Student External Awards for Research Training at Morningside Graduate School of Biomedical Sciences

Cardiovascular trajectories begin in early childhood and continue across the life course. Early recognition and management of cardiovascular disease (CVD) risk factors in childhood stand to improve these trajectories and prevent CVD risk factors in adulthood. One CVD risk factor which in conjunction with obesity has gained prominence in childhood and which affects about 1 in every 25 children in the U.S is hypertension. Health disparities which are intrinsically linked to social determinants of health (i.e., the circumstances that children live in), persist in the prevalence of hypertension with consistently higher rates of this condition seen in those of lower socioeconomic status and those of Black race and Latino ethnicity. The American Academy of Pediatrics (AAP) 2017 Clinical Practice Guidelines recommend regular screening and follow-up for the detection and management of hypertension. However, diagnosis of this condition is rare (~74% undiagnosed), and there are racial disparities in the likelihood of diagnosis as white children are more likely to have this condition diagnosed. Understanding the processes that lead to a diagnosis of hypertension (e.g., blood pressure screening and follow-up), since the release of the updated AAP guidelines, is lacking. Therefore, the goal of the present investigation is to describe the current state of pediatric blood pressure screening and follow-up according to the 2017 AAP guidelines. Using retrospective electronic health record data for children aged 3-17 years from the UMass Memorial Health Care System, a safety-net system, and the largest non-for profit health care system in Central Massachusetts, we will: (1) conduct a one year period prevalence study to quantify the prevalence of guideline adherent blood pressure screening and examine disparities in lack of receipt of guideline concordant care; (2) conduct a cohort study through which we will describe the cumulative incidence of guideline adherent follow-up after the detection of high blood pressure and disparities in the lack of receipt of guideline concordant care; and (3) conduct a qualitative study through which we will describe providers’ experiences with and perceptions of clinical practice guidelines for pediatric blood pressure screening and follow-up. Through the present work it is hypothesized that inequities in care, heterogeneity in follow-up, and challenges to guideline adherence will be identified to inform future efforts to improve clinical practice guideline uptake and pediatric preventive care. Supported by a robust academic environment at the University of Massachusetts Medical School, and a rigorous, tailored training plan, this F31 will position Ms. Goulding to become an independent investigator addressing CVD health disparities among youth.

Epigenetic inheritance is a process by which parental exposure to environmental factors influences offspring phenotype. This field of investigation has wide-ranging implications for human health. Epidemiologic studies have shown that exposure of parents or grandparents to starvation, trauma, cigarette smoke, or other stressors alters offspring susceptibility to cardiovascular disease, obesity, lung disease, or other conditions. Research with animal models has mirrored these findings and offers tools for disentangling the underlying mechanisms of epigenetic information transfer from parent to offspring. Such research has been greatly enabled by recent technological advances, including next generation sequencing and fundamental discoveries like microRNA biology. In vitro fertilization experiments demonstrate that sperm carry sufficient information to propagate epigenetic phenotypes across generations, and research with these paternal epigenetic inheritance models has identified sperm-associated small non-coding RNAs (sncRNA) as carriers of information from father to offspring. I have established an epigenetic inheritance model in which paternal influenza infection, with virus elimination and disease recovery prior to mating, results in an adaptive attenuation of disease severity (significantly decreased weight loss) in response to influenza infection in offspring, as well as a maladaptive altered glucose metabolism. While these phenotypes are robust, the underlying mechanism of information transfer to offspring remains to be determined. In preliminary experiments to address the mechanism I have found that influenza infection alters sperm-associated sncRNA. This proposal addresses the hypothesis that influenza virus-induced changes in sperm-associated sncRNA populations alter embryo development resulting in offspring metabolic and immune phenotypes. Aim 1 elucidates the underlying epigenetic inheritance mechanism through kinetic analysis of sperm sncRNA and early embryo development. Aim 2 determines the specificity of the offspring epigenetic inheritance phenotype to the paternal stressor both directly by challenging with a non-cross reactive strain of influenza virus, and indirectly by further metabolic phenotyping to determine if paternal influenza infection alters glucose homeostasis and liver gene expression in the offspring in ways similar to other paternal stressors. This research will provide valuable insight into the mechanism underlying epigenetic inheritance, and do so within the context of a novel epigenetic inheritance model with direct relevance to human health.

Alzheimer’s Disease (AD) is the most common neurodegenerative disease in the world and the 6th leading cause of death in the United States. Despite significant effort, current AD therapies are highly limited in both number and efficacy. Hope for new therapeutic interventions has emerged from recent genome-wide association studies (GWAS), which have identified genes whose mutations are linked to altered AD susceptibility. One of the strongest and most reproducible genetic associations with altered AD risk are members of the Membrane Spanning 4a (MS4A) gene family. In fact, current genetic data suggest that MS4A polymorphisms account for approximately 10% of all AD cases. However, a limited understanding of MS4As has hindered the development of therapies targeting these proteins. Single cell transcriptional profiling has revealed that the Ms4a genes genetically linked to AD are selectively expressed in a subset of microglia, the resident innate immune cells of the central nervous system. Furthermore, MS4A-positive subsets of microglia display a phenotype similar to microglia seen in neurodegenerative disease states, necessitating inquiry into the role of MS4As in microglia. We found that Ms4a-positive microglia are enriched for phagocytic machinery versus Ms4a-negative microglia, and animals deficient for individual Ms4a family members have significantly reduced expression of genes important for phagocytosis compared to wild-type (WT) animals. Microglial phagocytosis is a dynamic process by which brain debris, dying cells, unwanted synaptic connections, and toxic molecules are eliminated and disruption of this process is thought to underlie numerous neurological disorders, including AD. Thus, this proposal will test the hypothesis that MS4As regulate microglial phagocytosis and alter AD pathogenesis. To test this hypothesis, aim 1 will examine microglia phagocytosis of synapses, dead neurons, and amyloid-beta 42- one of the neurotoxic, pathogenic agents of AD- both in vitro and in vivo using Ms4a-deficient and WT microglia. These experiments will characterize the role of MS4As in microglia, and provide insight into how MS4As affect microglial phagocytosis. Aim 2 will investigate the role of MS4As in AD pathogenesis. Although GWAS studies have strongly linked MS4A polymorphisms to AD susceptibility, the role of MS4A genes in AD is unknown. Some MS4A alleles confer AD protection while others increase AD susceptibility, and MA4A polymorphisms are often located in non-coding regions of these genes. To investigate whether MS4As are protective against or contributive toward AD, mice that are homozygous deficient for individual Ms4a family members will be crossed to the 5XFAD mouse model of AD and pathological features of AD, including behavioral defects in memory, amyloid beta plaque formation, microgliosis, neuronal loss, and synapse elimination will be assessed. Together, these aims will provide fundamental new insight into the role of MS4As in AD and contribute to the development of therapeutic approaches targeting MS4A receptors.

Huntington’s Disease (HD) is caused by a CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT) gene that produces mutant HTT mRNA and protein. Expression of mutant HTT leads to progressive neurodegeneration via mechanisms that are poorly understood. Because HD is a genetically-defined disease, it is an ideal candidate for study and therapeutic intervention by small interfering RNAs (siRNAs) – which incorporate into the RNA-induced silencing complex (RISC) to target and degrade disease-causing genes. With the development of a divalent (di)-siRNA chemical architecture, siRNAs can be delivered throughout the central nervous system (CNS) of rodents and non-human primates. To ensure stability in the CNS, di-siRNAs require chemical modifications on every nucleotide. However, chemical modifications can affect siRNA activity and cellular localization – limiting the utility and flexibility of di-siRNAs in the CNS. The most common nucleotide modifications in siRNA replace the 2′OH of the ribose with 2′-Fluoro (2′F) or 2′-O- Methyl (2′OMe). Recent work suggests that incorporation of 2′OMe and 2′F at certain nucleotide positions may hinder siRNA loading into RISC or target binding/cleavage by RISC, and may alter the nuclear-to-cytoplasmic localization of siRNAs. Yet, the limited scope of this work has made it difficult to identify general design parameters for efficacious, compartment-specific siRNA. With guidance from Drs. Anastasia Khvorova (siRNA chemistry), Neil Aronin (HD), Phillip Zamore (RNA biochemistry) and Athma Pai (RNA sequencing), this proposal will systematically assess the impact of modification patterns on siRNA efficacy and cellular localization in the CNS to optimize siRNAs as an HD therapy and research tool for dissecting HD pathology. Aim 1 will characterize how siRNA chemical modifications impact RISC loading in vivo and target binding and cleavage in vitro. To measure how modifications alter RISC loading, a pool of differentially-modified siRNAs will be injected into the CNS of mice, RISC will be pulled down and loaded siRNAs will be sequenced. To determine the effect of siRNA chemical modifications on RISC-target interactions, target binding and cleavage kinetics will be measured for a panel of modified siRNAs using single-molecule total internal reflection fluorescence microscopy. Mechanistic insight into how modifications impact siRNA efficacy in the CNS will provide a framework with which to design optimized siRNAs to treat HD and other CNS diseases. Aim 2 will use the same modified siRNA pool from Aim 1 to identify optimal modification patterns for enhanced nuclear localization of siRNA in the CNS. These data will be used to design efficacious chemically-modified di-siRNAs targeting nuclear or cytoplasmic-only HTT RNA. These di-siRNA will then be injected into YAC128 HD mice and the effect on motor deficits, neurodegeneration, and striatal mRNA expression will be assessed. These results will provide valuable insight into the biology of HD and determine the potential of nuclear RNA-targeting siRNAs as a therapeutic paradigm for repeat expansion disorders with underlying RNA toxicity.

As animals explore their environment, they encounter millions of chemical signals that must be correctly interpreted to identify food and mates and to avoid predation. In mammals, the majority of this chemical information is detected by the sensory neurons of the olfactory system, (OSNs), which express more than one thousand distinct olfactory receptors (ORs), specialized chemoreceptors that sense volatile, chemical odorants. While many of these receptors have been molecularly defined, how they function to elicit appropriate behavioral responses remains largely unclear (i.e., how does a mouse know to run away from a cat and toward a piece of cheese). This proposal outlines a series of experiments aimed at taking advantage of the opportunities offered by the olfactory system to begin to characterize the mechanisms by which chemosensory information is detected and processed to generate specific, stereotyped behavioral responses. We have recently identified a new family of olfactory receptors encoded by the Ms4a family of genes, which our preliminary experiments suggest are required to mediate innate avoidance responses to stimuli such as predator-derived odorants that signify danger to the animal. However, how MS4A receptors function to elicit these behaviors is largely unknown, which significantly impairs our understanding of how the olfactory system interprets relevant sensory stimuli to elicit appropriate behavioral responses. To begin to elucidate MS4A chemoreceptor function, in Aim 1 I will use mutagenesis, electrophysiology, and calcium imaging to determine how MS4As bind ligands and signal their presence. In Aim 2, I will use ex vivo and in vivo preparations as well as mouse behavioral assays to determine the contribution of MS4As to transducing predator odor presence into appropriate innate avoidance responses. Together, the experiments proposed here will characterize a novel family of odorant receptor and how it signals the presence of ethologically relevant odors. Broadly speaking, these studies seek to illuminate basic principles of olfaction, provide insight into mammalian chemosensation, and shed light on the neural pathways associated with sensory perception.

The current U.S. opioid epidemic has fueled an increase in injection drug use and, in turn, an alarming surge in new hepatitis C virus (HCV) infections. Between 2010 and 2015, the incidence of HCV increased by 294% nationally, driven primarily by a rise in injection drug use and risky injection behavior – namely syringe sharing. This growing epidemic has disproportionately affected young people who inject drugs (PWID) in rural communities. There is an urgent need to implement tailored and effective harm reduction strategies to rural PWID who are disproportionately impacted by HCV. Although research has shown that syringe services and pharmacy syringe sales (i.e sterile syringe sources) are associated with a reduction in injection-mediated risks and HIV transmission, the evidence for whether these services reduce HCV risk among PWID remains mixed. This proposal will applying the risk environment model to evaluate the influence of sterile syringe sources on the HCV risk environment. Specifically, this proposal will evaluate whether spatial proximity to sterile syringe sources and receptive secondary syringe exchange are associated with HCV serostatus among rural PWID. The aims are: (1) To evaluate the association between road network distance to the nearest sterile syringe source (SSP or pharmacy that sells nonprescription syringes) and HCV serostatus; (2) To use egocentric social network analysis to evaluate the association between receptive secondary syringe exchange and HCV serostatus; (3) to explore and unpack rural PWIDs’ perceptions of and experiences with syringe acquisition and syringe sharing practices through in-depth interviews. These findings could help inform the development of future harm reduction interventions in rural New England, a region of the country that has been particularly hard hit by the opioid epidemic and related HCV infections.

After injury, axons begin to die via a process that is characterized by axonal fragmentation and disintegration of myelin sheath. This process is often termed Wallerian degeneration after Augustus Waller. Wallerian-like degeneration, which is morphologically similar to Wallerian degeneration, is associated with the early stages of many neurodegenerative diseases, including as Alzheimer’s, Huntington’s, and Parkinson’s Diseases. Wallerian degeneration was long thought to occur passively, but the discovery of proteins that actively prevent or promote degeneration negated this idea.

