severity at night. Though animal models have shown molecular circadian rhythm involvement in immune and inflammatory responses, little is known about how circadian rhythms impact responses in humans or diseases such as asthma. BMAL1/ARNTL, CRY1, NR1D1, and PER2 are the genes that form the “cellular clock” by which cells tell time. Our hypothesis is that core circadian gene expression is maintained in an expected, rhythmic manner in epithelial cells from donors with asthma. We use an ex vivo model with human airway epithelial cells cultured at an air-liquid interface in a temperature cycled incubator to mimic the epithelia of the human airway. After temperature cycling to synchronize cellular circadian cycles, RNA collection occurs every four hours over a 48-hour period. After RNA isolation, I perform reverse-transcriptase quantitative polymerase chain (RT-qPCR) on a planned eight donor lines (4 healthy/4 asthmatic) to measure the gene expression of the four clock forming genes. In three asthmatic donor lines, I have found that core circadian rhythmicity is maintained in asthmatic epithelial cells and resembles the circadian rhythm expression in eight healthy donor lines previously analyzed. Shown through a preliminary study conducted by the lab, genes linked to asthma in the IL-17 signaling pathway have altered circadian rhythms of gene expression. In the future, I will use qPCR to study immune and inflammatory genes to confirm the altered rhythmicity across a wider scope of donor lines. In addition, I will analyze gene expression in different subsets of asthma to investigate whether altered circadian regulation contributes to asthma subtypes, such as T2-low which has been linked to IL-17 signaling pathway dysregulation. Investigating the differences in asthma-related circadian gene expression is essential to the development of chronotherapeutics – therapies that take into account time of day.

El Niño is an atmospheric-oceanic phenomenon characterized by the periodic warming of the sea surface in the eastern equatorial Pacific. Profiling data from Argo floats in the eastern equatorial Pacific is used for this research. An Argo float is an underwater profiling technology that can record and transmit real-time data of various ocean parameters at different depths. This technology supports the analysis of temperature, salinity anomalies, and other nutrients. In addition, a numerical model will be developed to simulate the progression of El Niño and evaluate its regional oceanic impacts. With both observational data and modeling output, this research aims to enhance the understanding of the dynamics of El Niño-induced impacts on oceanic parameters at a broader global scale. Based on the current data, I have discovered a clear variation in temperature and salinity according to the annual average. The El Niño Southern Oscillation (ENSO) indicator also suggests that the 2023-2024 El Niño is very strong and still in its development phase.
 

Children diagnosed with Autism Spectrum Disorder (ASD), often experience co-occurring language impairments, including grammatical and lexical difficulties. Word segmentation, or the ability to identify word boundaries in continuous speech, is done through statistical learning and identification of speech cues. Recent studies in typically developing infants have shown a linear relationship between the ability to recognize words from continuous speech and the size of an individual's lexicon. The neural activity of the cerebral cortex, specifically the delta frequency band (1-4 Hz), contains the time scale of words and phrases. The delta band was found to track speech rhythm, along with semantic dissimilarity between successive words. Extracting acoustic features from sound signals and their linguistic representations (syllables, words, and phrases) is essential for speech comprehension. This study aims to look at the relationship between delta power recorded via scalp electroencephalogram (EEG) and communication skills in children with and without ASD. 193 participants (ASD = 96) from the NIH study on sex differences in autism were included in the sample. EEG recordings were collected while participants listened to phonemes that were statistically presented to result in 4 learned nonsense words. Parents completed a semi-structured interview on their child’s communication skills. EEG delta power was calculated over frontal, central and posterior regions of interest (ROIs). We expect children with ASD (compared to typically developing children) to have decreased delta power during nonsense word perception and lower delta power will be related to lower communication ability. This study will provide insight into the relation between neural perception of language and verbal communication in children with ASD.

Since their introduction to clean energy applications, organic-inorganic lead halide perovskites have received great attention for their potential to create highly efficient, manufacturable and cheap solar cell devices. To make effective perovskite solar cells, charge transport layers are used to remove electrons and holes from the bulk perovskite semiconductor, increasing current, voltage and power conversion efficiency. Phosphonic acid self-assembled monolayers (SAMs) are a common hole transport layer. The phosphonic acid binds to the transparent conductive oxide electrode while an organic head group forms the SAM/perovskite interface. This head group is key for charge transfer and voltage characteristics, but the structure-function relationship is still poorly understood. My project investigates the role that deposition techniques and electronic structure play in the optimization of this SAM/perovskite interface. Expanding from the standard two step spincoating SAM/perovskite deposition method, I explored whether the codeposition of the two layers or the addition of a SAM solvent wash step produced an improved interface. I also fabricated films using several different SAM compositions to test for performance trends and improvements compared to the current field standard SAM, Me-4PACz. I collected photoluminescence lifetimes, quantum yields and solar simulation measurements to evaluate film performance. Preliminary data shows that neither the washing step nor codeposition add any performance benefit, but the single step codeposition achieves a more streamlined manufacturing method. Two of the new experimental SAMs performed comparably to Me-4PACz. These results encourage codeposition of the SAM/perovskite interface as the most efficient method to create high quality devices and show promising alternatives to the industry standard Me-4PACz SAM.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease. Traditional therapies focus on treating the symptoms of the disease and do not directly treat the underlying functional changes. Myosin modulators are a novel class of pharmaceutical agents designed to treat patients with cardiomyopathies by directly modulating cardiac myosin function in the sarcomere. Compounds including Mavacamten (Mava) and Aficamten (Afi) reduce myosin function, measured through its ATPase activity. In this study, I investigate how these small molecules affect the multiple turnover kinetics of the ATPase cycle. Porcine cardiac heavy meromyosin (pcHMM) is rapidly mixed with a two-times excess of fluorescently labeled ATP (mant.ATP) in the presence of actin and fluorescence is measured over time. Calculating the length of time, tau (τ), until the fluorescence has returned to 50% of the peak value estimates the time taken to hydrolyze all the mant.ATP, and is used to calculate the rate constant of ATP hydrolysis (kcat). Both Afi and Mava increase τ and decrease kcat, while a lower concentration of Mava is required to reach similar inhibition as Afi. I also utilize the In Vitro Motility assay to measure the ability of myosin to move actin and compare the effects of Mava and Afi with stopped-flow data. Preliminary results indicate that both Mava and Afi inhibit actin filament velocity, with Mava requiring a lower concentration, similar to stopped-flow. I will discuss how each modulator affects ATP turnover with a direct effect on catalytic activity and extend those results to the functional consequences of these myosin inhibitors. Insight into the impact of these myosin inhibitors on the myosin actin cross-bridge cycle will help provide tailored treatments to patients who are impacted by HCM.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the deadliest pathogens in human history. The aim of this project is to begin probing the metabolic reprogramming that occurs in Mtb during treatment with isoniazid (INH), a commonly used frontline drug. Unpublished work from a collaborator has identified several common metabolites that change the minimum inhibitory concentration of INH needed to kill 99% of cells in a culture. The amino acid serine -- which is well known to be involved in one-carbon metabolism through interconversion with glycine -- was found to be of particular interest due to the increase in Mtb susceptibility to INH observed in serine-supplemented culture. To further explore this effect, I have generated timecourse growth curves using an avirulent Mtb (aMtb) model while under INH treatment with and without serine. I started cultures of aMtb at log-phase growth under the following conditions: without any additives, with serine, with INH, and with both INH and serine. I then plated aliquots of defined volumes on solid media to observe colony forming units (CFU) that assess how many cells were present in each culture. I repeated CFU plating 4 and 7 days after each culture was created, which gave me sufficient data to analyze the growth patterns of aMtb under these various conditions. My findings require validation but suggest serine supplementation may play a role in lowering INH tolerance in Mtb. If this result is found to be accurate, this may implicate one-carbon metabolism as a pathway with downstream effects on Mtb tolerance to INH.

