Student External Award for Research Training Details

Human immunodeficiency virus type 1 (HIV-1) remains a major global health challenge. While viral maturation has long been a promising target for inhibitor development, the emergence ofresistance to protease inhibitors continues to limit their effectiveness. As such, the Group-Specific Antigen (Gag) polyprotein presents an alternative avenue for therapeutic intervention, marked by the recent development of novel antiretrovirals such as bevirimat and lenacapavir, which target the immature Gag and mature capsid (CA). Gag encodes the core structural proteins that form the viral particle and undergoes cleavage by the viral protease during maturation. Among the domains in Gag, CA-SP1 cleavage is the key determinant for CA maturation, a process marked by CA undergoing drastic conformational changes. In Aim 1, I will elucidate how the stability of SP1 helices evolves during proteolysis. SP1 helices are arranged mostly in relatively stable six-helix bundles in the immature lattice but are bound to the protease extended and unfolded. It remains unclear how SP1 loses its secondary structure as it binds to the protease. I hypothesize that the initial cleavage of a single SP1 helix destabilizes the six-helix bundle, promoting the unfolding of neighboring helices and allowing for easier access to the protease. Using MD simulations, I will elucidate and compare the unfolding timescale and mechanisms of SP1 helices within fully intact and partially cleaved hexamers. I predict that SP1 helices in the cleaved hexamers will unfold more readily, whereas the intact hexamer will remain predominantly stable. These simulations will provide mechanistic insights into how SP1 proteolysis propagates structural changes through the hexamer and contributes to how the crucial last step of Gag proteolytic processing occurs. The expertise from the Schiffer lab provides me with the necessary training needed to perform robust structural modeling and molecular dynamics simulations. In Aim 2, I will investigate how SP1 cleavage mediates CA conformational changes that facilitate maturation. The destabilization ofthe six-helix bundle has been identified as the key structural switch for CA maturation. Using smFRET, I will visualize the conformational changes of single CA molecules within oligomeric assemblies. I predict that destabilization of the six-helix bundle following CA-SP1 cleavage induces conformational changes to the CA domain that are relevant for virion maturation. These findings will provide mechanistic insights into the allosteric communication between the CA-SP1 cleavage site and CA domains, revealing how initial cleavage events prime the hexamer for further structural transitions. The Munro lab has a proven track record and expertise in visualizing dynamic events in virions using single molecule fluorescence- based techniques. This study will significantly advance our understanding of the molecular mechanisms underlying HIV-1 maturation, potentially shedding light on the maturation processes of other retroviruses.

Cancer remains a leading cause of death in the United States and globally. Understanding the genetic mutations that drive cancer can help inform diagnosis, prognosis, and treatment options. Cancer driver mutations are thought to cause tumor formation and malignancy by leading to abnormal and uncontrolled cellular growth. To date, thousands of cancer driver mutations have been identified in hundreds of genes. Experimental models that precisely install cancer driver mutations are essential for investigating the mechanisms that underlie cancer. CRISPR-based prime editing offers a comprehensive way to generate a diverse array of cancer driver mutations, including substitutions, insertions, deletions, or combinations. In prime editing, a prime editing guide RNA (pegRNA) guides mutant Cas9 to nick one strand of the genome target site and serves as a template for reverse transcriptase (fused to Cas9) to copy new information into the genome. However, designing and validating effective pegRNAs is expensive and time-consuming. Preliminary experiments using a high-throughput screening approach based on Next Generation Sequencing identified pegRNAs that guide efficient editing. This screen enabled simultaneous testing of thousands of pegRNA designs for their ability to edit frequent mutations in hepatocellular carcinoma (HCC), the most prevalent form of primary liver cancer. This project aims to use these pegRNA constructs to further characterize HCC mutations and to generate a mouse model of HCC. Aim 1will characterize the cellular and molecular effects of HCC driver mutations. Utilizing a library of the highest efficacy pegRNAs from the preliminary screen, a secondary screen will be performed to identify which mutations confer a growth advantage in transformed primary human hepatocytes. Cellular fitness results from this screen will be validated using a competition assay. Cellular and molecular phenotypes of mutations that promote fitness will then be characterized. Colony formation assays will determine the ability of cells with cancer driver mutations to expand into a clonal population. RNA sequencing will reveal transcriptional profiles associated with the cancer mutation. Aim 2will model HCC driver mutations in vivo. Prime editing will be used to install cancer mutations in transformed primary human hepatocytes, and cells will be orthotopically implanted into the livers of immune-deficient mice via intrahepatic injection. After a period of growth, tumor formation and the histopathology and gene expression signatures of the tumors will be analyzed. This study will be piloted with pegRNAs (identified from the preliminary screen)that generated an activating mutation in the conserved -catenin gene CTNNB1.An activating mutation inCTNNB1combined with co-expression of the MYC oncogene was sufficient to drive tumor progression in mice. In vivo models of cancer driver mutations will be used to assess how tumors respond to treatment with first-line therapies for HCC. If successful, this project will generate a system to further characterize HCC driver mutations at the cellular level and in tumors, informing potential treatment approaches based on the genetic profiles of cancer.