Down syndrome (DS) is a common chromosomal disorder, occurring in 1 in 700 live births in the US. Half of newborns with DS begin life with congenital heart defects (CHD) requiring surgical correction, and there is evidence of potentially related vascular deficits. Children with DS are typically happy and sociable but have mild-moderate intellectual disability which can progress to severity in adults. High risks for other co-morbidities include pulmonary hypertension, autism, and almost inevitable early-onset Alzheimer Disease; any deficit in angiogenesis could contribute to these conditions. The Lawrence lab has pioneered a strategy to “silence trisomy”, by translating the basic mechanism of X-inactivation, the XIST gene, to chromosome 21 (chr21). Upon induction, XIST RNA spreads across and silences all ~250 genes across that Chr21, in cis, and this was shown to correct known hematopoietic pathogenesis from DS-induce pluripotent stem cells (iPSCs), in vitro. Using this tightly controlled system the lab recently showed that chr21 over-expression has a direct cell autonomous effect on endothelial cells which impairs micro-vessel formation in vitro and impacts gene networks important in both heart development and angiogenesis. The lab has now developed a modified method to silence just a small part of chr21, the proposed “Down Syndrome Critical Region” (DSCR). This now provides the opportunity to directly test the important hypothesis that a small cluster of genes, within the DSCR, cause major DS phenotypes. Using the available inducible iPSC system, I will test if silencing the DSCR enhances formation of micro-vessels by human endothelial cells in vitro. In addition, I am working to translate “trisomy silencing” with XIST in vivo to a mouse model (carrying human chr21) which exhibits CHD. This work utilizes an innovative approach with high significance for investigating the causes of CHD and vasculature in DS, but will also test a potentially transformative concept, relevant for other chromosomal disorders, which collectively are more common than DS and frequently impact heart structure or function. The work proposed will forward the prospects of “chromosome therapy” as we advance the basic biology of cardiac and vascular deficits in DS.

Glioblastoma multiforme (GBM) is the most frequent and aggressive primary brain tumor in adults. Despite significant progress being made in characterizing the genetic, epigenetic, and molecular drivers of GBM, effective therapies remain limited. A considerable hurdle between GBM research and translation into efficacious treatment is the extensive infiltration and molecular heterogeneity of GBM tumors, both of which cause tumor recurrence after treatment. Consequently, the average survival expectancy for GBM patients is less than 15 months after diagnosis. For therapies to be effective in treating these lethal tumors, they must overcome both GBM infiltration and heterogeneity. Antisense oligonucleotides (ASOs) – compounds that can modulate the expression of virtually any RNA molecule – offer distinct advantages for combating GBM infiltration and heterogeneity. Following local delivery, ASOs distribute throughout the brain, a necessary feat to reach infiltrative GBM cells. Moreover, as sequence- programmable agents, ASOs possess the specificity and flexibility required to modulate expression of multiple gene targets – an effective strategy to characterize and combat GBM heterogeneity. In 2016, the ASO drug, nusinersen, was FDA approved to treat spinal muscular atrophy, establishing the clinical efficacy of ASOs in the central nervous system. However, several ASO drug candidates for GBM have failed in clinical trials due to high toxicity and low potency. Identifying potent, well-tolerated ASOs for gene modulation in brain tumors would open the door to developing effective GBM therapies. The Watts lab has developed chemically-optimized, non-toxic ASOs with enhanced distribution and potency in the brain following local CNS delivery. However, their effect on GBM is unknown. The goal of this proposal is to identify ASOs that potently and safely silence GBM drivers, and assess the impact on tumor progression and resistance in vivo. With support from Drs. Jonathan Watts (oligonucleotide chemistry), Richard Moser (neuro- oncology), Sunit Das (GBM mouse models), Manuel Garber (bioinformatics), and Michael Green (cancer biology & therapeutics), Aim 1 will test the ability of chemically-modified ASOs to silence a clinically-relevant GBM driver (ATF5), inhibit cell proliferation, and induce cell death in molecularly-distinct patient-derived GBM cell lines. Lead compounds will then be evaluated for therapeutic efficacy in a GBM mouse model by measuring ATF5 silencing, tumor growth, and mouse survival following treatment. In Aim 2, the consequences of ASO-mediated silencing on GBM tumor biology will be investigated. ASOs targeting ATF5 will be injected into GBM tumors of mice. After treatment response, residual GBM cells will be isolated for single-cell RNA sequencing to characterize the transcriptome and determine how ASO silencing perturbs functional heterogeneity. This aim will establish a rational framework for drug combinations to minimize GBM tumor resistance. Collectively, the proposed project will advance ASOs as a novel GBM therapeutic and as a tool to dissect GBM progression.

All cells must replicate their genome once per cell cycle. To ensure proper duplication, cells integrate hundreds of factors that copy, surveil, and repair our genetic information. Proliferating Cell Nuclear Antigen [PCNA] and Rad9-Rad1-Hus1 [9-1-1] are ring-shaped clamps that act as master “conductors” that regulate many of the factors that replicate and maintain our DNA. PCNA is a homotrimeric ring that coordinates the replisome during DNA synthesis to work in tandem with DNA repair, chromatin remodeling, and cell cycle progression. When cells experience dsDNA breaks, they use the heterotrimeric clamp 9-1-1 to coordinate specific “SOS” repair factors. The collaborative efforts of both clamps are critical for genome stability. Many cancers are linked to inappropriate clamp coordination and changes in their expression. Because sliding clamps are central to many oncogenic pathways, we must address how they regulate themselves and their client partners. This proposal aims to address the following questions about sliding clamps: 1) How do sliding clamps coordinate their various partners? 2) Does the time sliding clamps spend on DNA influence genome stability? and 3) What determines site-specific loading of sliding clamps? I propose a multidisciplinary approach to address these questions about sliding clamps by investigating two-disease causing PCNA variants [PCNA-S228I [serine to isoleucine] and PCNA-C148S [cysteine to serine]] and the loading mechanism of 9-1-1. I hypothesize that sliding clamps control genome integrity via site-specific loading, proper partner interactions, and residence-time on DNA. I further hypothesize that PCNA-S228I and PCNA-C148S disrupt genome integrity by either promoting premature DNA dissociation or disrupting partner interactions. Finally, I hypothesize that the Rad17 subunit alters the clamp loader structure to specifically load the 9-1-1 clamp at sites of DNA damage. In aims 1 and 2, I will use PCNA-S228I and PCNA-C148S to address how clamps “choose” their partners and regulate their time on DNA. I will use x-ray crystallography, unfolding experiments, and a series of functional assays to determine how each variant compromises genome stability. In aim 3, I will determine the loading mechanism of clamp 9-1-1 to address how clamps are loaded to specific sites in the genome. I will use cryo-electron microscopy to determine how Rad17-RFC binds to clamp 9-1-1. Collectively, my work will broaden our insight into the factors that cause genome instability which may augment the development of personalized chemotherapeutics.

The mechanisms of pathogen sensing and immune effector induction in intestinal epithelial cells are not completely understood. Disruption in the mechanisms of pathogen sensing and immune homeostasis in intestinal epithelial cells can lead to dysbiosis and inflammation, as well as susceptibility to bacterial infection. Key insights into intestinal epithelial cell immunity and host-pathogen interactions have been made using the nematode C. elegans. Nematodes mount innate immune defenses against bacterial infection via conserved immune pathways, but the mechanisms of pathogen detection are unknown in this organism. In nematodes, the family of nuclear hormone receptors (NHRs) has dramatically expanded compared to other metazoans. NHRs are ligand-gated transcription factors that sense endogenous and exogenous signals to induce adaptive transcriptional responses. The C. elegans genome encodes 274 NHRs, of which 260 are homologs of human HNF4α. HNF4α is a key NHR involved in inflammatory bowel disease, though the mechanism through which HNF4α mediates inflammatory bowel disease in humans is unknown. The central hypothesis of this proposal is that C. elegans HNF4α homologs are an ancient family of pathogen sensors whose evolutionary expansion in C. elegans was driven by their function in detecting diverse pathogens. The following key findings support this hypothesis: (i) The nuclear hormone receptor, NHR-86/HNF4α, senses the cellular environment and activates C. elegans intestinal immune defenses; (ii) NHR-86/HNF4α is required for pathogen resistance and immune response towards the gram positive human pathogen E. faecalis; and (iii) A different C. elegans HNF4α homolog is required for pathogen defense and immune effector regulation against the gram negative pathogen P. aeruginosa. In this proposal, Aim 1 will define the role of C. elegans NHR-86/HNF4α in pathogen detection and immune effector induction during E. faecalis infection using a combination of transcriptomics, ChIP- sequencing, tissue-specific rescue and genetic epistasis. Aim 2 will characterize the function of a separate C. elegans HNF4α homolog in pathogen sensing during P. aeruginosa infection. The approach includes: transcriptomics, global NHR binding site identification, tissue specific rescue, and P. aeruginosa genetics. Collectively, these studies will characterize a fundamentally new paradigm of immune activation, which will solve a major conundrum of how pathogens are sensed in C. elegans. These findings will also establish NHRs as evolutionarily ancient pathogen sensors. Ultimately, the expectation is that detailed dissection of this mechanism will shed light on the role of HNF4α in mammalian pathogen sensing and inflammatory bowel disease.

The overarching goal of this project is to understand how non-apoptotic cell death is activated by the DNA damage response (DDR). In response to genomic insult, the DDR activates DNA repair and cell cycle arrest to resolve the damage and promote cell survival. Alternatively, in cases of severe damage, the DDR will activate apoptotic cell death. These critical pro-survival and pro-death responses are all regulated by p53. The centrality of p53 in the DDR allows cells to quickly and flexibly respond to different types of DNA damage. However, in the absence of p53, what outcome is predicted by this model? While we might expect that p53 removal abrogates both cell cycle arrest and apoptosis, many p53-mutated cancers are still able to execute cell death in response to DNA-damaging drugs. This suggests the presence of an additional and heretofore undescribed pathway linking the DDR to cell death. We found that DNA damage is also capable of inducing non-apoptotic cell death. Furthermore, non-apoptotic death is preferentially activated in cells that lack p53. Our strategy for characterizing this novel DNA damage-induced non-apoptotic death was to perform a whole-genome CRISPR screen. Genome-wide CRISPR screens do not typically identify death regulatory genes. To overcome this limitation, we devised a new experimental and computational method for calculating the drug-induced death rate of each single-gene knockout. Based on the results of our screen, in Aim 1 we will test the hypothesis that ROS and mitochondrial permeability transition (MPT) are required for DNA damage-induced death in the absence of p53. We will use CRISPR/Cas9 mediated knockout to compare DNA damage-induced MPT to canonical MPT. We will monitor activation of MPT using fluorescence microscopy, and use TEM to characterize mitochondrial morphologies. Our CRISPR screen also identified TGF-β signaling as a negative regulator of DNA damage- induced non-apoptotic death. In Aim 2, we will identify TGF-β pathway components that contribute to the suppression of non-apoptotic death, and determine the generalizability of this knowledge across cell lines. We will extend this exploration to an in vivo mouse model of cancers generated with and without functional p53. Our characterization of DNA damage-induced non-apoptotic death will improve our understanding of how p53- mutated cancers respond to chemotherapeutics. Ultimately, we hope that this work will improve our ability to predict which cancers will respond to DNA-damaging drugs, as well as which death pathways can be targeted to enhance treatment efficacy.

The apolipoprotein-B mRNA-editing catalytic polypeptide-like 3 (APOBEC3, or A3) family of cytosine deaminase enzymes forms part of the innate immune system - hypermutating cytosine (C) to uracil (U) in single-stranded DNA (ssDNA), as a first response to invading viruses. Specific A3 enzymes, including A3G, were first shown to potently restrict human immunodeficiency virus (HIV). However, expression of these enzymes is a delicate balance – low level deamination of the HIV genome provides a constant source of viral mutation, contributing to high viral fitness and rapid resistance to anti-viral drugs. Moreover, upregulation of other A3 enzymes, like A3A and A3B, is linked to many cancers, helping to expand genetic diversity in tumors to supercharge progression, metastasis, recurrence, and drug resistance. Abolishing activity of specific A3 enzymes may slow viral evolution and prevent tumor recurrence. Structural examination of A3 active sites reveal their close homology to that of human cytidine deaminase (CDA), which converts C to U in single nucleosides. Introducing CDA inhibitors (transition state analogues, or TSAs) in place of the target C in ssDNA enables delivery to the A3 active site. Yet, development of selective inhibitors against individual A3 family members remains a challenge. Recently, our lab solved the co-crystal structures for A3A-ssDNA and A3G-ssDNA, and have also discovered that both the tertiary structure and DNA sequence flanking the target cytosine confer specificity to different A3 enzymes. I hypothesize that by exploiting these preferences – through manipulation of DNA sequence, oligonucleotide tertiary structure, and rational placement of chemical modifications – I can create potent and selective A3 inhibitors. Leveraging the Watts lab’s expertise in nucleic acid chemistry and the Schiffer lab’s extensive experience with A3G, I will first develop A3G-selective inhibitors, and then A3A and A3B inhibitors.

Pulmonary lung fibrosis is a poorly understood process that can arise in pediatric patients with gain-of-function mutations that disrupt the regulation of the cytosolic double stranded DNA sensing pathway, cGAS-STING. This project will define the role that B cells play in mediating lung fibrosis in a mouse model of STING gain-of- function autoinflammation that recapitulates a human disease known as STING Associated Vasculopathy with onset in Infancy (SAVI). The expectation is that the results of these studies will offer insights into the mechanisms by which B cell contribute to fibrotic lung disease and assess, using murine models, whether targeting B cells is a valid strategy for prophylactically treating lung fibrosis.