The notochord is a rod-like embryonic structure that plays a critical role in transmitting spatial cues to surrounding tissues, serving as the defining feature of chordates and a vital component of vertebrate embryonic development. Understanding the molecular and cellular mechanisms that govern notochord formation and maintenance is a fundamental knowledge gap in developmental biology. Our lab has previously identified wnt16 to be the second wnt gene expressed in the developing notochord in vertebrates and have found that wnt16^w1001/w1001 zebrafish mutant embryos exhibit significant reductions in notochord height, length, and notochordal vacuolated cell size. The fluid-filled vacuolated cells are osmotically active structures that experience osmotic pressure and rapidly inflate along the confines of the notochord sheath, contributing to embryonic body axis development. Glycosaminoglycans (GAGs) are a family of complex polysaccharides that are predicted to play an important role in vacuolated cell and notochord development due to their regulation of cellular tonic conditions and generation of hypotonic/hypertonic stress. Our study aims to establish potential mechanisms for the maintenance of vacuole cells and notochord development, which we hypothesize to act through the crosstalk between the Wnt16 signaling and GAG biosynthetic pathways. To test this, I performed single-cell RNA sequencing analysis on a zebrafish notochord-specific cell population, identifying five GAG biosynthetic genes expressed in clusters alongside wnt16. By determining expression profiles of these GAG genes in wild-type and wnt16^w1001/w1001 mutants, I aim to uncover the underlying mechanisms of the previously observed notochord phenotype. Furthermore, the notochord directly contributes to spine development, persisting within the nucleus pulposus, a structure entrapped in developing intervertebral discs (IVD) and predicted to be important in maintaining IVD health. As such, the implications of our study extend beyond the bench and may contribute to advancements in treating spinal diseases, particularly intervertebral disc degeneration (IVDD).

Diffusible iodine-based contrast-enhanced micro-CT (diceCT) is a technique used to image soft tissue specimens using 3D x-ray microscopy. Staining soft tissues with iodine (I2KI) solution prior to scanning improves contrast for detailed visualization of internal organs, but iodine staining is also associated with tissue shrinkage which can interfere with quantitative analysis. It has been shown that stabilizing soft tissue with hydrogel can reduce shrinkage. We adopted these protocols for our lab, but, despite using hydrogel stabilization, we observed wrinkles in the external surfaces of E15.5 mouse embryos, qualitative evidence of considerable shrinkage. To quantitatively test for shrinkage, we compared the crown rump lengths (CRL) of mouse embryos measured from photos taken prior to the scanning process and then from diceCT scans. CRLs ranged from 12.4 to 20.0 mm in photos and 11.1 to 16.8 mm in scans. An average reduction of 12% resulted from the specimen preparation process and confirmed tissue shrinkage. Furthermore, the amount of shrinkage was not uniform across the specimens, complicating quantitative analysis based on diceCT. Our first hypothesis was that the iodine solution used to prepare the specimens was too acidic. We measured the pH of this solution and found a range from 4.5 - 6.4. To examine if a neutral pH reduced tissue shrinkage, we prepared specimens with a buffered iodine solution (pH 7.2). DiceCT scans of embryos in buffered iodine solution did not show reduced shrinkage compared to controls in the original solution. Further investigations will focus on other potential sources of shrinkage including the pH of other solutions and the time specimens spend in each step of the protocol. Continuing to investigate sources of tissue shrinkage in diceCT can lead to additional methods for shrinkage reduction, supporting more accurate quantitative analysis of diceCT.

Marine microbial communities produce and cycle organic matter in the ocean. Some of this organic matter is in the form of metabolites, small, organic biomolecules that are present both inside of microbial cells and dissolved in seawater. Studying metabolite dynamics provides insights into the fate of a significant portion of marine primary production as well as microbial community interactions that influence short and long term carbon storage in the ocean. Here I analyzed both dissolved (extracellular) and particulate (intracellular) metabolites from the 2019 Gradients 3 (G3) research cruise that were collected along a latitudinal transect. Sampling stations spanned the North Pacific Subtropical Gyre, an area with low nutrients and primary productivity, to the North Pacific Subpolar Gyre, an area with higher nutrients and primary productivity. I extracted particulate metabolites using a modified Bligh and Dyer extraction and dissolved metabolites using cation-exchange solid phase extraction. I acquired metabolite data using liquid chromatography mass spectrometry and processed the data using Skyline software. In both dissolved and particulate samples, metabolite pools were dominated by compatible solutes, compounds organisms use for handling osmotic stress, as well as amino acids. Metabolite abundances in both phases largely increased traveling northwards along the transect, reflecting increases in productivity, microbial biomass, and nutrients. However, certain metabolite concentrations did not follow this trend, suggesting that differences in microbial community composition or physiology may play an important role in regulating the synthesis of these compounds. These results show how varying environmental conditions affect the composition of organic matter produced by marine microbial communities. This information can be used in to predict how marine primary producers will store and use carbon in a future changed ocean.

Hypoxia-ischemia (HI), or brain injury caused by a lack of blood flow and oxygen to the brain, is a leading cause of infant mortality and morbidity. Contrasting between ages, the effects of HI tend to be more severe in younger neonates. Curcumin, a dietary compound derived from turmeric, exhibits anti-inflammatory, antioxidant, and antiapoptotic properties, but is not bioavailable in molecular form, thus may serve as a neuroprotective treatment when loaded into synthetic nanoparticles to allow for effective absorption and crossing of the blood-brain barrier, forming the treatment NanoCurc. Gestational ages of 37 weeks through 42 weeks are all considered term neonates, yet their brain continues to develop and differ significantly in response to treatments against HI. Using the rat Vannucci model of unilateral hypoxic-ischemic brain injury, we studied the in vivo effects of NanoCurc in neuroprotection, in P7, P10, and P13 rats, equivalent to 34, 38, and 42 weeks’ gestation, respectively. Tissue is collected 72 hours after unilateral carotid artery ligation surgery, followed by tissue staining and analyzed by tracing the healthy tissue versus damaged tissue, to calculate the average percent area loss in treated and untreated rats. I hypothesize that in all ages, neonatal rats treated with NanoCurc will have lower injury in comparison to those treated with saline (vehicle), while the treatment will be more effective in younger rats in comparison to older ages. In the future, NanoCurc treatment may be used as a neuroprotective agent in reducing the effect of HI in preterm and term infants. If NanoCurc provides a stronger neuroprotective effect in the younger population, it may serve to target infants most severely affected by HI, potentially creating personalized treatment for gestational ages.

Autism Spectrum Disorder (ASD) is a neurological disorder that affects social and behavioral development. Although there are no known biological markers of ASD, low birth weight and presence of an older sibling with an ASD diagnosis have been identified as elevating risk for ASD, such that between 7 to 15% of low-birth-weight infants and 20% of infant siblings will be diagnosed with ASD. One confounds to interpretation of outcome is use of clinician observational measures versus parent reports of behavior. For example, parent and clinician ratings showed strong correlation for motor skills but weak to moderate for communication skills. As altered communication skills are “red-flags'' for autism, understanding the relationship between sources of information is important. This project looks at the concordance between clinician administered and parent completed measures of communication in a longitudinal study of infants at elevated risk for ASD. Fourty-nine participants from an NIH funded longitudinal study of social and nonsocial development from 6 - 36 months were included. Infants were grouped as typical-likelihood for ASD (TL, n=24), or at elevated-risk for ASD, including, low birth weight (ER-LBW, n=17), and infant sibling of children with ASD (ER-Sib, n=8). At 12 and 24 months of age, parents completed the Vineland Adaptive Behavior Scales (VABS), a clinician administered parent interview that includes expressive language (EL) and receptive language (RL); and clinicians completed the Mullen Scales of Early Learning (MSEL) with the infant to assess EL and RL. We expect ER group to have the highest concordance between parent and clinician report and EL concordance to be higher than RL because it is easier for parents to assess their child’s spoken language compared to language understanding. If parent rating shows high concordance with clinician rating, this could inform the use of lower cost questionnaires for screening and diagnosis.

Prostate cancer is the most common type of cancer amongst men in the U.S. It relies on androgens that bind to the androgen receptor (AR), which increases the transcription of genes associated with the growth and proliferation of the prostate cells. For the AR-driven prostate cancer (ARPC), current treatments involve decreasing androgen levels (Androgen Deprivation Therapy) or inhibiting the ARPI (Androgen Receptor Pathway Inhibitors). However, around 15% of patients develop resistance to these treatments, resulting in a type of prostate cancer called neuroendocrine prostate cancer (NEPC). NEPC cells are no longer dependent on AR activity, which makes this subtype difficult to treat with the current treatment options in the clinic. To better understand the biology of NEPC, we focused on gene expression at the protein synthesis level and found that NEPC has a decreased level of a tRNA called Arg-TCT-1-1. Following Arg-TCT-1-1 tRNA overexpression in NEPC, we detected elevated expression of AR downstream targets via qPCR and western blot. NEPC with high Arg-TCT-1-1 also responded to an AR inhibitor called enzalutamide as measured by cell viability assays. To further investigate the role of Arg-TCT-1-1 in prostate cancer, we used shRNA-mediated knockdown of this tRNA in prostate cancer cells with high AR expression and measured changes in gene expression. This study will provide important insights on the role of Arg-TCT-1-1 during the differentiation process from ARPC to NEPC.

