Vanderbilt University

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Allergen-induced asthma is a chronic disease predominantly characterized by dysregulated type 2 inflammation in the lung. While the adaptive immune system has been implicated in asthma pathogenesis for many years, research over the last decade has recognized that Group 2 innate lymphoid cells (ILC2) are potent drivers of type 2 inflammation in the lung and major contributors to allergen-exacerbated asthma. ILC2 are similar in function to Th2-polarized CD4+ helper T cells in that they produce Type 2 cytokines like IL-5 and IL-13, but they lack rearranged antigen receptors and are instead activated predominantly by alarmin cytokines. While their pro-inflammatory potential and connection to asthma has been established, the precise mechanisms by which these cells are activated and proliferate in response to aeroallergens are ill defined. I have utilized the Collaborative Cross recombinant inbred mouse panel to map a quantitative trait locus (QTL) that associates with the number of lung ILC2, a function of activation and proliferation, in response to airway challenge with extract from the common environmental allergen Alternaria alternata (Alt Ex). The Collaborative Cross (CC) is an ambitious, multi-center mouse genetics project created to allow for the investigation of complex polygenic traits. The panel boasts over 50 recombinant inbred lines created from 8 founder strains (5 lab-derived, 3 wild-caught) that encompass >90% of the known genetic diversity in the Mus musculus species. I enumerated ILC2 after Alt Ex challenge in 45 unique founder and recombinant CC strains, and I successfully mapped a novel 0.343 megabase QTL associated with ILC2 number in the lung containing 72 protein coding genes. The goal of this proposal is to identify and define the precise function of the causative gene or genes within this QTL that contribute to ILC2 proliferation in response to aeroallergen challenge. I identified CD22, a Siglec family receptor that inhibits B cell proliferation, as the most biologically plausible of the 72 gene candidates. I hypothesize that CD22 is the causative gene driving the observed phenotypic differences among the CC lines and that CD22 signaling in ILC2 inhibits their activation and proliferation. I confirmed that CD22 is expressed on ILC2, and I observed differences in CD22 expression between CC strains that had high and low ILC2 number in response to Alt Ex challenge. I will further test this hypothesis with two specific aims. In Aim 1, I will perform experiments in our Alt Ex challenge model using CD22 knockout mice to investigate the effect of gene deletion on ILC2 proliferation in vivo. Further, I will utilize a CD22-blocking antibody to define the effect of receptor blockade on ILC2 proliferation. In Aim 2, I will continue to reduce the interval of my QTL to narrow the list of gene candidates through the phenotyping of additional mice. I will acquire Diversity Outbred (DO) mice and utilize my Alt Ex challenge model, increasing my fine mapping power and further defining my genes of interest. In completing these studies, I will characterize a previously unknown regulator of allergen-induced ILC2 number in the lung and potential therapeutic target in the treatment of allergen-exacerbated asthma.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Alzheimer’s disease (AD), a leading cause of morbidity and mortality in older adults, causes significant cognitive impairment, but current therapeutic approaches targeting core AD pathology have not provided clinically meaningful improvements in cognition. There is strong evidence suggesting parallel or ‘concomitant’ pathologies, such as vascular dysfunction, contribute to cognitive decline. Approximately 80% of AD patients at autopsy have evidence of co-occurring vascular pathology, and detectable vascular dysfunction predates detectable changes in traditional AD biomarkers. Associations between vascular risk and cognitive decline appear most pronounced in individuals with early or mid-life exposure to vascular risk factors prior to onset of overt cognitive impairment, suggesting that vascular risk may drive early cognitive decline and contribute to or exacerbate the effects of core AD pathology. To achieve clinically meaningful improvement in cognition, there is a dire need to develop multi-faceted approaches to prevention and treatment of cognitive decline to target concomitant risk pathways, such as vascular risk, alongside efforts targeting core AD pathology. Identifying novel plasma biomarkers predictive of adverse cognitive aging would greatly aid in these efforts by identifying novel therapeutic targets, but few plasma proteomic studies of AD to date have focused on early clinical changes, examined disease progression, or replicated results. This F30 proposal aims to fill those gaps by (a) focusing on participants with normal cognition and mild cognitive impairment, a prodromal form of AD, (b) examining longitudinal cognitive outcomes, and (c) replicating all results in a separate cohort. Leveraging these novel strengths, the applicant will take a two-pronged approach to (1) perform hypothesis-driven candidate protein analyses, based on prior literature and preliminary data, to characterize associations between ADAMTS13 and cognition, and (2) perform hypothesis-generating