Alzheimer’s Disease (AD) and Amyotrophic Lateral Sclerosis (ALS) are multifaceted, progressive neurodegenerative conditions that place a monumental burden on patients, providers, and the public healthcare system. No disease-modifying treatments are currently available for either AD or ALS. Although the etiology of each disease is well studied, strategies targeting characteristic features of disease pathogenesis— e.g., beta-amyloid in AD—show limited clinical efficacy. Identification of novel targets that modify progression of neurodegeneration is needed for innovative therapeutic development across neurodegenerative disorders. AD and ALS are caused by genetic and environmental factors that alter downstream pathways like lipid homeostasis. A critical player in systemic and central nervous system (CNS) lipid transport, that is also implicated in the onset and progression of neurodegeneration, is <em>apolipoproteinem> E (ApoE). Genetic deletion of ApoE reduces neuropathology in mice, but also causes atherosclerosis. Thus, despite its implication in disease, the diverse functionality of ApoE in its distinct biological “pools” (i.e. systemic and CNS) makes it a challenging therapeutic target. Reducing individual ApoE pools may circumvent this issue. However, the independent effects of systemic or CNS ApoE silencing on neurodegenerative diseases are unclear. The goal of this proposal is to determine the relationship between systemic and CNS ApoE pools, and their effects on disease progression in AD and ALS mice. The project will take advantage of chemically-stable, self- delivering small interfering RNAs (siRNAs) that enable sustained, <em>tissue-specificem> silencing of target mRNA. GalNAc-siRNAs specifically deliver to liver (site of systemic ApoE production), and divalent (Di)-siRNAs deliver throughout the brain and spinal cord after intra-cerebroventricular (ICV) injection. With guidance from Drs. Anastasia Khvorova (siRNA chemistry), Robert Brown (ALS), Evgeny Rogaev (AD models), Andrew Tapper (animal behavior), and Thomas Smith (neuropathologist), GalNAc- and Di-siRNA will be used to silence hepatic and CNS ApoE, respectively, and the effects on CNS and systemic ApoE pools, and AD and ALS phenotypes, will be examined. In Aim 1, GalNAc-siRNA targeting ApoE will be subcutaneously injected into mice. In Aim 2, Di-siRNA targeting ApoE will be delivered to the CNS via ICV injection. Both aims will utilize the APP/PSEN1 mouse model of AD and the SOD1G93A mouse model of ALS, and will measure systemic and CNS ApoE and cholesterol levels, and AD and ALS neuropathology and behavior two months post injection. These studies will advance the understanding of how ApoE pools interact in the context of neurodegeneration, and the effects on disease progression. Such findings will inform strategies for safe and effective therapeutic targeting of ApoE in AD, ALS, and age-related neurodegenerative disorders.

A glaring sexual orientation-related disparity is in the prevalence of disordered eating behaviors (DEBs), including severe calorie restriction, self-induced vomiting, laxative and diet pill use, and binge-eating. One in three sexual minority young people (lesbian, gay, bisexual, and other non-heterosexual individuals) engage in DEBs, a seven-fold higher odds than their heterosexual peers. There are considerable health consequences associated with these behaviors, such as metabolic and reproductive health issues, substance use, depression, and suicidality. However, research on DEBs lags behind that on other sexual orientation-related health disparities, with critical gaps including failing to consider both within-group diversity in risk and the upstream social determinants of the observed disparities. Importantly, experiencing multiple forms of social disadvantage has been shown to increase risk of eating-related pathologies, including DEBs. Gender nonconforming and higher-weight (i.e., overweight/obese) statuses are especially relevant dimensions of disadvantage to consider, as these groups experience high levels of appearance-based discrimination and may use DEBs as dangerous body-modification practices to cope. Sexual minorities who experience further marginalization through membership in these groups may encounter unique and/or compounding social stressors that exacerbate risk. Examining the intersectionality of sexual orientation, gender expression, and weight status is thus critical to addressing these research gaps. Using the Growing Up Today Study (GUTS), a national longitudinal cohort study of over 27,000 participants (~20% of whom are sexual minorities), the aims of this proposal are to: 1) Quantify the intersectional effects of sexual orientation, gender expression, and weight status on risk of DEBs among young adults; 2) Quantify the effects of interpersonal-level determinants (bullying victimization, weight-based harassment) on risk of DEBs by sexual orientation, and evaluate differences by gender expression and/or weight-status; and 3) Quantify the effects of structural-level determinants (discriminatory social conditions, state policies) on risk of DEBs by sexual orientation, and evaluate differences by gender expression and/or weight-status. The National Academy of Medicine’s 2011 landmark report on sexual minority health stressed the importance of adopting an intersectional framework for disparities research to inform the development of inclusive health equity efforts. Applying this lens through leveraging novel statistical methods will further understanding of a critically understudied sexual minority health issue and help identify high-risk subgroups and modifiable contextual risk factors. A tailored training plan accompanies this proposal and outlines the steps required to advance the Applicant’s career as an independent researcher focused on intersectional health disparities research.

CRISPR-Cas9 technology has profoundly advanced genetic research and gene therapy by enabling rapid, precise, and programmable genome editing to study gene function or correct mutations in cells and in vivo. Among CRISPR-mediated gene editing approaches, cytidine and adenine base editors (CBEs and ABEs) enable efficient and precise single-base transitions in dividing and non-dividing cell types. Base editors comprise a catalytically-impaired nickase Cas9 (nCas9) fused to a cytosine deaminase (CBE) or adenine deaminase (ABE) that is guided to a target site by a “guide RNA”. With the potential to enable all four nucleotide transitions in the context of a base-pair (C:G→T:A or A:T→G:C), base editors have the potential to cure a wide range of genetic disorders. Realizing this hope requires efficient and safe in vivo delivery methods. In vivo delivery of base editors relies on adeno-associated virus (AAV) vectors. Due to the limited packaging capacity of AAVs and the large size of nCas9 orthologs (e.g., SpyCas9 from Streptococcus pyogenes), delivery requires two AAVs encoding an intein-split nCas9-BE that fuses into a functional complex when co-expressed in cells. Dual AAVs encoding intein-split SpyCas9-BEs achieve therapeutically-relevant levels of base editing in pre-clinical disease models, including in the central nervous system (CNS). Nonetheless, dual AAV SpyCas9-BE delivery suffers from several limitations, including: toxicity from increased viral load; high vector production costs; immune response and off-target editing caused by sustained expression of nCas9-BE components; and limited ability of guide RNA to specify multiplexed edits. Under guidance from Drs. Erik Sontheimer (CRISPR), Miguel Sena Esteves (AAV delivery), Anastasia Khvorova (oligonucleotide chemistry), and Athma Pai (sequencing, bioinformatics), this proposal aims to develop flexible delivery approaches for efficient and safe base editing in vivo. This project will take advantage of established gene therapy modalities, including a single AAV vector encoding a compact Cas9 from N. meningitidis (Nme2Cas9), and chemically-modified oligonucleotides. Aim 1 will optimize and validate an all-in-one AAV encoding a compact Nme2Cas9-ABE and its guide RNA for efficient in vivo base editing in mice. The use of a compact ABE for AAV delivery will decrease viral load, production costs and may increase delivery efficiency. Aim 2 will develop chemically-modified Nme2Cas9 crRNA (target-specify portion of guide RNA) for co-delivery separate from an AAV encoding Nme2Cas9-ABE and tracrRNA (invariant portion of guide RNA). This approach will provide a way to control nCas9-BE expression and streamline multiplexed base editing via delivery of multiple crRNA. Although these delivery approaches are applicable to variety of tissues, this study will focus on the CNS, where AAV- and oligonucleotide-based therapies have shown some success, but a dire need for transformative therapeutics remains. Completion of this study will establish novel delivery approaches to advance the utility of CRISPR for in vivo applications, including functional genomic studies and base editing therapies.

As the most abundant and deadliest entities on earth, bacteriophage play an essential role in many biological environments. While there are well-developed phage model systems that have informed our understanding of phage in the past 60 years, these systems have structural limitations. Here, we use a unique thermophilic siphovirus, P74-26, with extraordinary strength and stability to fill in the knowledge gaps that remain and take a closer look at some of the fascinating abilities of a phage that developed under the evolutionary pressure of a hot spring. Double-stranded DNA (dsDNA) viruses, which include bacteriophage along with herpesviruses, adenoviruses, use a powerful ATPase motor to pump their genome into an immature structure called the procapsid. Genome loading leads to immense internal pressure, resulting in a conformational change from a spherical particle to an expanded, pressurized icosahedron. The extreme stability of P74-26 despite high temperature and pressure makes it a novel tool for elucidating the intricacies of phage assembly and thermodynamics. In this study, we will combine cryo-EM, SEC-multi angle light scattering, and mass spectrometry to examine physical and mechanistic aspects of P74-26. A structure of the uniquely long phage tail tube will provide perspective into the mechanism of DNA ejection and possible evolutionary advantages for such length. Additionally, we have found that the major capsid protein spontaneously assembles, allowing us to create a controlled system for determining the essential components for viral head stability.

A leading cause of Cystic Fibrosis (CF) is premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Suppression of translation termination at PTCs—i.e. PTC readthrough—to restore full-length CFTR protein may be a treatment strategy. Yet, current PTC readthrough drug candidates for CF are toxic (e.g. aminoglycosides) or ineffective (e.g. ataluren). Efficacy of PTC readthrough depends on efficiency of translation termination at the PTC. Thus, manipulating the molecular mechanisms of CFTR PTC termination to lower efficiency may improve PTC readthrough efficacy. However, strategies for such manipulations are limited in the absence of a detailed understanding of translation termination on CFTR PTCs. In the current model for normal termination, eukaryotic Release Factors 1 and 3 form a complex (eRF1•eRF3) that releases a newly synthesized protein from the ribosome. eRF1 recognizes a tetra-nucleotide stop codon at the end of an open reading frame, and catalyzes peptidyl-tRNA hydrolysis. Poly-A binding protein (PABP), which binds at 3′ ends of mRNA, recruits eRF3 and enhances termination efficiency. However, it remains unclear how PABP, eRF1, eRF3, and the tetra-nucleotide stop codon recognize the PTC to produce truncated CFTR protein. The goal of this proposal is to determine the biochemical and structural mechanism of translation termination. With guidance from Dr. Andrei Korostelev (expert in biochemical and structural mechanisms of translation), Dr. Allan Jacobson (expert in premature translation termination and PTC read-through), Dr. Phillip Zamore (RNA biochemist), Dr. Chen Xu (cryo-EM instrumentalist), and Dr. Nikolaus Grigorieff (expert in cryo-EM method development), release assays will be optimized to study the efficiency of translation termination mediated by eukaryotic release factors, and ensemble time-resolved (ENTIRE) cryo-EM will be used to capture structural intermediates of enzymatic reactions. Aim 1 will use defined mammalian translation systems to measure the individual effects of stop codon context, eRF1•eRF3, and PABP on the termination efficiencies (kcat/KM) of CFTR PTCs and the true stop codon. Aim 2 will visualize how the ribosome terminates on CFTR PTC G542X in its natural sequence context using ENTIRE cryo-EM. Collecting data at multiple time points will identify conformational changes and interactions between mRNA sequence, eRF1•eRF3, and PABP during termination. To reveal the termination mechanism on CFTR PTCs, structures and their progression intermediates will be compared with those recorded on the true CFTR stop codon. If successful, this study will reveal key molecular determinants of CFTR PTC termination, and may inform strategies to induce PTC readthrough for CF treatment.

The goal of this proposal is to dissect the molecular signaling between microglia and neurons that regulates synapse elimination in response to changes in sensory experience. Despite compelling evidence that microglia, the resident brain macrophages, play important roles in eliminating synapses in development and disease, the precise neuron-to-microglia molecular signaling that drives this process is poorly understood. I recently discovered a signaling pathway necessary for microglia-mediated synapse elimination by utilizing the well-described circuitry of the mouse barrel cortex circuit as a model to manipulate sensory experience and dampen neuronal activity. Here I found microglia robustly engulf synapses in the barrel cortex following either whisker lesioning or trimming, and that this engulfment is dependent on the microglial CX3CR1 receptor and its canonical neuronal ligand, CX3CL1, but not complement. Using single-cell RNAseq I also found that neuronal Cx3cl1 was not differentially regulated in the cortex following whisker removal, but the protease Adam10, known to cleave membrane-bound CX3CL1 into a soluble form, is increased following lesioning. Importantly, pharmacological inhibition of ADAM10 resulted in synapse elimination defects that phenocopied CX3CR1 and CX3CL1-deficient mice. These data suggest that post-translational modification of neuronal CX3CL1 by ADAM10 is required to regulate microglial synapse elimination in the cortex following whisker removal. Several exciting new questions have now arisen, which I will tackle in this proposal: 1) What is the cellular source of ADAM10 and is it localized to synapses (Aim 1)? 2) Do other subcortical synapses within the barrel circuit remodel via ADAM10-CX3CL1-CX3CR1 signaling and does this differ between whisker lesioning and trimming (Aim 2)? I hypothesize ADAM10 is derived from layer IV excitatory neurons to regulate microglia- mediated synapse remodeling and that ADAM10 signaling is specific for cortical synapse rewiring after whisker trimming and lesioning, but not for sub-cortical synapse remodeling. To test this hypothesis, I have acquired powerful in vivo molecular genetic tools to manipulate ADAM10 function in specific cells. I have also developed collaborations to learn and perform cutting-edge whole tissue clearing by iDISCO to assess structural remodeling of entire circuits. Finally, I have a strong mentoring team that includes my mentor Dr. Dorothy Schafer with expertise in microglial function within neural circuits, my co-mentor Dr. Andrew Tapper with expertise in structural and functional mapping of brain circuits, and collaborators with expertise in iDISCO. Together, I am in a strong position to molecularly dissect how ADAM10 modulates neuron-microglia signaling necessary for remodeling brain circuits. This could be highly relevant for neurodegenerative disease where microglial dysfunction, synapse loss, and ADAM10 have been implicated. In the process, I will receive training in a variety of microscopy and molecular genetic approaches that will provide a foundation for my future career as an independent principle investigator at an academic institution focused on dissecting functions for glial cells within neural circuits.

Germ cell immortality is essential for fertility and for species survival. To proliferate indefinitely, germ cells depend on mechanisms that maintain genome integrity and epigenetic programs. In animals, small RNAs that interact with Argonaute proteins of the PIWI family—called piRNAs—serve as a vanguard of transcriptome and epigenome integrity in the germline, by identifying and silencing transposable elements and by regulating germline gene expression. In many animals, piRNAs are essential for germline health. Defective piRNA biogenesis or function activates transposon expression and mobility, increases DNA damage, disrupts germ cell development, and reduces fertility. In Caenorhabditis elegans, small RNA pathways with opposing activities collaborate to maintain germ cell survival and fertility. Recent studies identified ZNFX-1 as a regulator of epigenetic inheritance in worms. ZNFX- 1 is a highly conserved UPF1-like helicase with C-terminal NF-X1-type zinc finger domains. znfx-1 mutants activate epigenetically silenced reporters, and in some cases they silence normally active reporters, indicating that ZNFX-1 balances opposing epigenetic programs. The targeting pattern of small RNAs redistributes in znfx- 1 mutants, suggesting that ZNFX-1 determines the origin of small RNAs required for silencing and anti-silencing pathways. Preliminary data also show that znfx-1 null mutations cause a mortal germline phenotype at elevated temperatures, suggesting that ZNFX-1 maintains balanced epigenetic signals essential for germline immortality. This proposal seeks to use genetic, computational, and biochemical approaches to test the hypothesis that ZNFX-1 is recruited to targets and identifies sites of small RNA biogenesis. Studies in Aim 1 will determine how ZNFX-1 regulates small RNA biogenesis by identifying how and where it binds to target transcripts, and if it unwinds or moves along RNA. Studies in Aim 2 will determine how ZNFX-1 regulates germline immortality by identifying functional domains of ZNFX-1, potentially redundant proteins with homologous domains, and small RNA and transcriptome features of germline immortality. These studies will reveal how animals transmit heritable epigenetic information and how epigenetic pathways maintain germ cell immortality. In addition, the proposed research will provide training in genetics and epigenetics, quantitative biochemistry, and computational approaches, and prepare the fellow for a postdoc in computational and systems biology and a future career as an independent investigator.