Bioconjugation, or the covalent linkage between a biomolecule and another chemical group, creates hybrid "conjugates" that exhibit the properties of both biomolecules and exogenous moieties. The N-termini of proteins often fall outside of their final fold, making the N-terminus an optimal site for conjugation while preserving a protein’s native folding and bioactivity. Consequently, N-terminal modification of proteins and peptides has been a long-standing goal in fields like drug delivery, biotherapeutics, and cellular imaging. However, the current techniques for N-terminal protein conjugation are limited by either the introduction of bulky protein assemblies at the conjugation site, the need for multiple costly and complicated steps, or low site selectivity. In this project, we aimed to develop an improved route for N-terminal bioconjugation. We created a generalizable platform for single-step purification and near-scarless N-terminal bioconjugation of proteins by leveraging the chemistry of the atypically split intein VidaL. To evaluate the effectiveness of our platform, we first examined the kinetics and reaction conditions of VidaL bioconjugation, confirming its ability to modify the N-termini of proteins successfully and selectively. Then, we used our platform to conjugate an alkyne, biotin, or FAM-biotin moiety to the N-termini of fluorescent proteins (EGFP and mCherry), a model enzyme (beta-lactamase), and a model growth factor (EGF). Through measuring fluorescence and conducting nitrocefin and proliferation assays, I found that, regardless of the moiety added, bioconjugation did not impact the native function or activity of these proteins. In the future, we expect that this platform's ability to easily N-terminally bioconjugate proteins with minimal impact on their functionality will find use across the growing fields of applied chemical biology.

Pregnant individuals infected with influenza A viruses (IAV) have higher risks of mortality, hospitalization, preterm birth, and stillbirth. The objective was to determine how the transcriptional program induced by IAV infection in the lung differs between pregnant and non-pregnant states. I hypothesized that a cluster of genes linked to aggravation of influenza disease would be upregulated in the pregnant lung early in IAV infection versus the non-pregnant lung. We used a non-human primate model [NHP; pregnant (N=10), non-pregnant (N=10); Macaca nemestrina, pigtail macaque] to investigate the transcriptional response in the lung of pregnant versus non-pregnant NHPs infected with the IAV CA/04/2009 (H1N1) strain. Maternal lung tissues were collected from the animals at necropsy 5 days after infection. mRNA-Seq was performed by first extracting mRNA from tissues, preparing mRNA libraries, and aligning raw sequencing data, using Spliced Transcripts Alignment to a Reference (STAR), to the macaque genome. I performed normalization of the raw gene count matrix using EdgeR in R Studio and alignment to the macaque reference genome. Next, I performed a single gene analysis using Limma-voom to determine differentially expressed genes (DEG). A total of 115 genes were significantly differentially expressed (>2-fold change, p<0.05) with 77 upregulated and 38 downregulated. Remarkably, genes linked to aggravation of influenza A viral disease, tissue injury, or acidification were upregulated in the infected pregnant versus non-pregnant lung 5 days after infection (MMP8, ATP12A, LGR4, NUP58, KBTBD6; log2fold change 1.28 - 2.8, all p<0.05). Next steps include gene set enrichment analysis and ingenuity pathway analysis to further investigate the gene networks linked to these upregulated genes. In summary, pregnancy was associated with upregulation of genes in the lungs 5 days after IAV infection that may predispose to greater tissue injury versus the non-pregnant lung.

Recent progress in synthetic biology has focused on utilizing probiotics as therapeutic production factories in the gastrointestinal environment to treat GI-related diseases. Although oral administration of probiotics is a convenient method for patients, a key challenge lies in the poor survival rate of probiotics in gastric and intestinal areas. Engineered living materials (ELMs), which are comprised of genetically engineered microbes embedded in a polymer matrix, present a novel formulation for orally-administered probiotics. Herein, we developed ELMs containing probiotics in a protein-based polymer matrix, aiming to enhance their viability in the GI tract. The ELMs’ photocurable polymer matrix allows us to 3D print our formulation into oral tablets. To form our protein-based polymer matrix, we functionalized bovine serum albumin with polyethylene glycol diacrylate. We then added a photoinitiator and E. coli Nissle genetically engineered to produce tryptamine (an anti-inflammatory agent) and subsequently photopolymerized this resin to 3D print probiotic tablets. We placed these tablets through a simulated gastrointestinal tract and observed cell escape using optical density measurements and cell viability through live/dead staining and fluorescence imaging. Liquid-chromatography mass-spectrometry was used to quantify the extent of therapeutic bioproduction in vitro by our ELMs over time. Overall, we found that the ELMs successfully delivered viable probiotic cells able to perform in situ therapeutic bioproduction. Furthermore, we observed that encapsulation of probiotics in ELMs yielded a higher survival rate of cells in the GI tract, suggesting that our polymer matrix formulation protected cells and allowed for extended proliferation and colonization in the colon. These findings are also supported by our observations that ELMs produced significantly higher amounts of tryptamine in the GI tract compared with non-ELM, free cells. The findings from our study can be applied to further development of orally-administered probiotic therapeutics, and show promise for future directions in drug delivery.

Malaria, a disease caused by the Plasmodium parasite, kills approximately 600,000 people per year. This disease consists of two stages, first an asymptomatic liver stage, followed by a symptomatic blood stage. The goal of our lab is to eliminate the transmission and spread of the parasite via the use of genetically modified parasites as vaccines. These parasites die in the liver, preventing blood infection and creating long-term immunity in the liver. Previous research in our group has shown that our vaccine model is effective in mice, with improved efficacy when the type-1 interferon response to the vaccine is disabled. Type-1 interferons are cytokines produced in an early immune response to a variety of pathogens. Yet, the mechanism in which these interferons are activated in a Plasmodium-infected liver cell is unknown. My project's goal is to identify and understand the mechanism in which this early immune response is turned on in response to parasite infection. To do this, I am developing an in-vitro system which can quantify the type-1 interferon response in Plasmodium-infected liver cells in culture. Utilizing various in-vitro techniques, we can identify the sensors, adaptors, and transcription factors that are most important in upregulating this early immune response. This knowledge will be used to inform methods to inactivate the type-1 interferon response, in turn improving our vaccine model. Our lab hopes to eventually use our model in humans for the goal of eradicating malaria from the modern world.

Lck is a lymphocyte specific tyrosine kinase involved in T cell activation in response to T cell receptor (TCR) mediated signaling. T cell activation is essential for the adaptive immune response, as it results in the proliferation of T cells after the detection of a peptide presented on a Major Histocompatibility Complex (MHC) and the production of cytokines necessary for immune response coordination. Lck activity is dependent on its global conformation, which is dynamically regulated via phosphorylation on its activation loop and C-terminus tail. Upon TCR engagement, active Lck phosphorylates the CD3ζ chains of the TCR complex, transducing the intracellular signaling events that activates T cells. Because Lck activity is dependent on its global conformation, we sought to map the conformational changes in Lck upon TCR simulation, as well as identify cysteine-reactive fragments that target and stabilize Lck in its conformational extremes. Lck has few endogenous cysteines, so we performed a yeast-growth-based deep mutational scan (DMS) of Lck–in which we utilized Lck’s toxicity to yeast to calculate the activity scores of ~5,000 Lck mutants–and identified 109 solvent-exposed, wild-type-like cysteine mutants of Lck. Expressing these wild-type-like cysteine mutants in T cells, and utilizing competition-based mass spectrometry, we can quantify changes in electrophilic reactivity of the cysteine side chains in the wild-type-like cysteine mutants upon T cell receptor (TCR) stimulation. Thus far, I have identified six wild-type-like cysteine mutants of Lck that are quantifiable using mass spectrometry and exhibit reactivity to our set of cysteine-reactive fragments, some of which show differential reactivity upon TCR simulation and fragment selectivity. Currently, I am using these mutants to map the dynamics of a hyperactive mutant of Lck. These quantifications provide insight into changes in the conformational flexibility of Lck, accessibility of the mutated residue sites, and intramolecular protein-protein interactions of Lck upon TCR stimulation.
 

