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External Awards for Research Training

Trainees planning applications to NIH or other funding agencies are encouraged to directly contact current and past awardees to request advice and/or copies of the funded applications.


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

    Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by amyloid beta plaques and neurofibrillary tangles in the brain along with inflammation both in the brain and systemically. This has led to the theory of microbial communities or infections as causative in the development of neuroinflammation, immunosenescence, and inflamm-aging seen in AD. Our own research has demonstrated a decrease in gut microbiota with anti-inflammatory properties and higher abundances of pro-inflammatory gut microbiota in AD elders. However, it is unclear how the AD microbiome exerts effects on the central nervous system. To address this gap in knowledge we have performed gut microbiome profiling, analysis of immune cell populations in blood, serum cytokine profiling, and cognitive assessments of AD elders at 90-day intervals. This analysis identified changes in B cell populations with an increased abundance of class-switched and decreased abundance of naïve B cells at levels of greater cognitive impairment. To better understand how the microbiome may control AD progression, we propose to investigate the connection between the AD microbiome and the adaptive immune system with a focus on regulation of the intestinal epithelium by commensal gut bacteria. Specifically, we intend to use stool and plasma samples collected from our AD cohort to measure makers of intestinal permeability and determine whether metabolites secreted by the AD gut microbiome cause disruptions in the intestinal epithelium. We will directly study the disruptive effects of AD stool by applying stool supernatants to intestinal epithelial cells, quantifying changes in epithelial permeability using established assays, and determining whether specific taxa depleted in AD are sufficient to cause epithelial disruption. In our previously published data, we have observed the loss of the phytoestrogen-metabolizing bacteria, Adlercreutzia equolifaciens (AE), in the microbiome of AD elders. My preliminary studies reveal that a metabolic product of AE, (S)-equol, prevents epithelial damage in the setting of inflammation. Therefore, we aim to determine whether AE or its metabolic products protect the intestinal epithelium. To untangle the role of the AD microbiome on our observed changes in class switched and naïve B cells, I have collected preliminary data which demonstrates that colonization of mice with the microbiome of AD elders promotes B cell class switching when compared with colonization of cognitively impaired elders without AD. This application proposes to expand this finding and characterize the changes in the adaptive immune system caused by the AD microbiome. This continuing work will further establish the connection between AD related neurocognitive decline, the microbiome, and immune system.

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  • Vincent N. Azzolino Headshot

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

    Certain viruses in the picornaviridae family, specifically enterovirus-D68 (EV68), have emerged as global health concerns over the last decade with severe symptomatic infections with EV68 able to result in long lasting neurological deficits and death. There are currently no US Food and Drug Administration approved drugs for any non-polio enterovirus, highlighting the need to develop strategies against these lethal enteroviral strains. One particularly attractive class of potential drugs are small molecules inhibitors, which can act as direct-acting antiviral (DAA) inhibitors towards the conserved active site of EV68 3C protease. This main viral protease is a cysteine protease conserved in the 3C family, responsible for cleaving eight sites along the viral polyprotein, which is essential for viral propagation. DAAs designed to target 3C proteases can potentially achieve robust inhibition across enterovirus species. However, as drug resistance in viruses can be prevalent, it is paramount to design inhibitors less susceptible to resistance mutations. It was demonstrated previously in the Schiffer Lab that when bound to protease, viral substrates occupy a conserved three-dimensional volume called the substrate envelope. It was also demonstrated that inhibitors that extend beyond the substrate envelope are more susceptible to drug resistance mutations. By utilizing the substrate envelope and cocrystal structures of the proteases, DAAs designed to fit within the three-dimensional consensus volume as naturally occurring substrates will interact primarily with functionally important residues and be less susceptible to drug resistance mutations. The central hypothesis of this proposal is that cocrystallization of EV68 3C protease with its natural substrates will enable the calculation of the substrate envelope to inform on substrate specificity, which will also aid in the design of robust pan-3C-protease inhibitors. In Aim 1, I will determine the cocrystal structures of EV68 3C protease bound to viral substrates. I will then use these structures to elucidate the molecular mechanism of substrate specificity for EV68 3C protease and calculate the substrate envelope. These data will aid in small- molecule design to create DAAs with improved resilience to mutations that can confer drug resistance. In Aim 2, I will design and test novel DAAs that target EV68 3C protease. I will first characterize previously designed inhibitors for other 3C and 3C-like proteases with the substrate envelope to establish a starting compound based on potency. Inhibitors based on the scaffold will be designed, synthesized, and tested in a FRET-based enzyme inhibition assay. Crystallization of novel potent compounds with EV68 3C and their characterization within the substrate envelope will assess inhibitors’ susceptibility to drug resistance mutations. Overall, this study aims to develop a robust, novel compounds with resistance-thwarting protease inhibition against the emerging pathogen that is EV68.

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

    Triple Negative Breast Cancer (TNBC) is an aggressive form of breast cancer with standard therapy involving neoadjuvant chemotherapy, surgical management, and radiation therapy. However, the high recurrence rate and low pathological complete response of TNBC suggest that radioresistance is a critical factor in the diminished therapeutic efficiency of the current treatment strategy. There is limited literature exploring the specific pathways responsible for radiation resistance in TNBC, but most data support the role of limiting reactive oxygen species (ROS) accumulation. Our lab has studied the role of Vascular Endothelial Growth Factor (VEGF) binding to Neuropilin-2 (NRP2) and initiating several cancer stem cell properties. Preliminary data indicate that radiation enriches for NRP2 expressing cells and using a function-blocking antibody specific to VEGF/NRP2 with irradiation decreases cell viability compared to either treatment alone in a TNBC organoid. The central hypothesis of this proposal is that VEGF/NRP2 induces radioresistance by altering redox homeostasis and can be targeted for better therapeutic outcomes in TNBC. This proposal will seek to investigate the possible role of NRP2 in regulating NOS2 transcription and its contribution to mitigating ROS accumulation. I will also use single-cell RNA sequencing technology to identify the subpopulations of TNBC that are radioresistant and whether they utilize the NRP2/NOS2 signaling axis. Another aspect of this proposal is to observe the effectiveness of a function-blocking antibody of NRP2 with radiation using an in vivo model. I plan to identify if this approach reduces the radioresistant clones in TNBC. The completion of this proposal will heighten the understanding of radioresistance in TNBC and identify a novel molecular pathway responsible for this phenotype.