The mammary epithelium is composed of diverse cell populations that are responsible for regulating key processes in mammary gland development, especially lactation. Alveolar progenitor (AP) cells are partially differentiated stem cells that produce alveolar/milk-producing cells and these cells have recently been implicated as cells of origin in breast cancer. Through analysis of published single cell RNA-seq data, integrin β4, a protein known to function primarily in basal mammary epithelial cells to maintain structural integrity of the gland, was found to be expressed in the AP population. This unexpected result suggests that β4 plays a novel role in the AP population and may regulate dynamic processes within the developing mammary gland. This goal of this proposal is to understand the functional role of β4 in the AP cells of the nulliparous and lactating mammary gland and elucidate the mechanism by which β4 regulates this population. Preliminary studies have revealed that β4 expression is necessary to promote progenitor potential of the AP cells and identified LacdiNAc, a novel glycosylation modification, on β4 that we hypothesize is essential for its localization in lipid rafts that promotes its function in the AP population. Using an AP cell culture model as well as a Cre-lox mouse model to conditionally knock out β4 from the AP population, the function of β4 in the AP population will be assessed in vitro and in the virgin and lactating mammary gland. Also, glycomics analyses and biochemical approaches will identify novel glycans on β4 and help in understanding how LacdiNAc affects function of β4 in the AP population. This approach will further our understanding of the novel role of β4 in the AP population and a new mechanism involving LacdiNAc-β4 localization to lipid rafts to regulate alveolar differentiation. These studies have to potential to define novel mechanisms that regulate the AP population during normal mammary gland development as well as breast cancer progression.

Metabolic diseases such as obesity have become significant health risks affecting one-third of the population worldwide and can have devastating complications. It is therefore imperative to understand the causes of metabolic disease predisposition in order to develop preventative strategies. Extensive genetic studies have failed to explain this ongoing epidemic. However, parents pass on not only genetic information, but also epigenetic factors, which can be modified in response to environmental stimuli, and can then affect gene expression. If information about parental environment can be recorded in the germline, it has the potential to be transmitted to the zygote and impact offspring health. This concept remains controversial in mammals and represents a large knowledge gap in the field of embryology. Previous work in the lab has demonstrated that sperm from fathers fed a low protein diet carry tRNA fragments and microRNAs that can modify gene expression in the embryo. Therefore, in Aim 1, sperm-derived RNAs will be purified and microinjected into parthenogenetically-activated oocytes, or parthenotes, which lack any paternal genetic content. Transcriptomic profiles of injected parthenotes will be acquired by single-embryo RNA-Seq to characterize resulting alterations to the embryonic transcriptome. Maternal transmission of epigenetic information has not been characterized to the same extent as the paternal side. Therefore, in Aim 2 a similar dietary paradigm will be utilized to address the question of whether information about maternal diet can be carried in oocytes and result in changes to the embryonic transcriptome. In vitro-fertilized embryos from mothers fed low protein, high fat, or control diet will be sequenced by single-embryo RNA-Seq. These embryos will be transferred to foster mothers to produce adult offspring, which will then be assessed for glucose tolerance and insulin resistance. The use of in vitro fertilization in this paradigm will ensure that any changes observed in the embryo originate from the oocyte and not from nutrient exchange during gestation. Completion of this research will elucidate effects of paternal and maternal epigenetic factors carried by gametes on the embryonic transcriptome. This work will be completed at the UMass Chan Medical School under the sponsorship of Dr. Oliver Rando. The fellowship training plan includes training in embryological techniques, such as microinjection and immunofluorescent staining of lineage markers, as well as metabolic phenotyping of adult mice. Opportunities to gain experience in science communication include participation in departmental seminars and local and national conferences. Furthermore, career development workshops are provided by the university, in addition to teaching and mentoring of first-year graduate students.

Perturbations in normal gene expression arising from defects in genome organization can lead to cellular dysfunctions linked to aging and various disease states. The mammalian genome is generally organized into chromosomes, compartments, topological associating domains (TADs) and loops. Although TAD and loop formation have been extensively studied, little is known about the processes that drive nuclear compartment formation. It has been proposed that microphase phase separation drives the association of genomic domains of similar chromatin state, resulting in the formation of either type A (active chromatin) or B (inactive chromatin) compartments. However, identifying factors involved has been limited by a lack of tools capable of quantifying the biophysical properties driving this phenomenon. Mammalian heterochromatin protein 1 (HP1) α and HP1β bind constitutive heterochromatin and are known to facilitate the bridging of nucleosomes, suggesting that these proteins play a key role in heterochromatin compartmentalization. Although a recent study has demonstrated that heterochromatin compaction is independent of HP1α, work from our collaborators suggest that this protein is required to stabilize interactions between heterochromatic loci. Interestingly, HP1 proteins and several of their interacting partners can bind RNAs. Independent of HP1 function, specific RNA transcripts are known to play important roles in the formation and maintenance of spatial genome organization and perhaps microphase separation, notably at nucleoli, speckles, and the inactive X chromosome of female cells. We recently developed liquid chromatin Hi-C (LC-Hi-C), which allows quantification of chromatin interaction stability measurements genome-wide. Briefly, isolated nuclei are subject to in situ restriction digestion. Digestion of the genome into a specific fragment size distribution results in the loss of low density/unstable interactions whereas higher density/stable interactions are maintained, which is quantifiable by genome-wide chromosome conformation capture (Hi-C). This technique reveals that the dissolution kinetics of chromatin interactions vary widely between A and B compartments as well as compartmental substructures. The development of “in situ LC-HiC” in Aim 1 will allow stability measurement on mitotic chromosomes, streamline the existing protocol and allow the study of smaller cell populations. Aim 2 will assess contributions of (HP1) α and HP1β to stability of heterochromatic interactions. In Aim 3, LC-Hi-C will allow identification of genomic regions destabilized by RNA depletion. Candidate factors contributing to stability will then be identified using in situ chromatin-associated RNA sequencing (iMARGI) and validated by perturbation followed by LC-Hi- C. Taken together, this study aims to measure the dynamics of chromatin interactions and to provide new mechanistic insight as to how the genome is organized throughout the cell cycle.

Atrial fibrillation (AF) is a cardiac rhythm abnormality that currently affects over 6 million Americans. This statistic is expected to double over the next decade given the increasing prevalence of AF risk factors such as advanced age and obesity. Atrial fibrillation confers a 5-fold risk of ischemic stroke, but can be treated effectively with anticoagulation therapy. Despite the efficacy of available treatment options, 1 in 5 patients with AF present with stroke as their initial manifestation of the arrhythmia. This is attributable to the significant challenge in diagnosing AF due to its episodic and sometimes asymptomatic nature. Existing AF monitoring strategies are burdensome or costly and invasive, and thus have low patient adherence and satisfaction. Recently, commercially available wrist-based wearable devices, or smartwatches, have shown to be accurate for AF detection, and may represent a promising tool for identifying AF. However, commercial devices are not primarily designed for use by older adults for arrhythmia detection, and there is a significant research gap in the feasibility of using smartwatches for arrhythmia detection in this population. Furthermore, no previous research has investigated the potential for implementation and integration of smartwatches into the healthcare system and infrastructure. Using data collected from the in-house randomized control trial Pulsewatch, and by conducting qualitative assessments in usability and implementation, this proposal addresses the evidence gap in the feasibility of smartwatches for AF detection with three specific aims: 1) to evaluate individual-level factors associated with adherence of using a smartwatch for AF detection, 2) to explore patient characteristics associated with acceptability of smartwatches and identify specific usability challenges and nuances for older adults, and3) to identify barriers and facilitators of implementing smartwatches for use in a clinical setting. We approach the problem with a user-centered focus and apply rigorous and systematic scientific methods in completing these aims. Knowledge generated from this proposal will provide future researchers and stakeholders with practical evidence in the potential use of smartwatches for detection of AF.

Physical frailty, characterized by decreased physiologic reserve and increased vulnerability to stressors, and cognitive impairment, ranging from mild impairment to dementia, often co-occur in older adults. Both are associated with considerable adverse health outcomes, high healthcare costs, and substantial caregiver burden, and are highly prevalent in U.S. community-dwelling older adults. However, for older adults receiving long-term care in nursing homes, data is scarce on the prevalence of the two conditions over their stay. Community-based studies suggest the heterogeneous clinical presentation of physical frailty, which may have implications for its management. It is unknown if such heterogeneity is similar in older nursing home residents and if it is influenced by cognitive impairment. Further, physical frailty and cognitive impairment share risk factors and predict the future onset of one another but the mechanism of this complex interplay remains unclear. Lastly, depression is strongly correlated with both conditions, yet findings regarding the impact of antidepressants on the progression of physical frailty and cognitive impairment are inconsistent. This proposed F99/K00 project seeks to address these gaps by two specific aims with population, longitudinal data, and advanced statistical methods. Aim 1 (dissertation research) focuses on older nursing home residents and will describe the prevalence of physical frailty and cognitive impairment; identify subgroups of physical frailty and examine the variation of subgroups by cognitive impairment levels, and delineate the developmental trajectories of physical frailty and cognitive impairment and examine the correlations between trajectories. Aim2 (post-doctoral research) expands to older adults in the community and will assess the reciprocal association between physical frailty and cognitive impairment; quantify the impact of cumulative exposure to antidepressants on trajectories of physical frailty, cognitive impairment, and depressive symptoms; and examine the effect of depressive symptoms as a mediator of physical frailty on cognitive impairment with causal mediation analysis. Methodological innovations include the use of latent class analysis, group-based trajectory models, structural equation models (autoregressive cross-lagged panel analysis; autoregressive latent trajectory model), and causal mediation. This proposal is directly relevant to the growing aging population in the U.S., including those residing in the nursing homes and those living in the community, since it uses the national nursing home database Minimum Data Set 3.0 (Aim 1) and the nationally representative Health and Retirement Study linked to Medicare Part D Drug Event Files and the Harmonized Cognitive Assessment Protocol (Aim 2). This project will shed light on the concurrent progression of age-related physical and cognitive conditions. Results will inform future work to develop diagnostic tools and prediction models to facilitate timely identification of older adults at risk for accelerated functional decline and implement care tailored to older adults’ needs to effectively delay the onset of negative health outcomes, enhance the quality of life, and foster healthy longevity.

It has become apparent that sperm are extensively remodeled during epididymal transit, with ongoing changes to the protein and RNA content of maturing sperm. In addition, an increasing number of studies have shown that epigenetic information is modified during the process of sperm maturation. During the first year of my PhD studies, I have discovered that cytosine methylation patterns are surprisingly dynamic during sperm maturation in the epididymis. Using whole genome bisulfite sequencing (WGBS), I identified widespread changes in DNA methylation as sperm move from the testes through the caput, corpus, and cauda of the epididymis. Given that sperm are a highly methylated and condensed cell type, it is surprising and novel that epididymal transit results in any DNA methylation reprogramming of sperm. I aim to address the question of how DNA methylation changes occur between different sperm populations. My approach is to recapitulate maturation-associated cytosine methylation dynamics in vitro to understand the mechanism by which these changes are occurring. Resolving what these changes mean for potential offspring and determining the mechanisms and factors leading to changes in methylation will have significant implications for both assisted reproduction, and for understanding how a father's environment impacts his children's well being.

Lung cancer is the leading cause of cancer-related death, accounting for approximately 1.3-million deaths worldwide. The most common type of lung cancer, non-small cell lung cancer (NSCLC), is frequently associated with oncogenic mutations in KRAS, a GTPase that regulates cell growth and division. Oncogenic KRAS mutations constitutively activate Kras protein and result in rapid cell division, even in the absence of growth signals, and thus play a critical role in tumor formation and maintenance. Genetic inactivation of oncogenic Kras reduces tumor size and metastatic potential, but Kras-independent tumors eventually recur and are more aggressive. Preliminary studies suggest that Kras-independent relapse may be mediated by the proto-oncogene, MYC. The MYC mRNA is a known target of the microRNA miR-34a, and treatment with ectopic miR-34a delays Kras-independent relapse. The goal of the proposed project is to understand the roles of Myc and miR-34a in Kras-independent tumor relapse in a mouse model of NSCLC. Aim 1 will investigate the role of miR-34a in delaying relapse. Endogenous levels of miR-34a will be quantified during tumor growth and regression, and during Kras-independent relapse. CRISPR/Cas9 genome editing will be used to mutate the miR-34a binding site in the Myc 3’ untranslated region to test whether miR-34a delays relapse by directly silencing Myc. Findings from this aim will provide insight into the use of microRNA-mediated inhibition as a potential therapeutic strategy to target Myc. Aim 2 will investigate how Myc controls relapse and glucose metabolism in Kras-independent NSCLC cells and mice. To achieve this, a novel doxycycline inducible dual shRNA system will be used to co-silence Kras and Myc expression in vitro. Using the seahorse bioanalyzer system, glucose metabolism will be monitored in both Kras-silenced and Kras/Myc co-silenced NSCLC cells to identify metabolic vulnerabilities of tumors. Using a mouse model of NSCLC, tumor burden will be monitored after Kras and Kras/Myc co-silencing. This aim will result in a novel dual shRNA based strategy to establish the efficacy of co-silencing Myc and Kras as a therapeutic strategy to induce tumor regression and prevent relapse in NSCLC. Taken together, findings from this study will elucidate mechanisms of tumor relapse induced by Kras silencing and identify regulators of tumor development, maintenance, and relapse. Ultimately, this work will aid in the creation of novel therapeutic strategies to improve NSCLC patient outcomes.