To perform its function as a tumor suppressor, breast cancer 1 (BRCA1) must dimerize with BRCA1-associated RING domain protein 1 (BARD1). Due to this critical interaction, pathogenic BARD1 variants are also associated with increased breast and ovarian cancer risk. Genetic testing has identified many rare single-nucleotide variants (SNVs) that cause missense amino acid substitutions in BARD1. Currently, 93% (1,692 of 1,819) of BARD1 missense SNVs are classified as a variant of uncertain significance (VUS) in ClinVar. A VUS classification prevents clinicians from using genetic test results to guide patient care. Consequently, there is a strong need to functionally assess BARD1 SNVs to help resolve VUS. We applied a multiplex assay for variant effect called saturation genome editing (SGE) to functionally assess all possible 12,000 SNVs and 2,300 3-base deletions in BARD1. In SGE, we use CRISPR-Cas9 to edit all possible SNVs into a region of BARD1 in haploid HAP1 cells. BARD1 is essential for cell growth, therefore cells edited with loss-of-function variants become depleted from the population. We track which SNVs become depleted from the population by sequencing. We then generate functional scores for each variant by calculating the change in the abundance of a variant in the original SNV library versus its abundance in the cell population after 13 days in culture. Thus far, I have generated reagents for all 14,300 variants and 2,400 have completed the full experimental pipeline. Functional scores for the functionally critical BRCA1 interaction domain show depletion of 94% stop-gain, 48% splice-site, and 21% missense variants relative to 5% synonymous and 6% intronic variants. This ultimately demonstrates SGE’s ability to accurately identify functionally normal and loss-of-function BARD1 variants. Generating functional scores for all possible BARD1 variants will provide the functional evidence needed for reclassifying BARD1 VUS and definitive test results for providers treating patients with BARD1 variants.

Budding yeast cultures grown in limited sulfate conditions are overtaken by cells with an inverted triplication of the gene SUL1, which encodes for a sulfate transporter. The extra copies of the sulfate transporter provide a selective advantage because these cells outcompete other yeast cells for the limiting resource. To explain the mechanism behind this type of amplification the Brewer and Dunham Labs proposed a model (Origin Dependent Inverted Repeat Amplification or ODIRA), which requires both a DNA replication origin and inverted repeats flanking SUL1. ODIRA starts with a DNA replication error involving replication fork regression that leads to an extrachromosomal DNA intermediate. This intermediate then replicates and recombines into the genome, producing the observed amplification. Because similar triplications are observed in the human genome, including in human disorders, the mechanism of ODIRA offers insights into human genome evolution and disease. While the yeast research is consistent with ODIRA, we still do not know which proteins are responsible for the process. I am testing whether the genes RAD5 and RAD54 — involved in fork regression and strand switching, respectively — are involved in ODIRA. To do so, I am measuring the ODIRA frequency in strains with each gene deleted compared to a wild-type control. If either gene deletion leads to a statistically significant change in ODIRA frequency compared to the wild-type strain, I can conclude this gene is involved in ODIRA. To measure ODIRA frequency, I grow the deletion strains under selection for DNA recombination events and use whole chromosome gel electrophoresis and Southern blotting to detect ODIRA events. Preliminary data analysis suggests that there is a reduction in ODIRA events when either RAD5 or RAD54 is deleted, indicating that these genes are likely needed for ODIRA. These results may provide insight into how inverted triplications may arise.

 âˆ†9-Tetrahydrocannabinol (THC), the primary psychoactive compound in Cannabis, is responsible for the experience known colloquially as “being high.” Considering its alarmingly high rates of usage, THC’s effects on movement behavior are insufficiently studied. My project addresses this crucial gap in our knowledge by investigating the dose-dependent effects of THC on movement behavior using mouse models in tandem with novel behavioral neuroscience techniques. My research aims to establish a preclinical model for THC-induced impairment, focusing on studying its impact on locomotor control. My main experimental tool is a behavioral linear track, which is a clear glass corridor with a 45 degree-angled mirror placed beneath it. The linear track allows us to create a standardized multi-dimensional environment in which mice are recorded after they are treated with either a control or variable doses of THC. The videos taken of the mice are then analyzed using SLEAP. SLEAP is a machine-learning, pose-estimation algorithm that I helped train to track individual points of interest on the mice, such as the nose, paws, and tail. Behaviors of interest, such as walking, rearing, and grooming, are classified by a random forest algorithm that analyzes SLEAP label data to output identified behaviors. This data is then tabulated and graphed to reflect the dose-dependent changes in behavior elicited by THC. These classifications are also used to further analyze metrics during a represented behavior. For instance, for a walk event, we can utilize positional data from SLEAP to calculate and measure kinematic features such as stride length and limb speed, allowing us to distinguish between an unimpaired and an impaired walk. This computerized analysis approach minimizes human bias, reduces error, and produces exhaustive data that can characterize subtle differences in behavior, like when comparing mice exposed to low THC doses of 0.1mg/kg and 0.3 mg/kg.

Chimeric Antigen Receptor T (CAR T) Cells are receptor proteins that can be modified to allow T cells to target specific antigens. CAR T therapy has shown promise in preclinical experiments in patients with solid tumors, such as glioblastoma, however limitations in distribution of CAR T cells in the brain limit the effectiveness of this treatment. Most commonly, CAR T cells are administered intraventricularly through a surgically implanted device and allowed to diffuse throughout the cerebrospinal fluid filled cavities and pathways to reach the tumor. However, there is little evidence supporting the effectiveness of current methods of administration, potentially due to a lack of target engagement. We hypothesize that the glymphatic system of the brain could be used to optimize delivery of CAR T cells to solid tumors. The glymphatic system is a network of perivascular pathways that facilitates the anatomically distinct movement of cerebrospinal fluid (CSF) into the interstitium of the brain, helps distribute solutes such as glucose, lipids, and neurotransmitters, and serves as a solute clearance system in the brain. Physiologically, glymphatic function is mediated by different factors such as arterial pulsation, vasomotion, and heart rate – which can be manipulated with anesthetics, pharmaceuticals, or even sleep. We proposed exploration of the difference in parenchymal distribution of CAR T cells when injected in the cisterna magna versus intraventricularly in mice. Non-tumor bearing mice were injected via the cisterna magna or intraventricularly with fluorescently labelled CAR T cells. We observed that at 1-, 4-, and 24-hours post-injection, CAR T cells were localized in the sub-ventricular regions similarly, regardless of injection site. In follow-up experiments, we will employ the same technique in tumor bearing mice, with and without pharmacological intervention to define the effect of glymphatic function on distribution and effectiveness of CAR T cells.

Androgen deprivation therapy (ADT), the pharmacologic reduction of testosterone (T), is a critical treatment for prostate cancer patients, improving cancer-related outcomes but markedly increasing the risk of cardiovascular disease development. Our recent findings suggest that patients with prostate cancer subjected to ADT have increased hypothalamic gliosis (the activation of astrocytes and microglia), a hallmark of central nervous system injury. Similarly, castrated mice fed a high-fat, high-sucrose with added cholesterol diet (HFHS) develop hypothalamic astrogliosis and atherosclerosis, which can be prevented through T replacement at the onset of castration. In this experiment, we tested whether established astrogliosis in hypogonadal mice can be reversed by restoring healthy T levels. Using a gonadotropin-releasing hormone antagonist (acyline) as ADT, we treated 3 groups of HFHS-fed C57Bl/6 wild-type mice with: 1) vehicle, 2) acyline for 4 weeks, and 3) acyline for 4 weeks followed by a 4 week period of reversal to vehicle. Hypothalamic brain sections were used for immunohistochemistry analysis to quantify levels of glial fibrillary acidic protein (GFAP) expression, a marker for astrogliosis, and neurokinin B (NKB) expression, a marker for reproductive function. As anticipated, ADT resulted in a significant reduction in testes weight and NKB expression, serving as surrogate measurements of low T production. ADT also induced a significant increase in hypothalamic GFAP expression, confirming that gliosis is exacerbated in hypogonadal conditions. Following the discontinuation of ADT, testes weight and hypothalamic NKB expression rebounded to normal levels, however, hypothalamic astrogliosis remained significantly elevated. Together these data suggest that once astrogliosis is established, the restoration of T via 1 month of ADT cessation is insufficient to reverse it. This finding is a critical step forward in clarifying the pathways between androgen signaling and cardiometabolic regulation, and raises an important clinical concern given the high incidence of morbidity and mortality associated with ADT.