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

    Liver transplantation is the only cure for liver failure, but many patients die waiting for a transplant due to a vast shortage of donor livers. Insufficient donor supply is further diminished by ischemia-reperfusion injury (IRI) during procurement and preservation of liver grafts. IRI-damaged grafts must be discarded because they are dysfunctional following implantation into recipients, causing patient death. Mitigating IRI is critically needed to increase the number of viable donor livers and improve patient survival. The IRI mechanism involves several pathways. After ischemic insult facilitates cellular injury, reperfusion triggers oxidative stress and innate immune pathways that converge to activate apoptosis, the major cell death mechanism in liver IRI. Hepatocyte apoptosis in IRI is mediated by Fas receptor (Fas), and leads to necrosis and inflammation, which cause liver dysfunction. In rats, Fas reduction prior to ischemia reduces liver damage. It may be possible to mitigate IRI-induced liver dysfunction by silencing hepatocyte-specific Fas during the transplantation process. The goal of this proposal is to use small interfering RNA (siRNA) to inhibit IRI and improve quality of liver grafts for transplantation. Chemically-modified siRNAs enable potent, sequence-specific silencing of any target gene in vivo. Modified siRNAs are delivered to hepatocytes when conjugated with N-acetylgalactosamine (GalNAc). GalNAc-siRNA technology is the basis of numerous FDA-approved liver disease drugs. With guidance from Drs. Anastasia Khvorova (siRNA), Paulo Martins (liver transplant), Athma Pai (RNA seq), Gyongyi Szabo (liver IRI), Jacob Bledsoe (liver pathology), and Matthew Gounis (in vivo imaging), this project will develop in vivo and ex vivo approaches using previously-validated GalNAc-siRNA targeting Fas to inhibit IRI and protect liver function. Aim 1 will determine how silencing Fas prior to ischemia perturbs IRI pathways (Aim 1.1), and explore if silencing Fas in combination with other mediators confers greater protection against liver damage (Aim 1.2). GalNAc- siRNAs targeting Fas, alone, or in combination with validated oxidative stress (Hmgb1) and inflammation (Tnfr1) mediators, will be injected into rats, and IRI will be induced using a liver clamp model. Post-reperfusion, target gene expression, liver damage, and IRI transcriptome changes will be assessed. Aim 2 will determine how silencing Fas in liver grafts (ex vivo) affects transplant outcomes in rats. Delivering siRNA during ex vivo preservation, by either static cold storage (SCS) or machine perfusion (MP), leads to uptake into liver grafts. Rat livers will be procured and GalNAc-siRNA targeting Fas will be delivered during SCS or MP. GalNAc-siRNA uptake, Fas expression, and IRI transcriptome changes will be assessed over 24 hours. This experiment will then be repeated, preserving grafts for maximal GalNAc-siRNA uptake/efficacy, transplanting grafts into rats, and measuring liver damage/function and recipient survival. Study findings will characterize how Fas silencing pre- and post-ischemia affects liver IRI pathways, identify in vivo and ex vivo approaches for maximal IRI inhibition, and help develop therapies that increase the donor pool and improve patient survival.

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

    Mitochondrial function declines during aging. The dysfunction is accelerated in age-associated diseases such as Alzheimer’s Disease and Parkinson’s Disease. Thus, therapeutic approaches to maintain or recover mitochondrial function may promote healthy aging or slow age-associated disease progression. Recently, we have shown that the mitochondrial network expansion that occurs during development is an emergent property of the synthesis of highly expressed mitochondrial proteins. Increased mitochondrial import of the highly expressed mitochondrial proteins outcompete the transcription factor ATFS-1, preventing it from entering mitochondria. This allows ATFS-1 to traffic to the nucleus and activate a mitochondrial network expansion transcription program known as UPRmt. These findings suggest an interplay between protein synthesis, mitochondria protein import capacity, and mitochondrial network expansion. The Integrated Stress Response (ISR) is a translation control pathway that reduces overall protein synthesis while preferentially increases translation of ATF-4 in response to diverse stressors including amino acid depletion, ER dysfunction and mitochondrial perturbations. The ISR is mediated by 4 kinases (3 in C. elegans) that all phosphorylate the translation initiation factor eIF2α, which in turn modulates protein synthesis. While considerable work has demonstrated that the ISR is active in response to mitochondrial perturbation, the functional outputs of the ISR related to mitochondrial biology remain unknown. I have obtained or generated several C. elegans strains in which the ISR is impaired. Quite surprisingly, these worms have increased mitochondrial mass and mitochondrial genomes. Intriguingly, these animals also live significantly longer than wildtype worms, suggesting that increased mitochondrial mass is sufficient to extend organismal lifespan. I hypothesize that the ISR matches mitochondrial network expansion with the physiological and environmental inputs that activate the ISR by antagonizing ATFS-1 function. Here, I focus on the role of ISR-dependent translation attenuation or ATF-4 synthesis as direct or indirect regulators of ATFS-1-dependent transcription via the following aims. 1. Determine the mechanisms by which the ISR regulates ATFS-1-dependent mitochondrial biogenesis. 2. Elucidate the mechanisms by which the ISR modulates longevity and healthspan.