Cognitive decline is a major hallmark of aging. Synapse loss and structural alterations are believed to be major contributing factors to normal age-related cognitive decline and are exacerbated in cases of neurodegenerative diseases such as Alzheimer's disease. The goal of this project is to identify conserved mechanisms responsible for synapse maintenance in aging animals. The short lifespan, invariant neuronal circuitry, and genetic tools available in the invertebrate Caenorhabditis elegans offer strong advantages for studies of synapse maintenance. While prior studies have largely focused on gross neuron morphological changes during aging, my work is focused on understanding age-related synapse changes. The Francis lab previously demonstrated that C. elegans GABAergic DD motor neurons have dendritic spines which share many characteristics with mammalian dendritic spines. My work focuses on understanding the mechanisms which regulate age-related synapse decline. My preliminary studies suggest that dendritic spine number is reduced in aging animals. My proposed work will define how aging impacts synapse structure and define the mechanisms required for synapse maintenance during aging (Aim 1). In Aim 2, I will identify potential postsynaptic targets crucial for synapse maintenance using proximity labeling and subsequent proteomic analyses. My proposed work will advance our understanding of the mechanisms regulating synapse maintenance in aging animals and potentially identify new gene targets for therapeutic intervention. Importantly, the proposed studies will also provide significant opportunities for new technical and professional training for the applicant.

The goal of this project is to enhance our understanding of how host genetic variation influences infection outcomes. Throughout human history, infectious outbreaks have demonstrated that identical pathogens cause dramatically different outcomes in individual patients, from asymptomatic to fatal. However, the impact of host genetic factors underlying this variation remains largely uncharacterized. Understanding how naturally occurring genetic polymorphisms influence infection outcomes is critical for predicting disease susceptibility and developing targeted interventions. The nematode Caenorhabditis elegans provides a powerful model to characterize the evolutionary forces that shape host-pathogen interactions. The critically important human pathogen Pseudomonas aeruginosa causes lethal infection in C. elegans, providing an experimental platform to study bacterial pathogenesis. The key virulence determinant of P. aeruginosa is the production of phenazine toxins—pyocyanin (PYO), phenazine-1- carboxamide (PCN), phenazine-1-carboxylic acid (PCA), and 1-hydroxyphenazine (1-HP). These toxins generate reactive oxygen species and kill host cells across multiple species. Unlike mammals, C. elegans lacks canonical pattern recognition receptors (TLRs, NLRs) and instead employs "patterns of pathogenesis.” This means C. elegans surveys for pathogen-induced disturbances in host physiology to mount specific immune responses. Previous work from our laboratory demonstrated that the nuclear hormone receptor NHR-86 functions as a non-canonical phenazine receptor, directly binding PCN to activate protective immune responses. This established that C. elegans can co-opt metabolic receptors as pathogen sensors. The central hypothesis of this proposal is that phenazines shaped the evolution of C. elegans immune surveillance mechanisms. I propose that environmental exposure to phenazine toxins fueled the evolution of mechanisms to detect their presence and identify bacterial infection. My preliminary research supports this hypothesis. First, I identified natural variations in susceptibility to bacterial phenazine toxins among wild-caught populations of C. elegans. Second, in a genome-wide association study (GWAS) of 130 natural isolates, I mapped this variation to a major quantitative trait locus (QTL) on Chromosome V (~1.7 Mb region) and four secondary loci. Third, I demonstrated that the Chromosome V QTL independently contributes ~16% to resistance through reciprocal effects using near-isogenic lines. The top candidate gene, nhr-243, encodes a nuclear hormone receptor with a leucine-to-valine substitution in its ligand-binding domain. This suggests convergent evolution where multiple nuclear hormone receptors may have been co-opted as phenazine sensors. This proposal seeks to identify and characterize the genetic determinants of natural variation in resistance to bacterial toxins (Aim 1) and define the genetic architecture of phenazine resistance (Aim 2).

