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MD/PhD Sponsored Awards

CURRENT AWARDS

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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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    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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    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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    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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    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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    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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    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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PAST AWARDS

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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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    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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    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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    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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    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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    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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