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  • Zachary Dyer - Ash Research Group - Funded by NIH

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

    Black, Latinx, and Indigenous populations in the US face a disproportionate burden of poor health outcomes. Progress toward eliminating gaps in health outcomes is minimal, despite increasing investments in and awareness of health inequities. Recognizing that those inequities are rooted in the conditions in which we live, grow, work, and learn, there has been increased attention toward social determinants of health. In the past several years, health systems and the federal government, through Medicare and Medicaid, have committed billions of dollars to address health-related social needs such as housing, nutrition, and transportation. Though increasingly recognized as the root cause of unequal mortality and disease burden, structural racism is infrequently considered, poorly understood, and inadequately measured. Using a structural racism framework, this study will create a neighborhood-level structural racism effect index by compositing publicly available data. Including data about housing, transportation, education, wealth and poverty, social cohesion, the built environment, employment, and criminal justice, the structural racism effect index will capture broad and interwoven effects of past and current racist policies. The index will assign a score of 0-100 to each census tract in the US and will be tested against publicly available outcome data such as average area life expectancy and prevalence of select health outcomes. The novel structural racism effect index may be used to predict costs and outcomes, direct resources, and inform decision-making about under-resourced populations. To illustrate the policy implications of a measure for the effects of structural racism, the index will be used to characterize the Medicaid population as a means of providing insight into where investments should be made. Using Massachusetts as a test case, this project will quantify the extent to which structural racism's effects modify the impact of a $149 million program to address the health-related social needs of the Massachusetts Medicaid population. 1

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

    During development of the human brain, neurons are forming a mature neural circuit, which requires major rewiring of synaptic connections. Developmental remodeling helps the brain integrate rewired connections, which when disrupted is linked to neurological disorders such as schizophrenia and autism spectrum disorder. The goal of this project is to identify mechanisms regulating the highly conserved process of synapse remodeling. I will be using the simple model system Caenorhabditis elegans because it undergoes a striking example of remodeling in GABAergic dorsal D-class (DD) motor neurons. Prior work on synapse remodeling has largely focused on the presynaptic side, while my preliminary work has focused on the post-synaptic domain. The Francis lab has identified dve-1 to act as a transcription factor of remodeling, specifically synapse elimination. Through bulk RNA-sequencing (RNA-seq) I have identified 2 downregulated targets of dve-1, the calcineurin-like EF-hand protein CHP1/chpf-1 and the regulatory subunit of CalciNeurin, PPP3R1/cnb-1. Preliminary data has shown a defect of synapse remodeling in both cnb-1 and chpf-1. Additionally, I will determine the functional requirement and site of action for cnb-1 and identify the contribution of calcineurin phosphatase function and the proteasome to the effect of cnb-1 on synapse remodeling. In aim 2, I will further characterize the role of cnb-1 in both synapse elimination and maintenance, while also identifying the effect of calcium binding on synapse remodeling. In aim 3 I will identify potential targets of dve-1 in the remodeling pathway by using neuron-specific RNA-seq. My proposed work will advance the understanding of remodeling and the mechanisms of regulation, potentially providing targets for future exploration.

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

    Ulcerative Colitis (UC) is a devastating disease characterized by recurring episodic inflammation of the colonic mucosa that imposes a significant health and monetary burden on the developed world. Currently a significant portion of patients with UC are treated with TNFα inhibiting antibodies. Such treatments are burdensome on the healthcare system financially and pose the risk of significant side effects and frequently lead to the development of anti-drug antibodies, and consequent infusion reactions, and treatment failure. Consequently, researching novel cost effective, low risk approaches for treating ulcerative colitis should be of high priority. One approach is to leverage the microbiome to restore and maintain a non-inflammatory state in the colon, instead of targeting the systemic immune system. Dysbiosis is a hallmark of ulcerative colitis and leads to consequent dysregulation of local host immune pathways such as neutrophil transmigration through the intestinal epithelium, which has been shown to be instrumental in the initiation of mucosal inflammation in UC and its perpetuation through disruption of the intestinal barrier. The dysbiotic microbes in the colon of patients with UC have been shown to decrease P-glycoprotein (P-gp) expression. Under homeostatic conditions P-gp inhibits neutrophil transmigration through maintenance of a transepithelial gradient of endocannabinoids, thereby preventing aberrant inflammation. Thus, increases in intestinal epithelial cell (IEC) P-gp expression promises to limit inflammation in UC by preventing neutrophil transcytosis. To this end we must understand the mechanisms by which intestinal P-gp is regulated. While previous work has shown the microbiome dependence of intestinal P-gp expression, the specific microbial signals and the underlying metabolic networks have not yet been explored. In this proposal I will design an optimized microbial consortium to induce P-gp in IECs and dampen colonic inflammation in ulcerative colitis. Additionally, I study the microbial signals and underlying microbial dynamics that induces P-gp. In Aim 1 I will determine candidate bacterial species with the potential to regulate IEC P-gp. I will then use these strains to design and optimize a commensal consortium to induce P-gp when transferred into mice. The use of such a consortium as a potential bacteriotherapeutic for dampening intestinal inflammation will be studied using murine inflammatory bowel disease models. In Aim 2 I will study the mechanisms by which microbes communicate with each other and the host epithelium to induce P-gp. I will use a targeted and an unbiased approach to determine the bacterial signals and metabolites that upregulate P-gp and study the interactions between bacterial species that encourage P-gp induction on IECs. Overall, this study will provide insight into how the human microbiome regulates neutrophil transmigration and consequently intestinal inflammation. The design of the commensal consortium will serve as a first step in the development of a bacteriotherapeutic for treatment of ulcerative colitis.