The brain’s ability to process sensory cues and adapt behavior occurs in virtually all animals and is crucial to survival. Understanding how individual neurons integrate multimodal inputs to orchestrate complex behaviors remains largely unknown. The goal of my work is to investigate molecular mechanisms regulating sensory input to a single neuron in the nematode, Caenorhabditis elegans. The robust genetics, and invariant neural organization of C. elegans allows for single-neuron mechanistic investigation in intact, behaving animals. My project aims to uncover molecular regulators encoding foraging behavior. Upon immediate removal of food, animals engage in area-restricted search (ARS) behavior, characterized by increased turning events to maximize re-entry to food. Previous work in the Francis lab has demonstrated that the NLP-12 neuropeptide, released solely from the interneuron DVA, drives ARS. The lab has further suggested that dopaminergic signaling to the DVA couples to NLP-12 release, although a direct mechanism has not been elucidated. My preliminary data suggest that the TRPN (NOMPC) non-selective ion channel, TRP-4, potentially inhibits NLP-12 release. This suggests that dopaminergic, and proprioceptive signaling via TRP-4, act antagonistically to modulate NLP-12 release and drive ARS. In Aim 1, I will investigate how dopaminergic signaling to the DVA drives DVA activity, NLP-12 release, and ARS. In Aim 2, I will uncover where TRP-4 regulates ARS, DVA activity, and NLP-12 release In Aim 3, I will investigate how disruptions in both dopaminergic and TRP-4 signaling regulate ARS, DVA activity and NLP-12 release. This project will define how sensory information encoding food availability and proprioceptive information are integrated to regulate neural activity, neuropeptide release, and behavior.

Innate immune responses rely on rapid and precisely controlled changes in gene expression to fight infection without causing harmful inflammation. How subtle changes in gene expression are regulated to enhance and finely tune responses to different pathogens remains elusive. Transcriptomic studies show extensive alternative splicing after immune stimulation, marked by global shifts in splicing patterns and increased isoform diversity. Although auxiliary splicing factors modulate processing of some immune transcripts, the mechanisms underlying genome-wide shifts in alternative splicing remain unknown. The evolutionary conservation across primates of global shifts in splicing following immune stimulation suggests coordinated regulation of the core splicing machinery, rather than gene-specific control. The core spliceosome comprises five small nuclear ribonucleoprotein complexes (snRNPs), each containing a small nuclear RNA (snRNA) and associated proteins. Altering levels of snRNPs influences splice-site choice and regulates alternatively spliced networks in mammalian cells. Additionally, human snRNA genes form large multigene families, producing variant snRNAs that may differ in splice-site recognition or catalytic efficiency. Preliminary data in LPS-stimulated macrophages show that immune stimulation increases transcription, transiently slows splicing rates, and upregulates a subset of snRNAs, suggesting that the core spliceosome undergoes remodeling upon immune activation. It remains unclear how changes in snRNA levels are regulated, and what the effects of spliceosome remodeling are on the immune cell transcriptome and signaling. This project will define how immune activation remodels core spliceosomal components and determine the consequences of such remodeling for alternative splicing and cytokine expression. The proposed research will provide rigorous training in RNA biology, innate immune signaling, biochemistry, and bioinformatics.

Cardiovascular disease (CVD) is the leading cause of death worldwide, yet the molecular mechanisms that connect metabolic dysregulation to cardiac pathology remain poorly understood. Conditions such as obesity, diabetes, and dyslipidemia disrupt energy balance and lead to metabolic stress, which drives tissue injury, fibrosis, and maladaptive remodeling. A central challenge is to identify the stress-responsive pathways and molecular signals that determine whether tissues adapt or decline under these conditions. Our recent Worm Perturb-seq (WPS) analysis mapped transcriptional responses to 385 metabolic gene perturbations in C. elegans. This dataset revealed lec-8, a galectin family gene, as one of the most strongly induced transcripts following disruption of eight metabolic pathways with established relevance to human CVD. Galectins are carbohydrate-binding proteins conserved from worms to humans. In humans, galectin-3 (LGALS3) is an FDA-approved biomarker of heart failure and is actively involved in cardiac fibrosis, remodeling, and inflammation. Despite this clinical importance, the upstream regulation, physiological roles, and biochemical ligands of galectin pathways under metabolic stress remain poorly defined. This proposal will address these gaps by establishing lec-8 as a model for dissecting conserved galectin pathways in vivo. I will determine whether lec-8 protects animal from metabolic stress or instead contributes to tissue injury. Then, I will identify the transcription factors and signaling pathways that drive lec-8 induction during cardiovascular-relevant metabolic stress. Finally, I will use untargeted metabolomics and glycomics to define the intracellular and secreted products, including advanced glycation end-products, that accumulate when lec-8 is absent. Together, these studies will provide mechanistic insight into how galectin pathways are regulated, how they shape muscle integrity under stress, and what molecular signals they respond to. The results will clarify why galectin-3 is such a strong biomarker of heart disease and whether it also contributes directly to pathology. More broadly, this work will establish a framework for understanding how metabolic imbalance engages conserved gene programs that influence cardiovascular health and disease.