Type 2 Diabetes (T2D) is a major public health issue in the United States with approximately 9.3% of the population suffering from the disease. Additionally, 86 million people have prediabetes and the economic impact is staggering, with 1 in 10 health care dollars being spent on T2D and its complications. T2D results from insulin resistance and reduced beta cell mass; thus, strategies to increase functional beta cell mass are critical goals for diabetes research. Although it is well established (from rodent models) that increased beta cell mass results from enhanced beta cell proliferation, new research suggests that beta cell dedifferentiation also contributes to reduced beta cell function. Some proteins involved in the G1/S transition of the cell cycle, especially Cdk4, are critical for the maintenance of beta cell proliferation and mass. Insulin receptor substrate 2 knockout (Irs2 KO) mice develop diabetes due to peripheral insulin resistance and reduced beta cell mass, and we previously found that in vitro re-expression of cyclin D2, which activates Cdk4, rescues the loss of proliferation in beta cells lacking Irs2. Therefore, we hypothesized that expression of a constitutively active form of Cdk4 (Cdk4 R24C) might be able to rescue the diabetic phenotype of Irs2 KO mice. Intriguingly, preliminary results suggest that Cdk4 R24C is able to completely rescue not only beta cell mass, but also insulin secretion and beta cell differentiation. Interestingly, recent studies show that the Cdk4 kinase plays many roles independently of its known activity in the cell cycle. Therefore, the goal of this proposal is to determine the mechanisms behind this rescue and determine what atypical roles cdk4 plays in the beta cell. In Aim 1, we will determine how Cdk4 rescues beta cell proliferation, focusing on both the canonical Cdk4-Rb- E2F pathway, and will also identify novel Cdk4 interactors in the beta cell using BioID. In Aim 2, we will determine if Cdk4 R24C rescues 1st or 2nd phase insulin secretion in Irs2 KO islets using both islet perifusion and hyperglycemic clamps studies. We will also perform molecular studies to determine whether the KATPase Kir6.2, which was previously reported to be a target of the Cdk4-Rb-E2F1 pathway, is increased and is sufficient to rescue insulin secretion in Irs2 KO islets. Finally, in Aim 3 we will explore how Cdk4 R24C is able to restore beta cell differentiation markers. This is surprising and interesting, since it goes against the data showing that when beta cells proliferate they lose differentiation markers, and I think the most likely explanation is that Cdk4 is having effects unrelated to its cell cycle actions. I will investigate how Cdk4 rescues Pdx1 expression, with a focus on FoxO1 and PPARγ, two transcription factors that regulate Pdx1 expression. Using in silico analyses and reading the primary literature, I found that both contain Cdk4 consensus phosphorylate sites. Therefore, I will determine whether Cdk4 acts via either or both of these to maintain beta cell differentiation. If Cdk4 plays atypical roles as a kinase to influence multiple aspects of beta cell biology, this may lead to better therapeutic options for preserving beta cell mass, function and differentiation in T2D.

Hepatoblastoma (HB), the most common pediatric primary liver tumor, affects children from infancy to five years of age. Surgical resection with adjuvant chemotherapy has saved many young lives. However, the five-year survival rate remains at 70%, and is worse for children with unresectable tumors. Meeting the clinical need for HB-targeted therapies requires a better understanding of how HB tumors are formed and maintained. The transcriptional co-regulator YAP1 is hyper-activated in 79% of HB cases, and recent studies suggest that YAP1 and the Wnt/β-catenin pathway act together to initiate HB tumors. But is YAP1 required to maintain HB tumorigenesis? Preliminary studies using a conditional mouse model of HB—driven by doxycycline-inducible hyperactive YAP1S127A and constitutively active β-catenin—suggest that YAP1 is essential for tumor maintenance. In the presence of doxycycline, YAP1 is expressed, and mice develop HB tumors; withdrawing doxycycline turns off YAP1, resulting in >90% tumor regression within 10 weeks. Transcriptional analyses revealed that hepatocyte differentiation factors and liver metabolic genes were induced in regressing tumors.

Breast cancer is the most common cancer diagnosis in women and is also one of the leading causes of cancer-related mortality in women who suffer from this condition. Tumors that originate in the breast consist of heterogeneous populations of cells. Breast cancer stem cells (BSCSs) are a subtype of tumor cells that have properties similar to normal, tissue stem cells such as the ability to divide slowly and give rise to differentiated cellular lineages. Furthermore, BCSCs have been implicated in tumor initiation, therapy resistance and metastasis to distant organs. Given this information, a greater understanding of the biological processes that sustain BCSCs will lead to the development of novel agents directed against this chemo- and radio-resistant population of tumor cells. The proposed work will explore the role of the α6 integrin splicing variants, α6Aβ1 and α6Bβ1, in the genesis of BCSCs, specifically addressing the mechanism of activation of the TAZ transcriptional coactivator. Integrins are a family of cell surface receptors that function in signal transduction and adhesion to the extracellular matrix. The α6Aβ1 integrin variant is expressed in differentiated, epithelial breast cancer cells and inhibits the acquisition of stem cell properties Conversely, the α6Bβ1 integrin variant is expressed in BCSCs and promotes tumor initiation by activating the Hippo signaling pathway transducer TAZ. TAZ has previously been shown to be important in the functioning of BCSCs, but the mechanism is unknown. Therefore, elucidating the relationship between the α6 integrin splicing variants and TAZ activation will provide insight into mechanisms of breast cancer progression. This proposal will use a cellular and molecular biology approach to establish the mechanism by which TAZ is suppressed in the α6Aβ1 expressing non-stem breast cancer cell population (Aim 1). Biochemical studies will also be undertaken with the purpose of connecting mechanisms of TAZ inactivation with classical Hippo pathway signaling in the non-stem breast cancer cell population (Aim 2). In summary, the studies included in this proposal will increase the understanding of integrin regulation of BCSCs. Our results can provide rationale in designing future targeted therapies for treatment resistant subtypes of breast cancer.

Small interfering RNA (siRNA) therapeutics are a promising class of drugs for the treatment of genetic disorders. Chemical modification of siRNA is necessary for delivery to the central nervous system, but may hinder the efficacy of some siRNAs. This project will define the relationships between siRNA sequence, chemical modification patterns, and efficacy to streamline the development of clinically-relevant siRNAs capable of treating genetically-defined neurological disorders.

Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship to Promote Diversity in Health-Related Research (Parent F31-Diversity). Smokers with mental health conditions (MHC) have increased risk of dying from lung and cardiovascular disease. The smoking rate among people with MHC greatly exceeds the rate in the general adult population. Although, these smokers are interested in quitting, their quit rates are much lower than the general population. Factors that act as quitting barriers for these smokers, include pro-smoking social norms and attitudes/behaviors of social network members, underuse of pharmacotherapies and behavioral strategies, and inconsistent treatment of tobacco dependency of mental health providers. Thus, leading researchers have called for innovative approaches to address smoking disparities in people with MHC. Family/peer-based behavioral interventions can be an innovative and effective approach to target smokers with MHC for several reasons. Family/peers influence smoking behaviors, and their importance in health behavior change is well-established. Families/peers are often a principal resource for persons with MHC in seeking and accessing health services. A small but consistent body of literature suggests that family/peers may influence the cessation behavior of smokers with MHC. Family /peers could augment other cessation interventions such as adoption of pharmacotherapies. However, interventions that attempt to harness family/peer support for long-term smoking cessation have underperformed. Knowledge gaps exist in our understanding of the mechanisms through which family/peers affect smoking behaviors, as well as how to involve family/peers in smokers’ cessation efforts. Guided by the social influence domain, as outlined in the Theoretical Domains Framework, my dissertation will address these knowledge gaps. My specific aims are to: 1) prospectively examine the effect of family/peer influences on smoking cessation among smokers with MHC using data from the Population Assessment of Tobacco and Health (PATH study), a nationally representative survey of US non-institutionalized individuals in which participants are interviewed annually, 2) evaluate relationships between smokers’ characteristics, family/peer influences, and smoking cessation among smokers with MHC using Structural Equation Models, and 3) qualitatively explore social and clinical barriers and facilitators to smoking cessation and inclusion of family/peer support among smokers with MHC and mental health care providers. This work in combination with the proposed training will facilitate my development into an independent research scientist committed to conducting research focused on tobacco prevention and control. I will be supported by an outstanding mentoring team with expertise in all the relevant areas: smoking cessation, mental health, implementation science, and biostatistics. My research directly addresses NHLBI’s objective of better understanding the causes of population health differences and identifying strategies to effectively address these differences.

Elaborate mechanisms exist to establish and maintain the appropriate balance of excitation and inhibition (E/I balance) in the brain. Defects in E/I balance are hypothesized to underlie many core clinical symptoms seen in ASD including repetitive behaviors and seizures. Concomitant with E/I imbalance are increased markers of inflammation in the periphery and brain. Central to this inflammation are microglia, a resident macrophage of the central nervous system. Whether microglial inflammatory state drives E/I imbalance in neuropsychiatric disease remains a critical open question. In this proposal, I will leverage my primary mentor’s (Schafer) expertise in using mouse models to study microglia function at synapses with my co- mentor’s (Frazier) expertise as a physician scientist studying neuroinflammatory processes in ASD patients to explore whether microglia-derived cytokine signaling modulates E/I balance. I will use a mouse model with altered inflammatory cytokine signaling to assess how microglial inflammatory cytokine production modulates neuronal excitability (aim1). Next, I will use human ASD functional imaging data and data from patient serum to identify pro-inflammatory cytokines that are dysregulated in ASD patients and assess how these ASD-specific cytokines affect E/I balance in our mouse models (aim2). To start, I already have one candidate TNF􏰀-alpha. The results from these experiments will help to identify novel targets for treating ASD with inflammatory modulation.

One out of every three children under the age of five in India are undernourished (48 million); to address this crisis, Indian government established a national program from 2005-2012. This study will apply advanced geospatial and multilevel methods to investigate 1) the changes in child undernutrition in India from 2005 to 2012, 2) individual, household, and community level predictors of child undernutrition, and 3) consequences of undernutrition on development during pre-adolescent (8-11) years. Results from this study can guide effective policymaking and implementation of intervention programs.

The novel NAD glycohydrolase, SARM1, is an active executioner in progressive axonal and neuronal degeneration1. This type of degeneration, termed Wallerian degeneration, defines a number of diseases, including neuropathies, traumatic brain injury and neurodegenerative diseases, yet no therapies exist. In fact, prior to the discovery of SARM1’s role in triggering Wallerian degeneration, the process was believed to occur passively.

Project Summary β-Thalassemia and sickle cell disease (SCD) are severe inherited anemias that result in defective β-globin expression. A common feature shared between both disorders is that the diseases can be mitigated by the production of fetal hemoglobin (HbF). Current treatments for both disorders are mainly supportive and focus on alleviating disease complications. However, management of these β-hemoglobinopathies is expensive, restrictive, lifelong, and associated with significant side effects. Currently, allogeneic hematopoietic stem cell therapy is the only curative treatment for β-hemoglobinopathies. However, finding human leukocyte antigen (HLA)-matched donors is highly challenging. Gene editing approaches that alter the expression of HbF should improve the health and quality of life of individuals with β-hemoglobinopathies. The goal of this project is to develop efficient, accurate, and safe CRISPR gene editing approaches as a universal treatment for β-hemoglobinopathies. Typically, a nuclease (SpyCas9) and guide RNA (gRNA) target a specific region (+58 enhancer of BCL11A) and creates a double-stranded break. Small insertions or deletions (InDels) are created when imprecise repair of the DNA break occurs, resulting in inactivation of the target gene or functional element. Current therapeutic editing approaches for β-hemoglobinopathies at the BCL11A locus focus on the mutagenesis of a single GATA1 recognition sequence within the +58 enhancer in CD34+ hematopoietic stem and progenitor cells (HSPCs), limiting the scope of their potential impact. This project will harness our recently developed Cas9-Cas9 fusion chimera, which is able to produce segmental deletions with defined junctions (precise deletions) at a higher efficiency than standard Cas9 nucleases. Furthermore, Cas9-Cas9 fusions are able to effectively target suboptimal PAM sequences, and thus these nucleases have a broader targeting range than standard SpyCas9. These properties make Cas9- Cas9 fusions ideal for the deletion of therapeutically relevant regulatory elements, such as the +58 enhancer of BCL11A. Our preliminary studies show that ribonucleoprotein (RNP) complexes of these Cas9-Cas9 fusions targeting this locus are functional in CD34+ HSPCs and result in robust induction of HbF. In Aim 1, I will define deletion products within the +58 enhancer that facilitate the maximum induction of HbF in erythroid model systems and optimize the Cas9-Cas9 fusions to efficiently and specifically produce these precise deletions with minimal collateral damage to the genome. In Aim 2, optimized Cas9-Cas9 fusion protein-sgRNA complexes will be delivered into CD34+ HSPCs and HbF induction levels will be quantified in erythroid progeny. Treated CD34+ cells will be engrafted into immunodeficient mice to measure engraftment potential and persistence of editing in long-term hematopoietic stem cells (LT-HSCs). The genome-editing tools generated from the proposed work will provide a path to improved autologous HSC therapies that will impact the lives of individuals affected by β-hemoglobinopathies.