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

    Huntington’s disease (HD) is caused by expanded trinucleotide repeats (CAG) in exon 1 of the huntingtin (HTT) gene. Therapies lowering the downstream mutant HTT protein show limited clinical success. New evidence reveals that repeat tract length in the HTT locus, not mutant HTT protein, correlates to disease onset/severity. CAG repeat length is inherited, but further expands due to somatic instability, which contributes to HD. Somatic expansion occurs in non- dividing cells like neurons after transcription, forming a slipped loop that activates mismatch repair (MMR). In MMR, nuclease complexes help recognize the slipped loop and cut the non-slipped strand to create a gap that is filled to expand the repeat. Polymorphisms in MMR complexes are linked to HD onset, and knocking out or altering activity of MMR proteins block expansion or induce contraction in HD models. Yet, the contribution of each MMR protein to CAG expansion, and the effect of their conditional CNS-specific reduction on HD outcomes, is untested. Also, mechanisms favoring contraction over expansion are unknown. This project seeks to define MMR complexes facilitating HTT CAG expansion/contraction using divalent small interfering RNA (siRNA)—which induce potent, CNS-specific silencing of target genes—and antisense oligonucleotides (ASOs)—which can disrupt specific protein-nucleic acid binding in the CNS. Aim 1 will use divalent siRNA to evaluate the effects of MMR silencing on HTT CAG repeat expansion and HD progression. Efficacies of siRNAs targeting each MMR protein have been validated in human and mouse cells. Furthermore, one of these siRNAs was delivered to CNS of an HD mouse model, BAC-CAG (carries human HTT with 120 CAG that undergo expansion), showing target MMR silencing and blocked somatic expansion 2 months later. In Aim 1, divalent siRNA targeting each MMR enzyme will be injected into BAC-CAG mice. Target silencing and HTT CAG repeat expansion will be measured 2 months later. Top siRNA that block expansion will be re- injected into BAC-CAG mice, and the impact on motor behavior, ventricular size, and HD pathology will be explored over 9 months. Aim 2 will develop HTT CAG-targeting ASOs to induce MMR-mediated contraction in HD cells and mice. An initial panel of ASOs targeting HTT CAG repeats was screened in non-transformed HD patient-derived fibroblasts (HDpFs) using a high-throughput format, and ASOs that increase contraction events were identified. To improve contraction rates, ASO chemistries and lengths will be optimized and screened in HDpFs using the same assay. HTT CAG repeat length/instability will be quantified over 40 days to identify leads. Leads will be delivered to HDpFs, in combination with validated siRNA targeting each MMR protein, to identify MMR proteins mediating ASO-induced contraction events. In parallel, in vivo efficacy of leads will be confirmed in BAC-CAG mice. This work will reveal somatic expansion/contraction mechanisms, inform HD therapy design, and provide the fellow with crucial training in therapeutic development, neurobiology, and bioinformatics.

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

    Infants with Rett syndrome (Rett) are born with loss-of-function mutations in the gene encoding MeCP2, a global regulator of gene expression. MeCP2 dysfunction in the brain severely affects neurons, leading to neurodevelopmental deficits of varying severity that manifest after 6 months of age. Current treatments can manage some symptoms, but correcting MECP2 mutations would more effectively restore patients’ quality of life. CRISPR gene editing has made this approach conceivable. Among CRISPR technologies, prime editing is the most flexible, utilizing an RNA-guided Cas9 nuclease fused to reverse transcriptase to “search and replace” mutations in post-mitotic cells. Thus, prime editing is a strong candidate for Rett treatment. Yet, prime editor has only been delivered to neurons via lentivirus (not clinically relevant), and its editing efficiency is low. Previous work demonstrates that mRNA-based delivery of gene editors is simple, safe, and supports robust editing in liver. Lipid nanoparticle-encapsulated mRNA delivers to brain, but efficiency of mRNA-based prime editing in neurons, and how it compares to that of lentiviral delivery, is undetermined. In addition, chemically modifying the guide RNA of other CRISPR systems can protect against nuclease-mediated degradation and improve gene editing rates in cells. The Watts lab recently developed a method to synthesize long, chemically modified prime editing guide RNA (pegRNA), something that had previously been unfeasible. However, the effect of pegRNA modification on prime editing efficiency has not yet been tested. With support from Drs. Jonathan Watts (nucleic acid chemistry), Michael Green (Rett neurobiology), Erik Sontheimer (prime editor biology), Scot Wolfe (gene regulation), and Athma Pai (bioinformatics), this project will establish and chemically optimize mRNA-based prime editors to correct MECP2 mutations and reverse their phenotypes in neurons. Aim 1 will establish baseline effectiveness of mRNA-based prime editor (vs. lentiviral) against the most common Rett mutation (a missense mutation) and two clinically severe nonsense mutations in HEK cells expressing each mutant MeCP2, patient-derived induced pluripotent stem cells (iPSC), and iPSC-derived neurons. This Aim will also probe neurons pre- and post-editing to understand the molecular phenotypes of each MECP2 mutation and extent to which editing reverses them. Aim 2 will iterate on the Watts lab’s pegRNA assembly method to optimize pegRNA yield and synthesis time, and identify editing-compatible pegRNA modification patterns using in vitro and in cellulo assays. The effect of pegRNA modifications on editing MECP2 mutations will be tested and optimized in HEK cells, iPSCs, and iPSC-derived neurons, as in Aim 1. Molecular phenotypes of prime edited vs. unedited neurons will also be characterized as in Aim 1. This work will offer insight into how MECP2 mutants affect severity of Rett phenotypes in neurons and inform development of a prime-editing platform to treat any form of Rett as well as other neurological disorders. The training provided from this research will prepare the fellow for a productive career in the gene editing and neuro-therapeutics field.