For people living with epilepsy (PWE), anti-seizure medicines (ASMs) are the primary treatment option. However, 30% of PWE are unable to control their seizures with ASMs because they have drug-resistant epilepsy (DRE). Pathological mechanisms that contribute to DRE are not currently understood. Nevertheless, both clinical and preclinical data indicate potential involvement of changes in the architecture of neuronal networks. I used a clinically relevant rat model of temporal lobe epilepsy, and novel medication in food delivery system to confirm DRE, or failure to reduce their baseline seizure frequency by 50% with two or more clinically used ASMs. I hypothesized that the DRE animals would have lowered neuronal cell density compared to the those with drug-sensitive epilepsy (DSE). All rats were euthanized at the end of a 6-week treatment period to process the brains for immunohistochemical labeling of mature neurons with the antibody, NeuN. Total percent staining area was quantified in the hippocampus, piriform cortex, and somatosensory cortex of brains. No differences in NeuN immunoreactivity were observed between DSE and DRE animals in any brain region. However, NeuN levels in animals with epilepsy, regardless of treatment outcome, trended lower than naïve animals without epilepsy in the CA1 and dentate gyrus regions of the hippocampus. Together, these data suggest that neuron density may not be driving pharmacoresistance. However, it is possible that the ratio between excitatory and inhibitory neurons may be disrupted in DRE. This underscores the need for future studies to quantify neuronal subtypes, providing a more nuanced understanding of the underlying mechanisms of pharmacoresistance. These studies play a crucial role in guiding future research into novel treatments designed for DRE.

The COVID-19 pandemic has reshaped our social dynamics, leading to a decline in in-person interactions across American society. This shift has raised concerns about increased loneliness and diminished social connections, with significant ramifications for mental health. College students, in particular, are likely to feel the impact of reduced social interaction, given its centrality to academic and campus life.The aim of our study is to determine the relationship between the amount of time University of Washington undergraduates spend interacting with others while feeling socially connected and their perceived levels of anxiety. Our cross-sectional study used an anonymous online survey to measure hours spent socializing per week, and self-perceived anxiety levels amongst 18–24-year-old undergraduate students. We will collect data through convenience sampling in February 2024 distributing our survey via social media and direct outreach. We will conduct our analysis including prevalence ratios, descriptive statistics and qualitative content analysis. We hypothesize that UW undergraduate students spend 35 hours a week interacting with others and that low social interaction would be correlated with high self-perceived anxiety levels, adjusting for >4 roommates and >40 hours spent working per week.Our findings could hold potential significance for public health initiatives aimed at addressing population-level mental well-being, reducing anxiety, and enhancing access to effective mental health care interventions.

Organs-on-a-chip (OOAC) are biomimetic structures that replicate the physiological environments of human organs. OOACs are growing in popularity as they provide control of parameters including shear stress, concentration gradient, and biological interactions between cells and biofluids; they can be used in pharmacokinetic, physiological, and toxicological studies. The Kelly-Yeung lab works with kidney OOACs to study toxicology and pharmacokinetics. An important component of the chips is the hydrogel that provides a tubular scaffold and biological substrate for the kidney cells. The hydrogel mixture typically consists of decellularized kidney matrix, Collagen I (Col-I), and two types of cell culture media (PTEC and 199x). The matrix mimics the kidney microenvironment, the Col-I is a stabilizer and tissue regeneration agent, and the two cell culture media are used to mimic the extracellular fluids. More matrix in the hydrogel is ideal since it will better mimic a kidney. However, the matrix by itself is not structurally stable, hence the need for a stabilizing agent. The goal of this project is to maximize the ratio of matrix to Col-I while maintaining a stable hydrogel. In order to determine the optimal ratio, stiffness testing of the hydrogels will be performed via AFM (atomic force microscopy) and a Parallel Plate Rheometer to find out how much matrix can be used before the stiffness of the hydrogel composition is compromised. At this stage, we have started testing the collagen hydrogel with the rheometer to gain base measurements before adding the kidney matrix. We aim to incorporate the kidney matrix, achieving measurements closely mirroring those obtained with the collagen hydrogel alone, while supporting healthy cell growth. Creating a more accurate OOAC based on optimized kidney extracellular matrices will help improve the models and recapitulate the effects of drugs, toxins, and diseases on human kidneys more precisely.

Introductory biology classes are often a stepping stone for students in various STEM majors, as prerequisites or major requirements. For this reason, it is vital that these courses properly prepare students for life beyond their classes, through the ability to apply the knowledge they are gaining to the real world. Because textbooks are often used as preparatory assignments or as the content on which a course is grounded, we decided to focus on this type of circular tool, in order to evaluate the level of humanization in introductory biology courses. We set out to find out the degree to which six popular and prominent introductory biology textbooks humanize science, by positioning the content in a social context. We did this by developing our definition of humanization, and based on this, a continuum of humanization. We then developed a rubric for coding the extent of humanization, with the lowest being scarce humanization and highest being content that centers justice. We also coded topics of biology, from ethics to the environment. The team went through page-by-page and line-by-line to apply the rubric to all six textbooks, including the text as well as questions. In terms of the questions, we found that out of 9262, only 236 questions were humanizing. For the text, out of 9670 pages, there were 1352 humanizing passages. Overall, we found the inclusion of humanizing content to be rare across the six textbooks analyzed, and humanization by inclusion of justice to be particularly rare. We end by giving suggestions to educators, a main one being to pose justice-centered problems to students - a “problem-posing” model of science education.

Age-related macular degeneration (AMD) is an acquired degeneration of the retina characterized by the presence of lipid-rich deposits, or drusen, underneath the retinal pigment epithelium (RPE). Development of drusen has been linked to degradation of extracellular matrices and aberrant RPE lipid metabolism. Mutations in tissue inhibitor of metalloproteinase 3 (TIMP3), involved in extracellular matrix (ECM) degradation, have been associated with Sorsby Fundus Dystrophy (SFD), an autosomal dominant inherited disease phenotypically similar to AMD. SFD and AMD share clinical features, such as the presence of drusen, geographic atrophy, and choroidal neovascularization. The aim of this project is to evaluate the hypothesis that increased ECM degradation results in reprograming of SFD RPE metabolism towards increased branched chain amino acid (BCAA) oxidation, resulting in lipid synthesis and deposition. We found that SFD induced pluripotent stem cells (iPSC)-RPE have increased apolipoprotein E deposits, and may have increased lipid metabolism. SFD RPE were found to have decreased levels of FABP7, a lipid binding protein that regulates lipid metabolism by increasing fatty acid oxidation, by proteomics and confirmed by Western blot. SFD RPE cells show increased BCAA consumption and upregulated expression of branched chain amino acid transaminase 1 (BCAT1), an enzyme that catalyzes the animation of BCAAs. Control RPE were treated with BCAT1 inhibitors, BCATc Inhibitor 2 and gabapentin. Treatment of RPE with 50 µM of BCATc Inhibitor 2 resulted in a greater than two-fold decrease in BCAA consumption at 48 hours, indicating effective inhibition of BCAT1. Results from this study will help determine whether enhanced BCAA oxidation results in activation of increased lipid synthesis and lipid deposits in SFD iPSC RPE.

 Alzheimer’s Disease (AD) has well-known brain alterations such as Tau protein build-up, beta-amyloid plaques, and neuronal cell death, yet the role of the brain’s microvasculature on the progression of this neurological condition has not been fully uncovered. My research examines changes in the microvasculature density, length, and function during normal aging using a well-established aging model in Brown Norway rats. This study contributes to the pantheon of previous microvascular research and forwards the field toward understanding AD development from another perspective. My hypothesis is that the density and length of these microvasculature are decreased in areas associated with learning and memory (i.e., the hippocampus and parietal cortex) before the development of AD symptoms and worsen as the disease progresses. To test this hypothesis, first in normal aging, 3 experimental groups of Brown Norway rats are employed: (n=6) young rats at 5-6 months, (n=6) middle-aged rats at 15 months, and (n=6) old rats at 20-24 months. Sagittal slices of the right hemisphere were fluorescently marked for their microvasculature, astrocytes, and cellular nuclei. The ImageJ analytical program was used to compartmentalize the areas of the dentate gyrus, CA1, CA2, and CA3 along with the parietal cortex into 900 x 900-pixel boxes for examination. The preliminary results show that the density of microvasculature within the 3 age groups were consistent while the distribution of the vessel lengths had more variability. The two leading postulates are increased tortuosity with increased age and/or rarefaction, where the microvasculature experience shortening with increased age. Further analysis is needed to examine this distribution among the 3 age groups.