The central nervous system is susceptible to many environmental insults and like many organs can be affected by alcohol. Alcohol impacts the brain in a variety of ways including short-term cognitive changes, development of dependence, memory deficits, neuronal loss and initiation of neuroinflammation. An emerging mechanism being studied in the field of central nervous system (CNS) inflammation, extracellular vesicle communication, has not yet been investigated in alcohol-related neuroinflammation and offers the potential for therapeutic intervention. Key components of alcohol-induced neuroinflammation, the cytokines IL-1β and HMGB1, are thought to be released from cells via extracellular vesicles. This study will explore the hypothesis that alcohol alters the release of extracellular vesicles within the CNS and that these vesicles contain content critical to the inflammatory process. Our Preliminary Data reveals that EVs are released by CNS cell types and can be taken up by unstimulated cells. First, we examined the effect of alcohol exposure on microglia and astrocytes in vitro and found that exosomes were stimulated for release at either 50 or 100mM alcohol. These findings were confirmed with western blot against exosome marker CD63 in the supernatant. Next, we used the membrane dye PKH26 to label membranes of microglia which were then stimulated to release EVs by alcohol. Those EVs were transferred to untreated/unlabeled cells and the dye was seen to incorporate in recipient cells, suggesting that those EVs were taken up by the untreated cells. Specific Aim 1 will investigate the effect of alcohol on extracellular vesicle release from primary mouse CNS cells (neurons, microglia or astrocytes) in single cell-type cultures in vitro. Nanoparticle tracking analysis will be used to measure released vesicles size, which will allow for quantification of the two types of released vesicles: exosomes (<150nm diameter) or microvesicles (150nm-1μm). Proinflammatory cytokines IL-1β and HMGB1 will then be measured in vesicles secreted from CNS cell types after alcohol exposure. These experiments will provide important knowledge regarding alcohol's impact on vesicle release as well as vesicle content. As extracellular vesicles are believed to transmit intercellular signals, Specific Aim 2 will explore the effect of transferring alcohol-induced vesicles onto naïve cells. First, extracellular vesicle uptake by primary CNS cell types will be measured. Next brain slices maintained in culture will be exposed to vesicles derived from alcohol-exposed cells and activation of inflammatory pathways will be examined. Finally, IL-1β or HMGB1 will be individually knocked down or overexpressed in CNS cell types and alcohol-induced vesicles will be transferred onto brain slices. These experiments will test the effect that alcohol-induced extracellular vesicles have on other cells, as well as the contribution made by cargo cytokines. Specific Aim 3 will elucidate the impact that alcohol-induced vesicles have on the brain in vivo. First, we will investigate the concentrations of EVs required for intracranial injection and uptake in the brain by using fluorescently-labeled vesicles. Next, vesicles will be stimulated in vitro from primary mouse CNS cells exposed to alcohol. After isolating those vesicles, they will be injected into the brains of naïve mice. Brain tissue will b examined for increases in immune cell activation and upregulation of inflammatory signals. This experiment will provide important information regarding the impact of extracellular vesicles on inflammation in vivo. The first year of this fellowship will be dedicated to quantifying and qualifying the vesicles released by CNS cells after alcohol exposure. Specific Aim 2 will be investigated in years two and three of the fellowship, while Specific Aim 3 will be completed in year three. The final two years of the fellowship will be dedicated to completing the clinical rotations for my MD training as well as any necessary follow up experiments needed for publishing this proposed work.

Approximately 7.2% of adults in the United States in 2012 suffered from an Alcohol Use Disorder, as defined by the American Psychiatric Association. Alcohol is responsible for a number of severe diseases, including alcoholic liver disease, alcohol related dementia and fetal alcohol syndrome. Past research has focused on organs such as the liver, or brain, but it has become increasingly evident that alcohol has significant effects on nearly all tissue in the body. One tissue that has garnered recent attention in part due to the rapid rise in obesity, is adipose tissue. Recent studies have provided compelling evidence relating the dysfunction of adipose tissue to the progression of many diseases, including liver disease. The interaction between ethanol and adipose tissue, particularly brown adipose tissue, has been understudied. There is little known about how ethanol impacts brown adipose tissue function, and further understanding of this relationship may prove crucial in elucidating origins of systemic changes seen in chronic alcoholics. This proposal will investigate the effects of ethanol on brown adipocyte development and function, elucidate mechanisms involved, and study the consequences of these effects on metabolic homeostasis. Both an in vitro and an in vivo model will be used. An in vitro model will be used to investigate whether ethanol interferes with the development of brown adipocytes. Brown preadipocytes isolated from wild-type C57BL/6J mice will be differentiated into mature brown adipocytes with or without exposure to 100mM ethanol. RNA and protein isolates will be collected from the differentiated cells. Markers of adipocyte character and function will be evaluated by qRT-PCR and western blot. Additionally, proteins involved in the mTOR pathway will be evaluated via western blot and immunoprecipitation to elucidate mechanisms that may be involved with brown adipocyte function. A similar approach will be applied to mature brown adipocytes. These studies are the focus of specific aims 1 and 2. An in vivo model will be used to study the effects of chronic ethanol intake on brown adipose tissue function, and to relate these effects with whole body metabolism. Wild-type C57BL/6J mice will be given a chronic ethanol diet. Groups of mice will be exposed to conditions that have been shown to mediate brown adipose tissue activity, including cold habitat, beta adrenergic injection, high fat diet (al 3 increase BAT activity), and thermoneutral habitat (decrease BAT activity). Weight and temperature will be recorded throughout the study, and mice will be sacrificed for collection of tissue. Various depots of adipose tissue will be collected, along with liver and muscle. These tissues will be evaluated for both functional markers, and markers of brown/beige/white adipocyte fate. These experiments are the focus of specific aim 3.

Dopamine (DA) neurotransmission is vital for behaviors such as movement and reward, as well as, cognitive functions including mood, learning and memory. Several neuropsychiatric disorders are linked to alterations in DA signaling including Attention Deficit Hyperactivity Disorder (ADHD), schizophrenia, Parkinson's disease, and addiction. The DA transporter (DAT) is imperative for temporal and spatial control of DA signaling. DAT is located at the presynaptic terminal of DAergic neurons and facilitates the termination of DAergic transmission by rapidly clearing released DA. DAT is the primary target of addictive and therapeutic psychostimulants, which compete for DA binding and block uptake through the transporter, preventing DA clearance and leading to the hyper-locomotive and rewarding behaviors associated with drug use. Given that DAergic signaling is highly sensitive to DAT function, understanding the molecular mechanisms that control DAT function and availability is a critical missing piece of the puzzle in understanding DAergic neurotransmission and dysfunction in DA- related disorders. Over two decades of research support that DAT surface expression is acutely regulated by endocytic trafficking. Protein kinase C (PKC) activation with phorbol esters stimulates DAT internalization and thereby decreases DAT surface expression and function. Although considerable progress has been made to define the molecular mechanisms governing basal and PKC-regulated DAT trafficking, there are significant gaps in our understanding of this process in bona fide DAergic terminals. It is not clear how DAT is regulated in response to the endogenous presynaptic receptors that are activated upstream of PKC, such as Gq-coupled receptors, and how the complex signal events stemming from Gq receptor activation integrate to acutely control DAT surface expression. It is additionally unknown whether regulated DAT trafficking is region-specific, or whether altered DAT surface expression impacts DAergic signaling in the striatum. The proposed studies will leverage chemogenetic receptors to test how Gq activation impacts DAT surface levels in a cell- autonomous manner, in both dorsal and ventral striatum. We will capitalize on a novel conditional, inducible, in vivo gene silencing approach to determine the endocytic mechanisms that are required for Gq-mediated DAT trafficking, by both chemogenetic and endogenous presynaptic receptors. We will further employ ex vivo fast- scan cyclic voltammetry to investigate how presynaptic DAT trafficking impacts DA signaling. I anticipate that at the completion of these studies, we will have gained a more in-depth understanding of the complex mechanisms underlying DAT regulation at presynaptic DAergic terminals, and its potential influence on synaptic DA homeostasis.

The goal of this project is fluorescently visualize ATP release and extracellular accumulation at the surface of stimulated microglia. The development of this innovative technology has the potential to enable spatiotemporal imaging of microglial extracellular signaling. For this project, I am exploiting the presence of the cell's glycocalyx to attach ATP-sensitive biosensors at the sites of ATP accumulation. There are two aims to this project: 1) to synthesize a novel, polyhistidine binding moiety that covalently modifies the glycocalyces of living cells and binds recombinant biosensors to measure ion and metabolite efflux and accumulation; 2) to visualize and measure ATP release from pannexin channels in C5a stimulated microglia. The completion of these aims will yield a transformative set of chemical-biological tools and methodologies to investigate the physiology and pathophysiology of pannexin-dependent activity in glia, and potentially in living animals.

Down syndrome (DS), or trisomy 21, is the leading genetic cause of intellectual disability in children, with approximately 1 in 700 live births carrying an extra copy of chromosome 21. Compared to less common single gene disorders, DS pathogenesis is still poorly understood. Treatment for DS would require either identification of molecular pathways to target with conventional therapies or, potentially, chromosomal therapy to silence the many possibly disruptive genes on the trisomic chromosome. One window of therapeutic intervention lies in the Alzheimer’s disease pathology that almost all DS patients suffer from in middle age. Recently, the extra chromosome has been silenced in an inducible manner by targeted insertion of a transgene for the XIST gene into human induced pluripotent stem cells (iPSC). XIST normally silences one X chromosome in females, providing a natural mechanism of dosage compensation. Chromosomal silencing in DS cells provides a powerful isogenic and isoepigenetic model for studying DS pathology and marks the first step towards the goal of chromosomal therapy for DS patients. The proposed work will investigate the effect of silencing the extra chromosome on DS iPSC-derived neuronal cells, investigating both DS and Alzheimer- specific phenotypes. Aim 1: In order to investigate the effect that chromosomal silencing has on DS neural phenotypes in vitro, iPSCs will be differentiated into neurons using conventional two-dimensional neuronal culturing techniques and three-dimensional organoids. Cerebral organoids are a recently-developed tool that have been shown to be a useful model for human brain development, and have been used to study disorders of brain development. Neurons derived from iPSCs with two and three functional copies of Chr.21 will be compared for phenotypes that DS neural cells are thought to possess. These include an increased glia:neuron ratio, altered dendritic spine morphology, and altered mitochondrial morphology. Three-dimensional cultures will be used to investigate less well-explored pathologies such as alterations in cortical lamination. This aim will also address the important therapeutic question of whether post-mitotic cells can support chromosomal silencing. Aim 2: The same chromosomal silencing system will be used to investigate Alzheimer’s-associated neuronal phenotypes. These include intra and extracellular amyloid deposition as well as intracellular hyperphosphylated tau deposition. Due to its relatively late onset compared to general intellectual disability, the Alzheimer’s disease component of DS is most suitable for therapeutic intervention. Studying the effect of chromosomal silencing on Alzheimer’s phenotypes provides a strong model for Alzheimer’s pathogenesis while also bringing this novel strategy one step closer to therapeutics. This proposal seeks to utilize a novel chromosomal silencing technique to better model human neural phenotypes in DS and associated AD.

The goal of this proposal is to understand how the Beclin 1/vacuolar protein sorting-associated protein 34 (VPS34) complex contributes to breast cancer progression and to determine how to sensitize tumor cells that are deficient in Beclin 1/VPS34 function to drugs that promote tumor cell death. Breast cancer is the most commonly diagnosed cancer among women worldwide and the second leading cause of cancer related mortality. Despite the availability of targeted therapeutics, the overall survival rate for stage I metastatic disease remains 23%. Therefore, there is a need to develop novel approaches for the treatment of advanced stage breast cancer. The applicant hypothesizes that one such approach could exploit Beclin 1/VPS34 function in breast cancer progression. Beclin 1 is monoallelically deleted in 40% of human breast cancer and there is an inverse correlation between Beclin 1 expression and poor prognosis in ER negative subtypes of breast cancer. In addition, low Beclin 1 expression serves as an independent predictor of patient survival. Beclin 1 interacts with and activates VPS34, the mammalian Class III phosphatidylinositol, to regulate multiple membrane trafficking pathways including autophagy, growth factor receptor trafficking and cytokinesis. The individual contribution of each of these trafficking pathways to cancer is unknown and needs to be investigated to understand the impact of Beclin 1 loss on breast cancer progression. Previous studies in the applicant's lab have shown that loss of Beclin 1 expression is associated with a sustained increase in AKT and ERK signaling downstream of the IGF and EGF receptors. In addition, loss of Beclin 1 expression in breast carcinoma cells leads to increased invasion. These preliminary findings suggest that VPS34 inhibitors, which are currently in clinical trials, may negatively impact tumor treatment. The work that the applicant proposes here will explore how the Beclin 1/VPS34 complex contributes to breast cancer progression. The applicant hypothesizes that inhibiting the Beclin1/VPS34 complex will lead to the upregulation of specific pathways that promote tumor growth and progression and that targeting these pathways in tumors with low Beclin 1 expression or in combination with VPS34 inhibition will suppress tumor cell viability. The goal with this proposal is to dissect out the roe of each Beclin 1/VPS34 complex in breast cancer progression with respect to tumor growth, metastasis, and tumor metabolism both in vitro and in vivo (Aim 1). Furthermore, mechanisms that sensitize breast cancer cells to death upon Beclin 1/VPS34 inhibition will be identified as a means to target Beclin 1 deficient tumors and optimize VPS34 inhibitors as potential therapeutic agents (Aim 2). The studies in this proposal will enhance our understanding of the role of Beclin 1 in breast cancer and can give insight into novel therapies for advanced stage breast cancer disease.