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

    Non-alcoholic fatty liver disease (NAFLD) currently effects around 30% of Americans and costs the U.S approximately $103 billion annually. However, the standard of care remains lifestyle changes and liver transplantation with currently no FDA approved therapies. NAFLD often correlates with obesity which is rising in both developed and developing nations. Mammalian target of rapamycin complex 2 (mTORC2) is emerging as a central hub for carbohydrate and lipid metabolism, with its activation having been linked to NAFLD in mice. These recent studies have highlighted a role for mTORC2 modulation during high-fat diets in mice, where hepatic mTORC2 ablation provides a protective effect against high-fat induced hepatic steatosis. However, the dietary supplements that have been most attributed to the rise of NAFLD in humans are carbohydrates, specifically fructose. While protective effects of mTORC2 ablation in the liver during high-fat diet have been investigated, its impact on high-carbohydrate diets has been neglected. Here, I will examine the ability for mTORC2 to protect against high-carbohydrate induced hepatic steatosis, as has previously been seen in the context of high-fat diets. Mechanistically, I will investigate the role by which hepatic mTORC2 regulates carbohydrate derived acetyl-CoA synthesis and utilization, subsequently promoting the pathogenesis of NAFLD. I will accomplish this by examining two distinct acetyl-CoA producing enzymes: ACLY and ACSS2. Not only will I investigate mTORC2’s role in regulating both ACLY and ACSS2 through phosphorylation, but also for the first time, investigate mTORC2 loss in an acute setting using an auxin degron system, which is a plant based endogenous degradation tag. With the completion of this proposed work, I strongly believe that a more detailed and mechanistic understanding of hepatic signaling, and metabolism will be obtained, providing targets which could ultimately be used to treat NAFLD and other metabolic diseases.

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

    Trisomy 21 is thought to broadly impact the development, transcriptome, and activity of many neural cell types. However, the extent to which DS alters each is conflicted and potentially overestimated by variability not due to trisomy. Reducing variability through “trisomy silencing” will increase the power to detect legitimate effects of trisomy 21 in different neural cell types, when during development changes occur, and which remain correctable.

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

    Duchenne muscular dystrophy (DMD)—a fatal inherited muscular dystrophy—is caused by loss of Dystrophin, a protein that maintains muscle integrity. Corticosteroids slow DMD progression but cause side effects. Addressing the root cause of DMD may improve patient health without needing corticosteroids. Many DMDcausing mutations disrupt the dystrophin mRNA reading frame, resulting in non-functional protein. Strategies that skip the out-of-frame exon to restore the reading frame and produce semi-functional protein for improved muscle function could correct 64% of DMD mutations. FDA-approved antisense oligonucleotide drugs can skip select exons in dystrophin mRNA, but require lifelong infusions and only work in a small group of patients. Using CRISPR to edit dystrophin would require just one treatment. CRISPR-mediated ablation of splice sites to cause exon skipping can increase Dystrophin in DMD models. Yet, editing in unintended tissues is a safety concern for Cas9 therapies. An ideal platform for DMD would restrict editing to muscle tissue to maximize therapeutic benefit. Efforts to achieve tissue-specific editing often rely on delivery via adeno-associated viruses (AAVs) with tissue tropism; yet, it is rarely absolute. Tissue-specific editing was recently achieved using tissue-specific miRNAs to regulate expression of Cas9 inhibitors [anti-CRISPR (Acr) proteins] via miRNA target sites (TS) in the 3’ UTR of Acr mRNA. When the platform is systemically delivered to mice via AAV, Acr-TS targeted by liver-specific miRNA allows editing only in the liver. Unlike tissue-specific promoters, this Acr-TS strategy could be adapted to one or multiple muscle tissues affected in DMD, as long as muscle-specific (myo)-miRNA can repress an Acr. With support from Erik Sontheimer (CRISPR, Acr), Eric Olson (DMD), Wen Xue (in vivo CRISPR delivery), Phillip Zamore (miRNA), Guangping Gao (AAV), and Zhiping Weng (bioinformatics), this proposal seeks to develop a muscle-specific editing platform to treat DMD. The myo-miRNA, miR-1, can repress an Acr in muscle cell lines to achieve muscle-specific editing. To fine-tune specificity of editing in muscle tissues for DMD, Aim 1 will test the ability of myo-miRs varying in abundance and muscle-type specificity to repress Acr and drive muscle-specific editing in mouse cell lines. The myo-miR construct supporting highest muscle-specific editing will be delivered to a DMD mouse model, and in vivo muscle function as well as dystrophin exon skipping, Dystrophin protein, and miRNA level in muscle tissues and liver will be measured. Aim 2 will test the compatibility of additional Cas9 orthologs in the Acr-TS system to enable targeting of more sequences, and develop a single AAV delivery system for improved safety. An Acr inhibiting the Cas9s to be tested has been identified. The ability of miR-1 to repress this Acr and drive muscle specific editing by each Cas9 will be tested in cells. A single vector encoding the AcrTS system will be designed and packaged into AAV, and muscle-specific editing will be compared to a dual AAV system in mice. This work will develop a flexible, safe, muscle-specific CRISPR platform with the potential to be used for any combination of muscle tissues to treat patients with DMD, or other genetic muscle disorders.

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  • Nicholas Harper, Lee Research Group, F31 Award from NIH

    Mechanism of cell lethality following loss of gene expression

    The goal of this project is to determine the mechanism by which cell death results from transcriptional inhibition. The consensus model in the field posits that cell death following transcriptional inhibition results from the loss of specific mRNA species and subsequent loss of protein. By targeting such a core cellular process, transcriptional inhibition is thought to overwhelm cellular control and lead to unavoidable cell death. This death process, defined as Accidental Cell Death (ACD), is not controlled by the cell, and does not result from the use of defined effector molecules. Contrary to the conventional model, we found that, rather than induce ACD, cell death following transcriptional inhibition results from a previously undescribed regulated apoptotic signal. Furthermore, we found that RNA Pol II degradation, rather than loss of mRNA production, resulted in cell death. Our data suggests a new model, whereby degradation of Pol II induces a signal that leaves the nucleus and is received by the mitochondria to initiate apoptosis. To identify genes that regulate a pro-apoptotic signal following transcriptional inhibition, we performed a genome-wide CRISPR screen. Genome-wide CRISPR screens often fail to identify death regulatory genes, making it difficult to elucidate mechanisms of cell death. To overcome this, we developed a novel experimental strategy that allowed us to identify genes whose knockout modulated the cell death rate following transcriptional inhibition. Based on the results of our screen, in Aim 1 we will test the hypothesis that the alternative splicing regulator PTBP1 facilitates altered splicing and nuclear export of regulatory pre-mRNA, and that this activity is required for cell death following transcriptional inhibition. We will use live cell microscopy to establish the functional role of PTBP1 nuclear export. We will use SLAM-seq and RIP-seq to quantify PTBP1 activity following transcriptional inhibition. Our screen also identified BCL2L12 as the critical apoptotic effector gene for transcriptional inhibition. In Aim 2, we will test the hypothesis that BCL2L12 activates apoptosis following transcriptional inhibition in an isoform-specific manner. We will perform a series of functional genetics experiments to characterize the role of BCL2L12 in the apoptotic response. By describing a new mechanistic model by which transcriptional inhibition induces cell death, we will improve our understanding of how to effectively use transcriptional inhibitors therapeutically. Ultimately, we hope our work will improve our ability to predict which patients will best respond to transcriptional inhibitors and help identify novel treatment strategies.