B-cell lymphoma-extra large (Bcl-xl) is a mitochondrial transmembrane protein that acts as an anti-apoptotic protein by sequestering the apoptosis-inducing proteins Bim, Bak, and Bad. This prevents the release of cytochrome c from the mitochondria, preventing activation of apoptosis pathways. Higher levels of Bcl-xl expression are commonly found in cancer cells. This contributes to the prevention of apoptosis in cancer cells, allowing them to proliferate uncontrollably. Bcl-xl is an incredibly important target for cancer therapeutics. A Bcl-xl binder would inhibit the interaction between Bcl-xl and apoptosis inducing proteins, allowing cancer cells to undergo apoptosis. In my research, I am using deep learning methods to design cyclic peptides that bind to Bcl-xl. To design the binders, I used RFDiffusion - a generative diffusion model - to produce thousands of cyclic peptide binder scaffolds bound to Bcl-xl. I then used a sequence-based deep learning tool to generate multiple sequences for each backbone design. The resulting binders were computationally validated with the highly accurate, machine-learning-based structure prediction tools AlphaFold and RoseTTAFold. Of the 40000 generated cyclic peptides, 2052 were predicted to bind to Bcl-xl based on standard metrics. Along with their excellent metrics, these designs show a high structural similarity and binding location to the known Bcl-xl binders Bim, Bak, and Bad. The designs were clustered by backbone into 350 unique clusters. We synthesized the top designs and identified which peptides display binding to Bcl-xl through a Homogeneous Time-Resolved Fluorescence (HTRF) assay. A successful Bcl-xl binder has the potential to serve as the basis for an effective and affordable cancer therapy.

We have observed that conventional respiratory pathogen sampling methods, such as pharyngeal swabs, elicit unpleasant experiences for both adults and children. Particularly for pediatric patients, having a non-invasive, enjoyable sampling approach is crucial to facilitate prompt diagnosis and treatment. In prior research, we introduced a novel saliva sampling device, the CandyCollect. This lollipop-inspired device, with its isomalt candy coating, is specially produced and surface-treated for capturing pathogens from saliva, providing a pleasant sampling experience to child patients. Clinical studies approved by multiple Institutional Review Boards (IRB) revealed an average candy dissolving time for CandyCollect (with a mass of 0.90g~1.10g) of 3.51 minutes, with a minimum of 1.25 minutes. To compete with original sampling methods which take up to 10 seconds, it is desirable for CandyCollect to have a shorter sampling time around 15–20 seconds. Therefore, this study aims to decrease the dissolving time by introducing a new CandyCollect recipe and design. For the new candy recipe, we replaced isomalt with a mix of glucose and sucrose. Additionally, baking soda (sodium bicarbonate) was added to increase the candy’s contact area with the tongue. An ongoing experiment will assess if baking soda affects PCR results for pathogen samples, and this modified recipe will be employed in a new clinical study. Concurrently, we have modified the CandyCollect design by placing the candy on the same side as the spiral, with a small candy reservoir beneath the spiral to decrease the mass to 0.06g-0.1g. To validate the efficiency of this new design, we plan to conduct another clinical study recruiting 30 younger participants and obtaining feedback about the new design. This collaborative study will provide valuable insights towards achieving a faster dissolving time, ultimately enhancing the viability of CandyCollect as an improved and more efficient replacement for conventional sampling methods.

Gastrointestinal symptoms are a common comorbidity of autism spectrum disorder (ASD), and individuals with the disorder tend to have a distinct gut microbial community composition and circulating metabolomes. My work in the Rajakovich Group focuses on a gut-derived metabolite, 4-ethylphenolsulfate (4-EPS), found in higher abundance in ASD mouse models and children with ASD. 4-Ethylphenol (4-EP), its precursor, is produced by gut microbiota before host-mediated sulfation, but the microbial biosynthetic pathway is unknown. A proposed metabolic pathway suggests the microbial stepwise conversion of plant-derived complex polysaccharides to 4-EP. My project goal is to identify a gut microbial enzyme responsible for the first step of this proposed pathway: a hydroxycinnamoyl esterase. I used literature searches and bioinformatics tools to identify characterized bacterial cinnamoyl esterases and candidate enzymes. I designed plasmids for two candidate enzymes (both from E. faecium, known to colonize the gut) and one characterized esterase (from L. plantarum). Currently, I am working on expressing the proteins in E. coli cells and purifying them by affinity chromatography. Once purified, I will assess the enzymes for their anticipated cinnamoyl esterase activity by incubating them with dietary hydroxycinnamic acid esters and detecting products with high-performance liquid chromatography (HPLC) and UV/Vis spectroscopy. Since the candidate enzymes are homologs of confirmed esterases and have conserved catalytic motifs, I hypothesize that they will have hydrolytic activity. If correct, I will see consumption of the substrate (no detection) and detect the anticipated products. Positive results from these assays would complement ongoing work by the lab to identify other E. faecium enzymes in this proposed pathway. Though it is debated if 4-EPS is causal to the disorder or simply a biomarker, elucidating its biosynthetic pathway and studying the biochemistry of gut microbes will contribute to detangling the gut’s role in ASD.

Cell co-culture systems are used to study intracellular interactions by culturing distinct cell-type populations within a shared environment. This in vitro method is more representative of the highly complex and diverse processes that occur in organisms, allowing accurate insight into mechanisms of cell signaling pathways, disease, drug interactions, etc. Existing designs can be two- or three-dimensional, with or without cell-cell contact, and use systems like microfluidics, solid supports, or transwells to control contact. Methods that physically partition individual cell-type populations often allow soluble factor transmission by using permeable material or flooding the compartments so the solvent is shared. However, this is less representative of the human body, where physical partitions do not divide different cell types. Thus, there is a need for a co-culture system with a partition allowing initial separation, that can later be removed to allow interaction without a physical barrier. We are developing this system by utilizing open microfluidic gel patterning techniques to test if a removable partition can be formed with enzyme-degradable polyethylene glycol (PEG). I first use computer-aided design to engineer a rail scaffold outlining two compartments, and fabricate these devices using 3D printing. I pipette PEG into an inlet in the rail, flowing along the scaffold channel due to spontaneous capillary flow and patterning the insert. The PEG polymerizes to form the insert with two distinct cell chambers on the well’s bottom surface, and the cells are seeded into their corresponding compartments. After the cell culture period is complete, sortase (SrtA) is added to completely degrade the PEG insert, allowing the cell populations to interact. We expect the PEG inserts to polymerize similarly to agarose, and leave no residue in the well once degraded. Future work will include utilization of this device for experiments using functionalized beads to monitor soluble factor signaling in co-cultures.

Half of the earth's photosynthetic activity occurs in the ocean. However, marine ecosystems generally have lower rates of carbon sequestration when compared to terrestrial ones. This is an opportunity to enable large scale carbon sequestration. The waters of the open ocean are nutrient deficient and can have low primary productivity. Supplying the limiting nutrients can theoretically enable rapid growth of photosynthetic cells but this growth must be contained or it will be lost to the ocean. By preparing these missing nutrients in hydrogels with efficient photosynthetic consortia, the growth process and inputs can be contained and the biomass harvested. The Winkler Lab is using this method to develope biological systems for carbon sequestration. I am researching the efficiency of microalgae and cyanobacteria consortia in seawater with native microbes. I aim to form cultures of photosynthetic marine microbes by inoculating hydrogels containing chlorella sp. in seawater samples. The objective is to optimize squestration with naturally occurring microbial consortia. Through multiple trials I have identified a mix of microbes and macroalgae cultured from the Puget Sound that exhibits rapid biomass production. Data is collected via microscopy, imaging and by measuring chemical oxygen demand and chlorophyll content. My aim is to compare this wild microbial mix to the Winkler lab's established mixes of cyanobacteria and microalgae and determine which is more effective in fixing carbon. Expected results will demonstrate this wild culture more efficient in low nutrient environments than the lab culture. Success in this project could help refine commercializable methods to remove atmospheric carbon dioxide and fight climate change. 