Melanoma is the leading cause of skin cancer death in the United States, with the 5-year survival rate of 20% for patients with advanced disease. Despite improvements in therapy, many patients receive minimal survival benefit and often develop resistance to standard-of-care therapies. Furthermore, a population of patients exist who do not have the appropriate mutations or tumor characteristics to be eligible for new targeted and immunotherapies. In order to provide adequate treatment options for patients who develop resistance or are ineligible for current cutting-edge therapies, new therapeutic targets must be identified. Our lab has discovered a novel melanoma oncogene, growth differentiation factor 6 (GDF6), a secreted bone morphogenetic protein (BMP) ligand that promotes melanoma by regulating expression of specific neural crest factors, which has the dual effect of preventing differentiation and suppressing apoptosis9. In addition to these specific factors, we found GDF6 more broadly promotes a gene expression signature that mimics that of the embryonic neural crest. Neural crest identity has previously been identified as a key feature involved in melanoma initiation, progression, and therapeutic resistance. Our studies show knockdown of GDF6 suppresses expression of many of these neural crest genes. Taken together, these data indicate GDF6 is an optimal target for melanoma therapy. As a secreted extracellular protein, GDF6 is amenable to targeting by antibodies. We produced a panel of monoclonal antibodies to target the C-terminal binding region of GDF6 and developed multiple in vitro and in vivo assays to assess candidate antibodies with the most potent action against GDF6 and evaluate their effectiveness as potential melanoma therapeutics. I hypothesize that a subset of antibodies will effectively block GDF6 activity leading to increase differentiation and cell death of melanoma cells in vitro and in vivo. I further hypothesize that blocking GDF6 will suppress neural crest identity in melanoma cells, leading to less aggressive tumor cell characteristics and sensitizing previously resistant cells to standard-of-care therapy. I will evaluate a pre- screened panel of 42 monoclonal antibodies for in vitro and in vivo activity against GDF6 in melanoma cells to identify top candidates with the most potent activity, in parallel with characterizing pharmacokinetic and dynamic properties of the antibodies in vivo. I will further characterize the effects of GDF6 inhibition by these antibodies on neural crest expression profiles and key features of advanced melanoma such as therapeutic resistance, invasiveness, and anchorage independent growth. Additionally, I will analyze potential combinatorial therapies in vitro and in vivo to assess changes in pathway activity for known therapeutic resistance mechanisms. Results of this study will identify lead candidate anti-GDF6 antibodies for first-in-human (FIH) studies and provide appropriate pre-clinical safety data for submission of an FIH application. Furthermore, these data will provide broad insight into the tumorigenic features that are connected to neural crest identity and the result of reversing neural crest characteristics in established melanomas.

The bacteria that live in our body (microbiota) help us metabolize different nutrients and chemicals from our diet, including the medications we take. Thus, these bacteria can influence our response to treatments, like chemotherapy, by converting drugs into more or less toxic forms. Understanding the mechanisms by which bacteria influence our response to drugs is critical to design better treatments that maximize therapeutic effects and minimize adverse effects. The nematode C. elegans and its bacterial diet provide a suitable model to explore host-microbe drug interactions because both host and microbe are amenable to high throughput drug screening and genetic screening. I propose to use the nematode C. elegans as a model to study host-microbe-drug interactions in cancer chemotherapy drugs. In Aim 1, I will test ~ 200 cancer chemotherapy drugs for developmental or fecundity phenotypes in C. elegans animals fed two different bacteria. Additionally, I will test the role of active bacterial metabolism in the observed host-microbe-drug interactions. Then, I will use genetic screening and metabolomics, in the host and in the bacteria, to characterize the mechanisms responsible for the observed drug response. In summary, this project will generate a set of host and bacterial genes that contribute to the response to cancer chemotherapeutic agents.

Promising emerging therapies for Huntington's disease target mutant but not wild-type huntingtin mRNA with small interfering RNAs. However, our limited understanding of allele-specific mRNA processing restricts the design of allele-specific therapeutics. The purpose of this project is to elucidate differences in wild-type and mutant mRNA processing to improve the specificity, and thus effectiveness, of small RNA treatments for Huntington's Disease.

Learning and memory are dynamic processes that require alterations in the connections among neurons. Synapses, the structures through which neurons communicate with one another, undergo biochemical and morphological changes in response to neuronal activity in a process known as synaptic plasticity. Disturbed synaptic plasticity is the basis for several diseases such as dementia, schizophrenia, and Alzheimer's disease. Local translation of mRNAs in dendrites is essential for regulating certain forms of synaptic plasticity. Impairment of this process is the cause of several neuropathies such as the Fragile-X Syndrome and other disorders linked to autism. Modulation of cytoplasmic mRNA poly(A) tail length is one mechanism that controls local translation in dendrites. It is estimated that ~7% of brain RNAs are regulated by this process in response to stimulation, which underscores its importance in synaptic plasticity and therefore higher cognitive function. Because poly(A) tails lengthen as well as shorten, there are likely to be several enzymes involved in this process. Several factors responsible for poly(A) elongation have been identified, but the enzyme(s) responsible for shortening the poly(A) tail remains unknown. The proposed research seeks to identify this deadenylating enzyme, which is likely to act as a negative regulator of dendritic translation and learning and memory. The proposed research focuses on CNOT7, a major mammalian deadenylase, in the brain and seeks to test whether (1) knockdown or mutation of CNOT7 alters poly(A) tails in cultured neuronal dendrites, (2) CNOT7 controls specific mRNA deadenylation in dendrites, and (3) knockdown of CNOT7 alters various forms of synaptic plasticity. This work will further our understanding of the process of learning and memory and disorders affecting these important processes.

About 1.2 million Americans are hospitalized annually with acute coronary syndrome (ACS), and most are discharged alive. Although post-ACS mortality and clinical morbidity have been improving patients may be living longer but not better. In fact, many patients suffer substantial declines in quality of life and functional status after discharge with ACS. Because of critical gaps in our understanding how health status evolves over time for ACS patients, important opportunities for prevention and intervention are potentially being missed. The proposed research takes a systematic approach to examining the association of demographic, psychosocial, clinical, and neighborhood factors on trajectories of health-related quality of life after discharge for ACS. Our study will leverage the availability of rich data already collected for the NHLBI-funded TRACE- CORE, a longitudinal prospective cohort study of 2,183 patients hospitalized with ACS. This study includes data from interview, medical record abstraction, linked administrative databases, and geo-coded census tracks. Specific aims are to: (1) Determine associations between individual level socio-economic, clinical, in- hospital and psychosocial factors and trajectories of patient health status post-ACS discharge, both generic (SF-36) and disease specific (Seattle Angina Questionnaire with domains of physical limitations, angina stability, angina frequency, treatment satisfaction and angina specific quality of life); (2) Determine how neighborhood deprivation is associated with trajectories of patient health status; and (3) Identify the extent to which trajectories of generic quality of life and disease-specific quality of life at baseline, one month, 3 months and 6 months predict mortality or readmission 6 months to 1 year post-ACS discharge. This pre-doctoral fellowship proposal also includes a carefully training plan for me to become an independent physician scientist able to fully exploit the potential of patient-reported outcomes to improve the lives of patients with cardiovascular disease.

In many types of cancer, less differentiated tumors are more strongly associated with a poor patient prognosis. These tumors tend to be more aggressive: they have higher proliferation rates, a greater propensity for invasion and metastasis, and increased resistance to therapy compared to more differentiated tumors. Less differentiated tumors, by their nature, share characteristics with their embryonic cells of origin. In melanoma, these less differentiated tumors are associated with a neural crest identity that is acquired during early stages of tumor initiation and is present through tumor progression. Previous studies have shown that acquisition of a neural crest identity is a necessary step during initiation of early melanoma lesions and supports fundamental properties of aggressive tumors such as invasion and metastasis. However, the mechanisms of generating a neural crest identity are unknown. Recently, our lab has identified growth differentiation factor 6 (GDF6) as a novel melanoma oncogene. GDF6, a bone morphogenetic protein (BMP) ligand, is recurrently amplified in both human and zebrafish melanomas, and expressed in tumors but not normal melanocytes. We have shown GDF6 acts to prevent differentiation and suppress apoptosis in established melanomas, both in vitro and in vivo. Additionally, we have found that GDF6 regulates expression of multiple neural crest and melanocyte factors previously implicated in melanoma. Upon knockdown of GDF6, we observed downregulation of select neural crest factors coupled with upregulation of melanocyte differentiation factors, leading to melanoma cell differentiation and ultimately cell death. These results suggest that GDF6 plays a role in regulating oncogenic neural crest identity. Here, we look to identify the role of GDF6 and BMP signaling in establishing a neural crest identity during melanoma initiation and explore oncogenic characteristics imparted by GDF6 during melanoma progression. I hypothesize that GDF6-depenent BMP signaling acts to initiate a neural crest identity in melanomas to promote tumor initiation and aggressive tumor characteristics. I further hypothesize that loss of BMP activity leads to differentiation (and in tumors death of differentiating cells), making GDF6 an attractive target for differentiation therapy in melanoma.

“Alcohol abuse has been attributed to 3.2% of the world's disease burden. Chronic abuse manifests as reversible steatosis, steatohepatitis and/or irreversible cirrhosis. Regardless abstinence, 5-15% of patients with steatohepatitis still progress to cirrhosis and hepatocellular carcinoma (HCC). Studies have demonstrated the role of miR-122 as a mediator of hepatic metabolism, cellular differentiation and the development of HCC. Our laboratory has shown decreased miR-122 in a four-week chronic-alcohol mouse model. The role of miR-122 in ALD is unknown. Bioinformatic miRNA target prediction tools suggest Hypoxia-Inducible Factor 1-� (HIF1�) is a primary target of miR-122. HIF1� is critical to the progression of ALD. Specific Aim 1 will study the role of miR-122 in the pathogenesis of chronic alcohol-induced hepatitis. C57Bl/6 mice will be transfected using a hepatocyte-tropic adeno-associated virus serotype 8 (rAAV) vector to either knockdown or overexpress miR-122 via tough decoy (TuD) or the miR-122 respectively. Mice will be maintained on Lieber-DeCarli diet for 28 days. On day 28 mice will be withdrawn from alcohol and given 150- mg/kg acetaminophen (APAP). Mice will be sacrificed on day 30 to assess regeneration. Livers sections will be analyzed histologically for morphological changes, lipid accumulation, and regeneration. In addition, we will determine the impact on miR-122 modulation on hepatic inflammation and differentiation through expression analysis of inflammatory cytokines and cell cycle regulators associated with ALD. MiR-122 has been correlated with a network of Liver Enriched Transcription Factors (LETFs). Collectively, these LETFs function as master regulators of hepatic function. HNF6�, specifically, has been shown to function in a positive feedback loop with miR-122. Specific Aim 2 will examine the effect of miR-122, its overexpression and knockdown, on HNF6� in a chronic-alcohol model. We will also examine if miR-122 can reduce ALD through enhancement of HNF6� and the LETF network. The rAAV8 vector stated above will deliver anti- HNF6� shRNA or rHNF6 in vivo. The mice will be treated and assayed as described above. During the first year of this fellowship we aim to examine the effect of miR-122 on the severity of ALD development using gene therapy. The remaining two years of my Ph.D. (funding yrs 2 & 3) will be spent studying the effect miR-122 and HNF6� regulation of the LETF network in the progression of ALD. During years 4 and 5 of funding I shall complete my M.D. training while finalizing any work required for publication. Collectively, we propose to examine two potential avenues by which miR-122 may serve as a potential therapeutic modality. First, is the development of steatosis though inhibition of HIF1� and secondly, is the regeneration after alcohol and APAP-induced liver injury.”

Hepatitis C virus (HCV), a pathogen that infects over 150 million people worldwide, is the leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is a genetically diverse virus with 6 known genotypes with genotypes 1 and 3 being the most prevalent. This genetic diversity makes HCV infection difficult to treat. In the last few years, the advent of direct-acting antivirals (DAAs) has remarkably improved therapeutic options and treatment outcomes. However, despite highly potent inhibitors against multiple proteins, drug resistance is a major problem in all drug classes. Drug resistance is a loss of inhibitor potency while maintaining substrate processing. Though NS3/4A protease inhibitors are highly potent, they are not efficacious against all genotypes and are susceptible to drug resistance. Underlying differential inhibitor potency are the molecular mechanisms of drug resistance and genotypic differences. Elucidating these are key to developing protease inhibitors that avoid drug resistance and are effective against all HCV genotypes. Specifically most protease inhibitors in clinical development contain P2 moieties that contact unessential residues of the protease, which while increasing potency also increases their susceptibility to single site mutations that confer drug resistance. I hypothesize that protease inhibitors that avoid contact with these residues while leveraging contact with unexploited areas in the active site will result in inhibitors with enhanced potency and higher barriers to drug resistance. To investigate this hypothesis, using computational techniques, I will design a panel of novel protease inhibitors with extended P4 groups. I will then synthesize and enzymatically assay these protease inhibitors. Top leads will be co-crystalized with the protease and structurally analyzed to optimize the computational designs and initiate iterative rounds of inhibitor design. This project will provide molecular insights about the mechanisms of drug resistance as well as new strategies for the design of novel protease inhibitors for the effective treatment of HCV infection.

The respiratory mucosa employs the innate and adaptive immune system to protect normal respiratory function from invading organisms. However, when there is a defect in one of these defenses the host becomes vulnerable to Aspergillus fumigatus (Af). This ubiquitous fungus enters the airways as a spore, or resting conidium but is generally cleared by intact respiratory defenses. However, when individuals are immunocompromised, Af conidia are more likely to germinate and form filamentous structures called hyphae. As this morphotype, Af can become invasive particularly in persons with low neutrophil counts. An estimated 200,000 people are diagnosed with invasive aspergillosis annually worldwide, and up to 90% die from the infection. Af can also colonize the airways of those who suffer from asthma or cystic fibrosis causing allergic bronchopulmonary aspergillosis (ABPA), which affects over 4 million people annually worldwide. Studies proposed here seek to further understand the intact innate immune response to Af conidia, particularly the involvement of interleukin-23 (IL-23) and interleukin-17 (IL-17). A better understanding of this response may uncover nuances associated with the host defects that predispose to infection with Af, as well as potentially inform rational vaccine design and immunotherapies against Af. Af conidia elicit IL-23 and IL-17 production from the host airways within the first 24 hours of infection. These cytokines are known to be important for the adaptive TH17 response. However their role in innate immunity against Af is largely unknown. IL-23 has been shown to augment IL-17 production, and in turn IL-17 elicits neutrophil recruitment. The relationship between IL-23 and IL-17 is referred to as the IL-23/IL-17 axis and this proposal aims to systematically characterize each portion of this axis in the innate response against Af conidia, and test whether this response is required for protection against this mycosis. In order to characterize the temporal production pattern of IL-23, we will measure levels of this cytokine at regular intervals in the fist 72 hours of infection by ELISA. From a preliminary screen, we have uncovered candidate cell types that may be involved in the production of IL-23. We aim to confirm these sources by in vivo and ex vivo intracellular cytokine staining. To test whether IL-23 production is protective against mortality in Af infection, the survival rates of wild-type (WT) mice will be compared to a functional IL-23 knock-out strain (IL-23p19-/-). Finally, we propose to create mixed bone marrow chimeras to test whether any specific cellular source of IL-23 is required for protection against Af (Specific Aim 1). The temporal pattern of production for IL-17 and its source will also be characterized in the first 72 hours of infection with Af using methods described above. To test whether IL-23 augments IL-17 production innately in response to Af, IL-17 levels will be assessed in IL-23p19-/- mice and WT mice. In addition, we have evidence that IL-23 and IL-17A are co-produced by one innate cell type in response Af, we propose to test and dissect any potential autocrine mechanisms in this cell type. Finally, the role of IL-17 in protection against f infection will also be tested by monitoring the survival of IL-17RA-/- mice and WT mice (Specific Aim 2). Because many at risk for IA are transplant patients who are iatrogenically immunosuppressed, knowledge of the factors leading to protection against aspergillosis could also inform development of targeted immunosuppressive agents that keep defenses against opportunistic infections intact.