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    The role of Neurexin in serotonin synaptic function and social behavior

    The goal of this proposal is to examine how presynaptic Neurexins (Nrxns) at serotonin (5-HT) synapses impact 5-HT signaling and social behavior. Extensive 5-HT axon terminal innervation throughout the brain corroborates 5-HT’s modulatory role in numerous behaviors including social behaviors, reward, emotion regulation, and learning and memory. Abnormal brain 5-HT levels and function are implicated in Autism Spectrum Disorder (ASD). While 5-HT therapeutics are often used to treat ASD, variable improvements in symptomatology require further investigation of 5-HT-mediated pathology. Many different genes contribute to increased ASD susceptibility and clinical presentation variability. Notably, synaptic dysfunction, specifically dysregulation of synaptic excitation and inhibition, remains a hallmark of ASD pathogenesis. Nrxns are presynaptic cell adhesion molecules that are well characterized in maintaining synapse function for proper neural circuit assembly. The three Nrxn genes transcribed from two promoters (α and β) express six principal Nrxn isoforms (αNrxn1-3, βNrxn 1-3). Additionally, mutations in Nrxn1 and Nrxn2 genes have been reported in ASD. In the current literature, the role of Nrxns at 5-HT synapses has yet to be investigated. Given that aberrant Nrxn and 5-HT function independently contribute to signaling pathology and social behavior impairments, it is critical to understand how Nrxn-mediated 5-HT neurotransmission participates in pathological mechanisms underlying the core deficits of ASD. Here, I will explore how 5-HT signaling mediated through Nrxns regulates social behaviors (Aim 1) and how Nrxns regulate 5-HT circuits relevant to social behaviors (Aim 2). Our group has created a novel mouse model in which the three Nrxn genes are selectively deleted in 5-HT neurons. My preliminary studies indicate that the loss of Nrxns at 5-HT synapses impairs social recognition memory and social reward preference. The hippocampus and nucleus accumbens, respectively, are crucial in these behaviors. In Aim 1, I will determine whether 5-HTergic Nrxns are critical for social behaviors through completion of social (and other complex) behavior studies. In addition, I will explore (i) if and (ii) how 5-HT is necessary for social behaviors using (i) 5-HT therapeutics to augment 5-HT function prior to social behavior studies and (ii) in vivo microdialysis to measure extracellular 5-HT levels during social behavior. In Aim 2, I will perform a mouse breeding and lentiviral rescue approach to determine whether specific Nrxns control social behavior. Furthermore, I will use immunohistochemical and electrophysiological approaches to identity how Nrxn proteins regulate excitatory and inhibitory synapse distribution and physiology. A close examination of Nrxns in 5-HT synaptic function is necessary to shed new light on social behavior disturbances in ASD.

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  • Samantha Tse, Pukkila-Worley Lab, Funding provided by National Institutes of Health

    Detection of intestinal pathogens through host surveillance of bacterial toxins

    Although commensal and pathogenic bacteria can be recognized by host pattern recognition receptors, intestinal epithelial cells target protective inflammatory responses towards pathogenic organisms through mechanisms that are incompletely understood. Additional mechanisms of pathogen sensing must exist that allow intestinal cells to target inflammatory defenses towards bona fide pathogens during an infection, and not harmless commensal bacteria. Pathogenic bacteria can express virulence determinants. Phenazine toxins are a family of redox active virulence determinants that are produced by a variety of human pathogens, including P. aeruginosa. P. aeruginosa can colonize the intestines of immunocompromised patients and cause fulminant septicemia and subsequent death. The mechanism by which intestinal epithelial cells detect P. aeruginosa, and whether this involves the surveillance of phenazine toxins, is not known. Nuclear hormone receptors (NHR) are transcription factors that program adaptive host responses following recognition of specific exogenous or endogenous ligands. In particular, HNF4⍺ is an NHR expressed in the intestine. In the model organism C. elegans, the HNF4⍺ homolog NHR-86 is required for the transcriptional activation of innate immune effector genes that protect against P. aeruginosa infection. The central hypothesis of this proposal is that intestinal epithelial cells detect infection through the surveillance of pathogen-derived phenazine toxins by NHR-86/ HNF4⍺, which directly activates protective anti-pathogen defenses in the intestinal epithelium. The following key preliminary findings support this central hypothesis: i) P. aeruginosa mutants that cannot make phenazine toxins do not activate C. elegans innate immune defenses; ii) synthetic phenazine toxins can activate immune genes; and iii) induction of immune genes by phenazine toxins is dependent on the expression of NHR-86/ HNF4⍺. In this proposal, Aim 1 will characterize the C. elegans immune response towards bacterial phenazine toxins, and Aim 2 will define the role of intestinal NHR-86/HNF4⍺ in detecting P. aeruginosa infection in C. elegans. The research proposed here will define a new concept of immune activation in intestinal epithelial cells and will also attribute a novel role for NHRs in pathogen sensing in the intestine. Insights from these findings may identify unexplored approaches for the development of anti-inflammatory and anti-infective therapies.