TBC1(Tre2/Bub2/Cdc16) Domain-Containing Kinase (TBCK) is a pseudokinase with proposed involvement in the endocytic pathway. Kinases are proteins that can post-translationally modify other proteins through the addition of inorganic phosphate from ATP to serine/threonine/tyrosine residues. TBCK, being a pseudokinase, lacks critical residues that allow ATP binding and, therefore, cannot catabolize ATP. Pseudokinases, while catalytically inactive, have been shown to have protein scaffolding properties as well as modulate the activity of other kinases. Whether pseudokinase TBCK plays a role in any of those functions has yet to be discovered. Via the TBC1 domain, TBCK interacts with Rab proteins, a class of membrane-binding proteins involved in multiple cellular pathways that coordinate intracellular vesicle transport with GTP active and GDP inactive states. TBCK functions as a Rab GAP(GTP-hydrolysis activating protein), hydrolyzing Rab bound GTP and leaving an inactive GDP-bound Rab. Mutations in TBCK have been found to be clinically associated with a rare neurological disorder, TBCK syndrome, characterized by delayed development, intellectual disorder, and hypotonia. The interactors and Rab substrates of TBCK are under researched and still poorly understood; we aim to illuminate those interactions through immunoprecipitation (IP) and mass spectrometry. Early attempts at this goal involved co-transfection of various Rab protein targets with TBCK WT and TBCK R511H (a TBC1 inactive mutant) in HEK 293T cells and subsequent co-IP, enriching for TBCK and interacting Rab proteins. These preliminary results, in combination with live imaging and immunofluorescence of TBCK and its mutants with Rab proteins and other membrane markers, proved inconclusive. Therefore, we are now performing crosslinking immunoprecipitation mass spectrometry to allow the identification weakly interacting protein complexes through mass spectrometry. These experiments will provide insight into the fundamental biology underlying TBCK’s role in neurodevelopment and how its dysfunction contributes to disease states.

The use of pesticides in the Pacific Northwest is essential in the process of safeguarding public health, most notably by mitigating pests, protecting our food supply, and aiding in distribution to supermarkets, restaurants, and our homes. However, long-term exposure to pesticides can result in illness for those handling the substances as well as their families. Prior research has shown that current pesticide application methods play a role in accelerating illness. Newer methods, such as aerial drone spraying and “smart” sprayers, involve the use of emerging technologies that are poised to change the landscape of the agricultural industry and health outcomes of farmworkers. Under the supervision of the Pacific Northwest Agricultural Health and Safety (PNASH) Center, my project will be assessing thoughts regarding adoption of these technologies. Through the creation of an electronic REDCap survey, I will be obtaining a variety of responses from agricultural workers, farm decisionmakers, and others involved in the application of pesticides on farms. Once the survey is deployed, I will analyze responses both quantitatively and qualitatively using Dedoose and R statistical methods, respectively. From these responses, I will work with the PNASH team to evaluate the adoption of current and emerging pesticide technologies among Northwest fruit growers, as well as their impacts on occupational health and safety. Through this project, I hope to collect a wide range of perspectives and thoughts regarding the implementation of new pesticide application technologies, particularly unique opinion points (positive and negative) I did not otherwise consider in my initial research with the PNASH Center. The main objective of my research project is to capture the attitudes of the pesticide application technologies to inform policy, regulations, and decision-making regarding their uses.

The mechanisms underlying the development of neurodegenerative diseases such as Alzheimer's Disease (AD) are not well understood. The characteristic accumulation of pathological protein plaques and tangles has led AD research to focus primarily on abnormal protein function and metabolism contributing to the pathology. Tau is one such protein that forms toxic aggregates in those with AD and various other neurodegenerative diseases. Our lab researches the mechanisms of tau toxicity using the nematode C. elegans. Recent research has implicated improper lipid metabolism as another potential contributor to neurodegeneration. Mutations in a gene known as gba-3, which is critical for lipid metabolism, are risk factors for the development of Parkinson’s Disease (PD), but little is known about the relationship between gba-3 mutations and tau toxicity in AD models. My research project will investigate the role of gba-3 in a C. elegans model of tau toxicity. I have crossed strains with various mutations in the gba-3 gene into two strains with tau toxicity. I will perform motor performance assays, measure the accumulation lipids, quantify the amount of tau, and measure oxidative stress to investigate whether gba-3 mutations modulate tau toxicity. Preliminary results suggest some gba-3 mutations slightly rescue some tau models but not others, but further investigation is needed to validate these findings. Ultimately, this project will provide further insight into the complex mechanisms underlying neurodegenerative diseases such as AD and may guide future development of treatments targeting lipid metabolism.

In the 20th century, the discovery and widespread use of antibiotics became humanity's primary weapon against pathogenic bacteria. Overuse of antibiotics has unfortunately given rise to antimicrobial resistance, weakening us in this evolutionary arms race. Proteolysis-targeting chimeras (PROTACs) have been proposed as a strategy for development of novel therapeutics. By tagging target proteins with ubiquitin, targeted protein degradation (TPD) can occur via a eukaryote's own molecular machinery. Due to prokaryotes lack of ubiquitin, research has shifted to the development of PROTAC-like molecules to achieve proteolysis and cell death in bacteria, called BacPROTACs. However, to eventually experiment with these small molecules and see their mechanism of TPD, off-target effects, and changes in host and bacterial proteomes, we must be able to profile the degradation of proteins in an unbiased manner. Proteomics and mass spectrometry can identify and measure thousands of proteins simultaneously, enabling systems-level and mechanistic understanding of these novel therapeutics. In order to ensure reliability, reproducibility, and overall accuracy in analyzing a cell’s proteome, an optimized proteomics workflow is imperative. Our lab sought to understand the downstream impacts of different sample preparation protocols, differing in the material used to capture precipitated protein. Here, I present an evaluation of three proteomic sample preparation methods used on triplicates of reduced and alkylated aliquots of human cell lysate: "SP3" (single-pot solid-phase sample preparation), uses magnetic carboxylate beads as a substrate for protein aggregation; "SP4", omits a capture substrate in favor of centrifugation; and "S-Trap/S-Tip," captures protein on a borosilicate glass fibers filters. Following a Trypsin and LysC digest and desalting, samples will be run on an Orbitrap Eclipse mass spectrometer. Analysis and subsequent optimization with more complex human samples will ensure the selection of a protocol providing the highest proteomic coverage for further research.

Perineuronal nets are extracellular matrix structures comprised of chondroitin and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs). Histological imaging of brain PNNs is achieved using Wisteria floribunda agglutinin (WFA) labeling of PNN CS/DS-GAGs, while composition can be determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although these methods are used to determine PNN CS/DS-GAG abundance and composition, it’s unknown whether brain fixation or processing influence these outcomes. We first explored whether tissue processing, using cryosectioning (CRYO) or paraffin embedding (PE), influence PNN analyses. Ten mice were perfused with PBS and post-fixed in 4% paraformaldehyde (PFA). Brains were cut sagittal, and one hemisphere was prepared as floating tissues (CRYO) and the second hemisphere was processed as direct mounted tissues (PE). Histochemical analyses show a 78.9% reduction in hippocampal WFA+ PNNs in the PE processed hemisphere compared to CRYO processed side. LC-MS/MS analysis of hippocampal CS/DS isomers also showed differences between each method. In a second cohort of mice, we determined that fixative (4% PFA vs 10% formalin) did not influence hippocampal WFA or CS/DS isomers between groups, suggesting tissue processing (not fixative) influences PNN analyses. We then explored whether we could correct for these CS/DS baseline differences. By comparing CS/DS isomers isolated from CRYO vs PE processed tissues within each mouse, we discovered reproducible correction factors for each isomer. Adjusting the CRYO group using these factors normalizes baseline compositional differences between CRYO and PF groups. To determine translational relevance, we compared hippocampal CS/DS isomers between three CRYO vs PE prepared non-human primate (M. nemestrina) tissues and observe similar baseline CS/DS differences. Adjusting the CRYO prepared group using corrections factors normalizes the baseline composition. These results provide strong, translational evidence that tissue processing greatly influences both PNN glycan histology and composition analyses, and that corrections must be made to account for baseline differences before comparing groups.

Multiple Sclerosis (MS) is a chronic central nervous system (CNS) disease in which the immune system attacks the myelin surrounding nerve cells, resulting in decreased communication between the brain and the rest of the body, and severe physical and neurological defects. Gut metabolites produced by gut microbiota play a significant role in maintaining host immune homeostasis, and research has shown that short-chain fatty acids (SCFAs) have therapeutic potential. When tested in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), delivering butyrate-conjugated block copolymer micelles to the distal gut ameliorated EAE progression by increasing the amount of regulatory T cells and cytokines, and strengthening intestinal barrier functions. For prolonged SCFA delivery to the distal gut, a SCFA-conjugated hybrid lipid-polymer nanopolymer carrier is required. A nanoparticle is required because butyrate has a strong odor/taste and requires many pills per day to have significant suspension. To synthesize this nanoparticle, we optimized the nanogenerator to determine its effects on size and charge. This included modulating the total flow rate, flow rate ratio, input concentration ratio (lipid:polymer). We hypothesize that larger size, smaller surface area-to-volume ratio, and anionic surface charge will prolong retention and time in the distal GI. Once the nanoparticle is optimized, the potential of SCFA-conjugated nanocarriers to ameliorate EAE progression will be measured by comparing EAE mice and healthy controls, using the same outcome measures. This will allow us to select which polymer-conjugated SCFAs to incorporate into the optimized nanocarriers. We believe the SCFAs will inhibit proinflammatory macrophage activation, induce anti-inflammatory Treg cells, and promote demyelination and axonal degeneration to reduce the effects of MS in mice. In humans, delivering these nanoparticles as a drug will serve as a much more efficient means of treating MS when compared to the current treatment procedures.