Breast Cancer is the most frequent malignancy diagnosed in women in the United States and the second leading cause of cancer related mortality in women. Metastasis to distant organs continues to be the greatest obstacle to eradication of this malignancy. Greater understanding of the biological processes that contribute to tumor progression will lead to development of effective therapies against this disease. The insulin receptor substrate (IRS) proteins are important signaling intermediates downstream of the Insulin-like growth factor (IGF-1) receptor and they play a crucial role in the response of tumor cells to IGF-1 stimulation. The two IRS proteins expressed in mammary epithelial cells, IRS1 and IRS2, play different roles in breast cancer. Specifically, tumors that express IRS2 are highly metastatic in comparison to IRS-1 expressing tumors. In addition, IRS2 staining at the membrane in patient tumor samples correlates with decreased overall survival. Studies from our group have identified an IRS2-specific interaction with the microtubule cytoskeleton and demonstrated that disruption of microtubules leads to a decrease in PI3K/Akt activation downstream of IRS2. These findings in conjunction with our preliminary data suggest that the subcellular localization of the IRS proteins plays an important role in their cellular functions. Te work the applicant proposes will explore the potential role of IRS2-microtubule interactions in breast cancer progression, specifically addressing the requirement of this interaction for invasion, glycolysis, PI3K/Akt signaling and tumor metastasis. Our goal with this proposal is to take a molecular biology approach to elucidate the importance of IRS2-microtubule interactions in IRS2 mediated functions (Aim 1). Furthermore, we will use animal models to study the significance of this interaction for tumor progression to metastasis and the impact of IRS2 function on tumor response to IGF-1 receptor inhibitors (Aim 2). The studies proposed in this application will enhance our understanding of the importance of IRS2 in breast cancer progression. Additionally, our results can provide the rationale for the development of novel therapeutic approaches for the treatment of IRS2-dependent malignancies.

This proposal has two primary aims: (1) to improve the understanding of the association between patient- reported functional health, comorbidity, and sociodemographic factors and other cause mortality in older men newly diagnosed with prostate cancer; (2) to develop a prototype tool for calculating individualized risk of other cause mortality in this population. Prostate cancer is the most common non-cutaneous cancer in American men and primarily afflicts those age 65 and older. However, most men are diagnosed at early stages with tumors that most often have an indolent course. Guidelines recommend that patients only pursue aggressive treatment if they have >10 year overall life expectancy. Within 5 years of diagnosis, only 11% of American men die due to their prostate cancer, while the majority of patients diagnosed with prostate cancer die of other causes. While validated calculation tools have been developed for clinical use in predicting prostate cancer related mortality, no validated tool has been developed from existing models that identify variables associated with the risk of dying of other causes (OCM). As men in the U.S. live longer and the population above age 65 is rapidly growing, individualized predictions of life expectancy are necessary given the substantial heterogeneity in individual health status. Nearly three quarters of this aging population may have multiple comorbid conditions. Previous work has shown that decrements in patient-reported functional health may be more strongly associated with OCM than the presence of most individual comorbidities. These findings have promise for improving the approach to accounting for the impact of multiple conditions. This predoctoral research training proposal seeks funding to explore the generalizability of prior work demonstrating the association of patient-reported functional health and OCM. The proposed work will help to identify the variables most strongly associated with OCM in older men with prostate cancer, utilizing data from 4,510 subjects in the linked Surveillance Epidemiology and End Results- Medicare Health Outcomes Study (SEER-MHOS) database. This database contains detailed information on cancer characteristics, treatment, cause of death, baseline comorbidities, sociodemographic information, and functional health measures. This proposal will evaluate and improve upon the performance of other cause mortality prediction models through modern statistical techniques to assess predictive model performance. After identifying key predictors of OCM, we propose to develop a prototype clinical risk-calculation tool that estimates personalized risk of 10-year OCM, adapting validated techniques for developing risk calculators. This study will help to establish the utility of patient- reported functional health measures in improving the accuracy of OCM risk estimation in older men newly diagnosed with prostate cancer and will make progress towards a clinically useful OCM risk estimation tool. Completion of this work will help to better identify older patients who would most likely benefit from aggressive treatment vs. those who may not, as they may be more likely to die of other causes.

Adverse health consequences of tobacco use are the leading cause of preventable mortality worldwide, resulting in approximately 6 million deaths per year. The addictive component of tobacco is nicotine, a tertiary alkaloid that binds and activates nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels that are normally activated by the endogenous neurotransmitter acetylcholine (ACh). Neuronal nAChRs are pentamers assembled from various combinations of receptor subunits and different subunit combinations confer different affinities and functionalities to the receptor subtypes. Eleven subunits, ¿2- ¿7, ¿9, ¿10 and ¿2- ¿4, have been identified in mammalian neuronal nAChRs. Interestingly, chronic nicotine or cigarette smoke exposure results in the upregulation of nAChRs in the brain, including structures within the mesocorticolimbic dopaminergic (DAergic) pathway that is implicated in reward and addiction. While not completely understood, nicotine- induced upregulation of nAChRs is thought to contribute to addiction by altering the neural network, possibly resulting in increased tolerance or altered sensitivity to nicotine. While there are many proposed mechanisms for nAChR upregulation, it is largely believed that multiple forms of posttranscriptional regulation is responsible for this phenomenon. Currently, there is not much known about posttranscriptional regulation of mammalian nAChR subunit expression by microRNAs (miRNAs), small single stranded RNA molecules that function as negative regulators of gene expression. However, there is emerging evidence that miRNA expression is decreased in various rodent tissue types in response to nicotine exposure. In addition, recent studies have found that miRNA dysregulation in response to exposure to various drugs of abuse, including cocaine, can influence rewarding properties of the drug and alter addiction-associated behaviors. We have recently generated preliminary data suggesting that a novel regulatory mechanism involving miRNAs may be at work in the nicotine-mediated upregulation of nAChRs. Preliminary experiments from our lab have identified several miRNAs that are predicted to target nAChR subunit mRNA transcripts, in particular miR-494 and miR-542-3p that target ¿4 and ¿2 transcripts, respectively. In Aim 1, I will determine if ¿4 and/or ¿2 are modulated by miR- 494 and/or miR-542-3p in primary midbrain neuronal cultures. In Aim 2, I will determine if miR-494 and/or miR- 542-3p are modulators of nicotine reward-associated behavior in mice. Through these aims, I hope to achieve a better understanding of the role of miR-494 and miR-542-3p in nicotine reward-associated behaviors, possibly revealing new targets for the development of tobacco cessation aids.

External Awards for Research Training

CURRENT AWARDS

Understanding the regulation of the intestinal epithelium in Alzheimer’s disease by commensal bacteria and the role it plays in preventing neurocognitive decline

Characterization of Enterovirus 68 3C Protease For the Development of Robust and Potent Direct-Acting Antiviral Inhibitors

VEGF/Neuropilin-2 Signaling and Radioresistance in Triple-Negative Breast Cancer

Identification of a putative mitochondrial solute carrier that regulates mitophagy

Investigating siRNA-mediated inhibition of ischemia-reperfusion injury during the liver transplantation process

Examining the Role of a Pathogenic HTT Isoform, HTT1a, in Somatic Expansion and RNA Aggregation in Huntington's Disease

Modulation of mitochondrial biogenesis by the Integrated Stress Response (ISR)

Outlining Shadows of Structural Racism Using Publicly Available Social Determinants of Health Data

Implementation of Medications for Opioid Use Disorder in Massachusetts Jails

Investigating the Role of cnb-1 and chpf-1 in GABA DD Motor Neuron Remodeling and Synapse Maintenance

Bacterial targeting of the P-glycoprotein/endocannabinoid axis for reducing intestinal inflammation in ulcerative colitis

Structure-based Antiviral Design against HTLV-1 Protease

Investigating the Role of Endosomal Toll-Like Receptors in Remyelination

Modulation of Somatic Repeat Expansion as a Therapeutic Approach to Huntington's Disease

Establishing and optimizing a prime editing method in neurons for treatment of Rett syndrome

Elucidating the role of hepatic mTORC2 as a key regulator of carbohydrate metabolism in non-alcoholic fatty liver disease

Investigating Trisomy 21 Impact on Human Neural Cell Development and Function Using "Trisomy Silencing" in vitro

Muscle-Specific CRISPR/Cas9 Exon Skipping for Duchenne Muscular Dystrophy Therapeutics

Care Integration, Supportive Housing, and Outcomes for Medicaid Accountable Care Organization Enrollees with Behavioral Health Conditions

Mechanism of cell lethality following loss of gene expression

The role of Neurexin in serotonin synaptic function and social behavior

Detection of intestinal pathogens through host surveillance of bacterial toxins

Discovery of a novel role of VPS13D in Autophagy

Targeting dormant leukemia-initiating cells in T-cell acute lymphoblastic leukemia

Investigating mechanisms of neurodegeneration

PAST AWARDS

Adherence to Clinical Practice Guidelines for Screening and Management of Pediatric High Blood Pressure within a Massachusetts Safety-Net Health Care System

Mechanism of epigenetic inheritance in a mouse model of acute paternal stress

Investigating the role of MS4As during Alzheimer's disease

The Impact of Nucleotide Modification Patterns on Therapeutic Small Interfering RNA Activity in the Central Nervous System

Characterization of MS4A Chemoreceptive Function

The Influence of Spatial Proximity to Sterile Syringe Sources and Secondary Syringe Exchange on HCV Risk Among Rural People Who Inject Drugs

Investigating the Activation Mechanism of SARM1 during Axon Degeneration

Investigating the impact of trisomy 21 silencing on cardiovascular development and function

Development of Advanced Oligonucleotides for Glioblastoma Therapeutics

Investigating sliding clamps and their contribution to genome stability

Pathogen sensing by nuclear hormone receptors in C. elegans intestinal epithelial cells

Activation of non-apoptotic cell death by the DNA damage response

Structure-based design of potent and selective chemically modified oligonucleotide inhibitors for APOBEC3 enzymes

Investigating the role of B cells in pulmonary fibrosis resulting from STING gain-of-function autoinflammation

Tissue-specific modulation of Apolipoprotein E in neurodegeneration

Intersectionality of Sexual Orientation, Gender Expression, and Weight Status on Risk of Disordered Eating Behaviors

Engineering Synthetic Guide RNAs and Compact Base Editors for Enhanced In Vivo Delivery

Elucidating the structural and mechanistic features of a thermophilic bacteriophage

Elucidating premature translation termination in Cystic Fibrosis

Dissecting ADAM10 function in microglia-mediated synapse elimination

Investigating how the conserved ZNFX-1 protein regulates epigenetic inheritance and germline immortality in Caenorhabditis elegans

Integrin Regulation of Mammary Gland Development

Characterizing effects of sperm- and oocyte-derived epigenetic factors on early embryonic gene expression and offspring metabolic function

Stability of the folded genome

Feasibility of Smartwatches for Atrial Fibrillation Detection in Older Adults

Concurrent trajectories of physical frailty and cognitive impairment among nursing home residents and community-dwelling older adults

Replication-independent DNA methylation dynamics during post-testicular sperm maturation

Exploiting RNAi-based silencing of Myc and metabolic vulnerabilities to prevent relapse afer Kras inhibition in lung cancer

Mechanism of cdk4 diabetes rescue in IRS2 knockout mice

Investigating YAP1 control of differentiation and metabolism in Hepatoblastoma

Integrin Function in Breast Cancer Initiation

Defining the Rules for Designing Fully Chemically Modified siRNAs to Treat Genetically Linked Central Nervous System Disorders

Smoking Cessation in Persons with Mental Health Conditions: Exploring the Role of Family and Friends

Microglia-derived neuroactive cytokines governing neural circuit excitatory-inhibitory balance

Trends, Predictors, and Consequences of Child Undernutrition

A chemical biology approach to studying the role of SARM1 in a novel neuronal degradative pathway

Amelioration of Beta-hemoglobinopathies by efficient precise deletion of the +58 BCL11A enhancer using orthogonal Cas9-Cas9 chimeras

The Role of Extracellular Vesicles in Alcohol-Induced Neuroinflammation

Ethanol's Effects on Brown Adipose Development and Function

Gq Receptor Regulation of Striatal Dopamine Transporters

Fluorescent visualization of complement-dependent pannexin activity in microglia

Modeling Down Syndrome Neural Phenotypes with Chromosomal Silencing

Impact of Beclin 1 Loss on Breast Cancer Progression

Targeting BMP signaling to treat advanced melanoma and suppress therapeutic resistance

C. elegans as a model for host-microbe-drug interactions

Investigating the Mechanism and Effect of Disease-Associated Increases in the Huntingtin Long 3'UTR Isoform

The Role of 3' End Formation in Synaptic Function

Patient and Social Determinants of Health Trajectories Following Coronary Events

Examining BMP signaling as a regulator of neural crest identity during melanoma initiation and progression

The Role of miR-122 in Alcoholic Liver Disease

Structure-based design of robust cross-genotypic NS3/4A protease inhibitors that avoid resistance

The Role of Innate IL-17 Responses to Aspergillus fumigatus