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    Discovery of a novel role of VPS13D in Autophagy

    Defects in autophagy, the self-degradation of cellular components, are linked to multiple disorders such as cancer, diabetes and neurodegenerative diseases. Autophagosomes, containing cargo marked for degradation, fuse with lysosomes to recycle cell resources, such as protein aggregates and damaged organelles. However, we know little about the mechanisms that regulate the association between autophagic cargoes and autophagosome formation. Here, I investigate the role of vps13d, an essential gene with relatively unknown function, in context-specific autophagy and cell death in the developing Drosophila intestine. Proteins that regulate autophagy and cell death are of particular interest given the roles they play in tumorigenesis. Previous studies of VPS13D identified a role in the clearance of mitochondria by autophagy, also known as mitophagy. Intriguingly, VPS13D also appears to be involved in dissolution of membrane contacts that are associated with autophagosome formation. Furthermore, little is known about the role of VPS13D in associating autophagy-bound cargo with the site of autophagosome formation, despite having links to the core autophagy machinery. I hypothesize that VPS13D facilitates context-dependent autophagy by associating ubiquitinated cargo with the autophagic machinery and disassembling membrane contact sites at the phagosome assembly site (PAS). Here I propose to determine if VPS13D functions as an autophagy receptor for ubiquitinated cargo and determine the relationship between VPS13D and membrane contact modulator Vacuole Membrane Protein 1 (VMP1). The association of VPS13D and mutations in other factors that regulate autophagic cargo recruitment with human disorders illustrates the importance of studying VPS13D function.

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  • Kevin O'Connor, Kelliher Lab, Funding Provided by National Institutes of Health

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

    Therapy resistance is a major barrier to long term remission in pediatric T-cell acute lymphoblastic leukemia (T-ALL). The prognosis for children with relapsed or refractory disease is dismal. Leukemia-initiating cells (L-ICs) regenerate disease upon transplantation into mice. They also recapitulate the immunophenotypic complexity of the parent leukemia supporting that, as in normal hematopoiesis, there is a cellular hierarchy among leukemic cells. Our laboratory has previously demonstrated that the L-IC is a committed thymocyte progenitor and resides in the leukemic DN3 population, however, only a fraction of DN3 cells can give rise to disease. L-ICs rely on NOTCH1-induced MYC signaling for survival. Recent studies identified dormant, therapy resistant L-ICs in both murine models and T-ALL patient samples. The role of cell cycle restriction in L-IC latency is incompletely understood. In an effort to uncover pathways that govern L-IC function, we performed single cell RNA sequencing on thymocytes at varying stages of T-cell leukemogenesis using our transgenic Tal1/Lmo2 model. This approach identified a dormant DN3 cluster, marked by low Ki67 expression, which is observed in other murine T-ALL samples. Dormant DN3 cells exhibit high Notch1, but low Myc expression. The transcriptional signature of these cells shows enrichment of genes previously implicated in leukemia initiation or leukemia stem cell function. Dormant DN3 cells show enrichment of the non-canonical Wnt receptor Ryk, which is reported to maintain hematopoietic stem cell self-renewal by limiting proliferation and promoting quiescence. RYK is overexpressed in primary pediatric T-ALL and in Tal1/Lmo2-induced murine T-ALL compared to healthy thymus. This indicates that RYK may not be restricted to this rare subpopulation and moreover, there may be a therapeutic window for RYK inhibition in relapsed T-ALL. The central hypothesis of this proposal is that dormant DN3 cells are quiescent L-ICs that retain proliferative and differentiative capacity, which permits their therapy tolerance and subsequent expansion during relapse. This proposal will identify a gene signature of dormant DN3 cells and uncover the potential role of these cells in T-ALL relapse by evaluating their L-IC function and chemoresistance (Aim 1). Aim 2 will define the non-canonical WNT/RYK signaling network in T-ALL and uncover the role of these pathways in dormant DN3 cells and L-IC function by testing whether inhibition of RYK reduces the L-IC frequency of murine and patient T-ALL cells. Collectively, these studies will provide critical insight to TALL heterogeneity and will lay the foundation for development of L-IC targeted therapy for relapsed disease.

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    Investigating mechanisms of neurodegeneration

    Disease and injury cause a catastrophic loss of cognitive and motor abilities in the nervous system. Preventing neurodegeneration is critical to maintaining neuronal function. To develop effective treatments, we first need a greater understanding of the genetic and cellular mechanisms that determine whether the nervous system degenerates in response to various insults. Caenorhabditis elegans is a highly tractable model to dissect conserved molecular and cellular mechanisms. The goal of this proposal is to take advantage of the C. elegans model to identify regulatory mechanisms of axon degeneration. To reach this goal, I will apply my developing skills in detailed genetic analyses, laser axotomy, imaging, sequencing, and bioinformatics. The impact of this project is significant. In addition to providing a critical advance in understanding the fundamental mechanisms of neurodegeneration, it will also inform future therapeutic approaches that can be manipulated to protect the nervous system from degenerating.

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

    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.

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    Investigating the role of MS4As during Alzheimer's disease

    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.

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

    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.

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    Characterization of MS4A Chemoreceptive Function

    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.