Neurogenic bladder is a common condition associated with traumatic spinal cord injuries (SCIs), which results in inhibited detrusor function, bladder-sphincter dyssynergia, and scarring of the bladder walls and muscle. Care for neurogenic bladder is aimed at reducing abnormally high pressures, which left untreated lead to hypertrophy and tissue fibrosis of the bladder wall, as well as upper urinary tract complications. Current treatments target the neurotransmitter release of acetylcholine, utilizing anticholinergic drugs to counter the overactive bladder. However, these drugs can have negative/deleterious side effects, and have broad symptoms influencing unintended targets around the body. Targeted chemodenervation that uses Botulinum toxin (Botox) to interfere with nerve conduction is often reserved as a second line of defense to treat neurogenic bladder. Unfortunately, this late provision concedes irreversible damage to the detrusor muscle. We hypothesize that administering early chemodenervation can prevent the development of neurogenic bladder, improve bladder compliance post SCI, and increase the overall quality of life of SCI affected patients. Using a rat model, the Khaing lab administers a controlled contusion injury to the T8/T9 vertebrae, simulating a spinal cord injury in humans. The recovery of the rats is tracked with behavioral observations, cystometry data collection, and histological stains. My team and I are working to determine the effective therapeutic time post SCI for Botox injections, and the optimal doses for treatment. Our results thus far support that acute chemodenervation with Botox reduces bladder overactivity, and bladder wall thickness.

Age-related macular degeneration (AMD), a multifactorial eye disease, is distinguished by drusen formation and thickening of Bruch’s membrane. Early onset macular drusen (EOMD) is a rare inherited retinal degeneration with clinical similarity to AMD. EOMD results from genetic variants that cause decreased protein expression levels of complement factor H (CFH) and factor H-like protein 1 (FHL-1). The precise mechanism of drusen formation is unknown, although there are multiple lines of evidence that complement dysregulation and inflammation play a major role. RPE cells derived from EOMD patient induced pluripotent stem cells (iPSCs) can serve as in vitro models for understanding the effects of altered local complement. The complement system is a cascade of proteins and reactions that modulate inflammatory responses for the removal of pathogens. Specific components, such as C3a, could be involved in inflammatory responses that contribute to drusen formation and AMD. The purpose of this project is to observe how increased C3a impacts inflammatory cytokine secretion in EOMD RPE cells. iPSCs were generated from peripheral blood mononuclear cells collected from two patients in an EOMD family and differentiated into RPE cells. Western blot analysis was performed on EOMD and control RPE cells to quantify C3a, CFH, and FHL-1 levels to determine baseline expression levels. Control and EOMD RPE were treated with C3a, and inflammatory cytokine (IL-6 and IL-8) secretion was measured by ELISA. Decreased CFH/FHL-1 secretion and increased local C3a were observed in EOMD RPE. Preliminary data indicate that increased C3a levels may alter cytokine secretion by RPE, indicating that increased complement may play a role in local inflammatory responses that contribute to the pathophysiology of EOMD and AMD.

Cannabis is the most commonly used drug of abuse used by approximately 2.5% of the world's population. Tetrahydrocannabinol (THC) is the major psychoactive component of cannabis. Cannabidiol (CBD) on the other hand is a pharmacologically active component of cannabis that is not psychoactive. THC is metabolized via cytochrome P450 enzyme-mediated oxidation to 11-hydroxy-THC (11-OH-THC) and subsequently to 11-carboxy-THC (11-COOH-THC). 11-COOH-THC then undergoes glucuronidation to form an acyl glucuronide (AG), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide (11-COOH-THC-Glucuronide). CBD is also metabolized via an analogous pathway to THC, where CBD is oxidized to 7-hydroxy cannabidiol (7-OH-CBD) and then subsequently to 7-COOH-CBD and ultimately glucuronidated to form the AG 7-COOH-CBD-Glucuronide. The THC and CBD AGs are important because they circulate at high concentrations in biological samples. Thus, identifying the enzymes responsible for the formation of cannabinoid AGs is important for understanding inter-individual variability and disease effects of cannabinoid exposures and pharmacological effects. Glucuronidation is governed by UDP-glucuronosyltransferases (UGTs). The goal of this study is to identify and characterize the UGTs that form THC and CBD AGs, to define in what organs these glucuronides are formed, and to determine whether disease states may alter 11-COOH-THC and 7-COOH-CBD glucuronidation. To accomplish this I incubated human liver microsomes (HLMs) and recombinant UGTs with 11-COOH-THC (5 µM) or 7-COOH-CBD (5 µM). The incubations were done at pH 7.4, 37℃ for 5 (THC-COOH) or 45 (7-COOH-CBD) minutes. Formation of the THC and CBD AGs was detected in HLMs and with UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT2B7. UGT2B4 and UGT2B17 were specific to THC AG formation and UGT1A10 was specific to CBD AG formation. Kinetic assessments such as intrinsic clearance measurements and inhibition assays will further the understanding of the importance of the specific UGT isoforms responsible for cannabinoid acyl glucuronide formation in vivo.

The modification of tRNA plays a significant role in the efficiency and accuracy of translation during protein synthesis. A modification that plays a direct role in reading cognate codons of mRNA in E. coli is the 5-oxyacetic acid methyl ester (mcmo5) modification. This modification occurs on the uracil base at position 34 (U34). The biosynthetic pathway of this modification is initiated via a hydroxylation reaction. Previous in vivo studies demonstrate the enzyme TrhP, tRNA hydroxylation protein, performs this hydroxylation reaction in anaerobic conditions. No in vitro work has been done to study this enzyme and its mechanistic function. TrhP is known to coordinate an iron-sulfur cluster, a metallic cofactor known to contribute to a variety of critical cellular processes, however, the necessity of an iron-sulfur cluster for a hydroxylation reaction is unique to this newly discovered protein family. The goal of this research project is to spectroscopically characterize TrhP’s iron-sulfur cluster to understand the importance of the FeS cluster. Site-directed mutagenesis is utilized to study the coordination of the iron-sulfur cluster. Changes to iron-sulfur cluster coordination are monitored via UVVIS, electron paramagnetic resonance (EPR), and colorimetric assays. These experiments determine how the loss of cysteine, a known iron-sulfur cluster ligand, impacts the iron-sulfur cluster coordination. Coordination of a [2Fe2S] cluster by 4 conserved cysteines is expected, and UVVIS data agrees with that hypothesis. Colorimetric assays show the cysteine to alanine mutants contain less iron than wild-type TrhP, indicating each cysteine has a significant role in cluster binding. Learning more about the specific coordination will establish the site of cluster-binding within TrhP and shed light on the cluster’s role in TrhP’s stability, geometry, and redox properties which all contribute to the enzyme’s modification activity.

As we receive spatial and temporal information, our brain develops sequential patterns to store events, giving us the ability to learn and store relationships. For learning and memory, these rapidly-encoded sequences are reactivated as “replay” sequences after experiencing the original trajectory, as often observed in the hippocampus. This brings up the question: what biological mechanisms enable us to build, encode, and trigger these relationships and replays? The goal of this project is to model sequential replay in spiking neural networks to explore and understand various biological mechanisms that produce the acquisition of such sequences. We are using NEST Simulator, a spiking neural network simulator software, to model large-scale neural networks. Then, we explore how changing dynamics such as non-random structure of the network and interactions between excitatory and inhibitory cells can contribute to sequence generation, as well as the salience and speed of such sequences. We have observed the significance of interplay between particular parameters, such as the widths of spatial Gaussian distributions for neuron connection strengths, by analyzing generated spiking raster plots. Recent work has also suggested an important role of long-term potentiation of intrinsic excitability in sequential replays, which we are integrating with the aforementioned dynamics by building a unique synapse model within the simulation software. This is a novel method to introduce excitability in a network, which is important to determine how changing excitability through potentiation, rather than plasticity, facilitates network formation and propagation. This research is significant because it highlights the components of neural networks that could be crucial to quickly generating and maintaining sequences for learning and memory, therefore helping us understand the brain’s mechanisms for storing spatiotemporal relationships.