The goal of this F30 Fellowship is to investigate novel mechanisms of metastasis, a significant goal in cancer biology with high clinical relevance. This is particularly significant for triple-negative breast cancer (TNBC), which has extensive intratumor heterogeneity and a propensity to metastasize rapidly, for which effective therapies are lacking. Most solid tumors display wide intratumor heterogeneity and harbor a subpopulation of slow-cycling quiescent cells which act as metastasis initiating cells (MICs). A major challenge MICs face is the considerable amount of oxidative stress encountered in systemic circulation that they need to overcome to form metastases. These cells need to acquire mechanisms that enable them to evade ferroptosis, a non-apoptotic, iron-dependent cell death mechanism, in which excessive oxidative stress leads to lipid peroxidation of cellular membranes and ultimately cell death. Cell biological characterization of the quiescent MIC population revealed an enrichment of ferroportin (SLC40A1), the only known iron exporter, relative to non- MICs. Furthermore, the labile iron pool (LIP) was decreased in MICs relative to non-MICs, indicating that MICs have a lower level of bioavailable iron, an essential reactant required for ferroptosis to occur. Thus, I hypothesize that MICs can evade the ferroptotic stress encountered during the metastatic journey by the ferroportin-mediated export of iron that maintains a LIP below the threshold needed for ferroptosis. Although increased iron metabolism has been implicated in cancer growth and progression in many studies, the hypothesis that a minority population of cells that is responsible for metastasis initiation needs to maintain a low concentration of intracellular iron to survive the metastatic journey has not been considered. There are significant therapeutic implications to these findings, as ferroportin can be inhibited using VIT-2763, a drug that has passed Phase 1 clinical trial for ß-thalassemia with no significant toxicity. To test this hypothesis, two specific aims are proposed: First, to investigate a causal role for iron export mediated by ferroportin in metastasis linked to ferroptosis resistance and second, to investigate the therapeutic potential of inhibiting ferroportin as a method of sensitizing MICs to ferroptosis and reducing metastasis formation. Using both in vitro molecular characterization of MICs and models of experimental and spontaneous metastases in vivo, the role of ferroportin in MICs and the therapeutic potential of inhibiting its function will be rigorously assessed. This novel therapeutic approach has the potential to reduce metastasis formation and sensitize MICs to ferroptosis by exploiting their vulnerability while in circulation. Furthermore, when paired with existing ferroptosis inducers, this therapeutic approach could reduce the burden of previously established metastases, providing the foundation for new therapeutic strategies for targeting metastases.

Ataxia telangiectasia (AT) is a rare neurodegenerative disorder caused by mutations in the ATM gene and is characterized by progressive cerebellar degeneration. Increasing evidence suggests that somatic mutations accumulating in individual neurons may contribute to selective neuronal vulnerability, yet the rate, spectrum, and functional consequences of these mutations in human brain tissue remain poorly understood. This project proposes to apply single cell genomic approaches to quantify somatic mutation rates in cerebellar neurons from individuals with AT compared to neurotypical controls. By leveraging high resolution single cell sequencing and computational analysis, the study aims to identify molecular mechanisms driving mutagenesis, determine how these mutations affect neuronal function, and assess their contribution to neurodegeneration. Results from this work will provide critical insights into how genomic instability impacts neural health and may reveal novel pathways relevant to neurodegenerative disease pathogenesis.

Opioid Use Disorder (OUD) is an ever-increasing public health concern. Despite available treatments, most patients relapse within the first few months. Therefore, there is a substantial need to understand the etiology of OUD and what mechanisms lead to increased opioid use and relapse. Notably, many patients with OUD have reported severe and persistent disruptions to sleep and circadian rhythms. Indeed, long-term opioid use is associated with increased sleep and circadian rhythm disruption, as well as intense cravings and negative affective states leading to vulnerability to relapse. Importantly, the Logan lab has shown that subregions of the striatum, specifically the nucleus accumbens (NAc), display altered molecular rhythms in OUD. Furthermore, preliminary data from the lab suggests that immune signaling in the NAc is altered due to OUD. Microglia, the primary innate immune cells in the central nervous system, have recently been implicated in OUD. Interestingly, microglia exhibit specific molecular rhythms that are dysregulated in OUD, suggesting a potential role for these cells in OUD-related sleep disruption. However, the role of microglial molecular rhythms in the NAc in OUD has yet to been fully elucidated. Thus, this proposal seeks to investigate the molecular rhythms of microglia in the NAc in OUD and how these mechanisms affect opioid-related behaviors. In the aims of this, I will use snRNAseq to characterize circadian rhythms in NAc microglia from human OUD samples and fentanyl-administering mice of both sexes. Additionally, I will disrupt molecular rhythms in microglia using cell type-specific conditional knockout (cKO) of Bmal1 in mice, a master regulator of circadian rhythm, to determine whether inflammatory gene expression and chromatin accessibility is altered. Furthermore, I will examine whether Bmal1 cKO mice display altered drug-taking behavior using fentanyl self-administration. Results from these studies will provide novel insights into microglial molecular rhythms across species and how they contribute to OUD-related behaviors.