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    Investigating the Activation Mechanism of SARM1 during Axon Degeneration

    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. One such protein is SARM1. SARM1 is a NAD+ hydrolase that cleaves NAD+ to nicotinamide, ADPR, and cyclic ADPR; generation of these products ultimately leads to axonal degeneration. Moreover, SARM1 knockout delays degeneration in animal models of Wallerian-like diseases, including traumatic brain injury and peripheral neuropathy. Given the critical role of SARM1 in Wallerian-like diseases, the central hypothesis of this proposal is that SARM1 inhibition would prevent the pathophysiology of axon degeneration associated with neurodegenerative diseases. However, development of SARM1 inhibitors is limited by the lack of knowledge surrounding the regulation, structure, and mechanism of this enzyme. As such, the goal of this proposal is to understand SARM1 regulation in the context of Wallerian degeneration, and this goal will be achieved by pursing the following Specific Aims. Aim 1 focuses on identifying proteins that regulate SARM1 activity. Proximity dependent labeling will also be used to identify proteins that interact with SARM1. The impact of SARM1 interacting proteins on NAD+ hydrolase activity and SARM1-mediated axon degeneration will also be assessed. These experiments will identify intermolecular events that regulate SARM1 during axon degeneration. Aim 2 will focus on understanding the structure and function of TIR-1, the C. elegans ortholog of SARM1. Here, we will solve the TIR-1 structure and characterize the enzymatic mechanism of this enzyme. These studies will complement recent structural and kinetic studies of SARM1 and will yield insights into the intramolecular characteristics of SARM1/TIR-1 that contribute to its degenerative capacity. Investigation into the regulation of SARM1, both inter- and intramolecularly, is a rapidly growing field in the context of neurodegenerative diseases. As such, completion of this work will significantly enhance our understanding of the fundamental molecular mechanisms that control axonal degeneration. These studies will yield insights into the role of SARM1 in axon degeneration, which will have broad implications in the development of therapeutics for neurodegenerative diseases.

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  • Melissa Goulding, Lemon research group, funded by NIH

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

    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.

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

    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.

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

    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.

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    Development of Advanced Oligonucleotides for Glioblastoma Therapeutics

    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.

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  • Joseph Magrino - Kelch Research Group - Funding provided by National Institues of Health

    Investigating sliding clamps and their contribution to genome stability

    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.

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

    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.

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  • Megan Honeywell, Lee Research Group, Funding provided by National Institutes of Health

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

    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.

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

    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.

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

    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.

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    Tissue-specific modulation of Apolipoprotein E in neurodegeneration

    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.

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  • Ariel Beccia, PHARE Study Group, Funding provided by National Institute on Minorty Health and Health Disparities

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

    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.

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

    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.

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  • Emily Agnello, Kelch Lab, Funding provided by National Science Foundation

    Elucidating the structural and mechanistic features of a thermophilic bacteriophage

    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.

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    Elucidating premature translation termination in Cystic Fibrosis

    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.

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    Dissecting ADAM10 function in microglia-mediated synapse elimination

    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.

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

    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.

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    Integrin Regulation of Mammary Gland Development

    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.

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

    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.

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  • Denis Lafontain, Dekker Lab, Funding provided by National Institutes of Health

    Stability of the folded genome

    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.

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    Feasibility of Smartwatches for Atrial Fibrillation Detection in Older Adults

    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.

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  • Yiyang Yuan, MPH, MS, PHARE Study Group, Funding Provided by National Institutes of Health

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

    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.

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

    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.

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

    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.

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    Mechanism of cdk4 diabetes rescue in IRS2 knockout mice

    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.

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  • Jordan L. Smith, Xue Lab, Funding provided by National Cancer Institute

    Investigating YAP1 control of differentiation and metabolism in Hepatoblastoma

    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.

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    Integrin Function in Breast Cancer Initiation

    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.

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

    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.

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

    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.

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

    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.

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  • Apurv Soni, Allison Research Group, Funded by NIH

    Trends, Predictors, and Consequences of Child Undernutrition

    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.

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

    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. SARM1’s causal role in Wallerian degeneration demonstrates that it is an attractive therapeutic target that could prevent disease progression. However, the design of therapeutics targeting SARM1 is limited by the dearth of knowledge surrounding its inherent NADase activity. In order to evaluate SARM1’s therapeutic efficacy and design potential SARM1 inhibitors, the proposed research will study its structure, enzymatic mechanism and cellular activity. Solving the structure by leveraging the benefits of crystallography and cryoEM, determining the enzymatic mechanism via a series of assays and analyzing the in vivo activity with activity-based probes will fill in important gaps. Revealing these properties would enable the design of SARM1 inhibitors that could ultimately treat Wallerian-type diseases. Moreover, demystifying the role SARM1 plays in neurodegeneration would also allow for a better understanding of these disease types, the enzymatic capabilities of toll/interleukin receptor (TIR) domains and the involvement of NADases in numerous disease states.

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

    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.

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    The Role of Extracellular Vesicles in Alcohol-Induced Neuroinflammation

    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.

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    Ethanol's Effects on Brown Adipose Development and Function

    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.

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    Gq Receptor Regulation of Striatal Dopamine Transporters

    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.

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    Fluorescent visualization of complement-dependent pannexin activity in microglia

    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.

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    Modeling Down Syndrome Neural Phenotypes with Chromosomal Silencing

    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.

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    Impact of Beclin 1 Loss on Breast Cancer Progression

    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.

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  • Alec K. Gramann, Ceol Lab, Funding provided by National Institutes of Health

    Targeting BMP signaling to treat advanced melanoma and suppress therapeutic resistance

    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.

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

    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.

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

    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.

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    The Role of 3' End Formation in Synaptic Function

    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.

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  • Lisa Nobel, Allison Research Group, Funded by NIH

    Patient and Social Determinants of Health Trajectories Following Coronary Events

    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.

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  • Alec K. Gramann, Ceol Lab, Funding provided by Melanoma Research Foundation

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

    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.

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    The Role of miR-122 in Alcoholic Liver Disease

    “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.”

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

    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.

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    The Role of Innate IL-17 Responses to Aspergillus fumigatus

    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.

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    The Impact of IRS2-microtubule interactions in the progression of breast cancer

    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.

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  • Daniel Frendl, Ware Research Group, Funded by NIH

    Functional Health Predictors of Other Cause Mortality Risk in Prostate Cancer

    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.

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    Modulation of Nicotine Reward-Associated Behaviors by MicroRNAs

    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.

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