discovery analyses to identify novel biomarkers for adverse cognitive aging. Pursuing two distinct but complementary approaches offers a unique training opportunity to develop skills and methodologic approaches for working with ‘omics data and will directly contribute to efforts to identify novel biomarkers and therapeutic targets for adverse cognitive aging. The proposed research will leverage the rich resources of the Vanderbilt Memory & Alzheimer’s Center. The research will be guided by an interdisciplinary mentorship team, including experts in neuropsychology, neuroscience, plasma proteomics, cardiovascular medicine, and AD. The parallel training plan will facilitate the candidate’s acquisition of the necessary knowledge and skills to propel her into a successful career as an independent physician-scientist bridging clinical cardiovascular medicine and cognitive aging research. Findings from this F30 proposal will provide valuable insight into the role of ADAMTS13 in cognitive decline and aid in the identification of novel plasma biomarkers predictive of early cognitive decline.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Huntington’s disease (HD) is a chronic, fatal neurodegenerative disease characterized by impairments in multiple domains of neurocognitive functioning, particularly executive dysfunction (e.g., inhibitory control, working memory). Notably, levels of impairment in executive functioning within HD are highly heterogenous and the specific underlying mechanisms driving declines are not well understood. Evidence from seminal reviews and longitudinal empirical studies (e.g., Slavich & Irwin, 2014; Zainal & Newman, 2022), have identified chronic stress and inflammation as two important mechanisms that contribute to executive functioning deficits in other populations. However, little to no research has investigated how stress and inflammatory mechanisms may be contributing to declines in executive functioning in HD. The proposed study will fill this gap in the literature by examining the contribution of chronic stress, inflammation, and disease processes to impairments in executive functioning in a sample of adults with HD. The proposed study will leverage access to participants in an ongoing NIH grant (R01HD104188; m-PI’s: Compas & Claassen) testing the longitudinal associations among social connectedness and health behaviors in HD patients and their families as compared to a community sample of families. The primary goals of the proposed study are three- fold. First, this study will improve the quality of the measurement of stress in individuals with HD by adapting and using state-of the art methodology (i.e., stress interviews with a blind rating component) to assess general and disease specific stressful events in a sample of individuals with HD compared to a community sample of adults. It is hypothesized that individuals with HD will exhibit a greater number of general acute and chronic stressful events on average compared to a community sample of adults. Further, variability in disease specific acute and chronic stress within the HD sample will be associated with relevant demographic and disease characteristics (e.g., disease progression). Second, the proposed study will investigate two candidate mechanisms, chronic stress and CAG by Age Product (CAP) scores (a standardized marker of disease burden; Warner et al., 2022) that may contribute to heightened inflammatory processes in a HD sample. Lastly, direct and indirect path analyses will examine associations of stress, inflammatory cytokines, and CAP scores with impairments in executive functioning in a sample of individuals with HD. Because the proposed is embedded within a larger, longitudinal grant, a portion of analyses for the final aim will be tested both cross- sectionally and prospectively at 6-month follow-up. Training for the applicant will include development of new skills with biological methods (i.e., inflammatory cytokine assays), data management and analyses, along with an opportunity to expand knowledge on the biological underpinnings of stress and disease processes on an important indicator of functioning, executive function. Finally, evidence from this proposal yields potential important ramifications on treatment and intervention targets for supportive care in this population.

NIH Research Projects · FY 2026 · 2023-12

PROJECT SUMMARY Neurodevelopmental disorders are a serious health problem affecting more than 3% of children worldwide. More than 1,000 genetic variants in synaptic proteins are linked to neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. While the symptoms of these disorders differ, each of them affects transmission between synapses in the brain, altering how information is passed throughout the neural network. To understand what causes these disorders and how to treat them, researchers are striving to learn the mechanism behind how these genetic variations affect synaptic function at the cellular and molecular level. This study aims to help answer foundational questions about synaptic transmission using the emerging framework of macromolecular assemblies. Transmitting information efficiently from one synapse to another requires transcellular nanocolumns (TNCs). TNCs span the synaptic cleft and align the neurotransmitter release site of one neuron with the receptors on a neighboring neuron. Unfortunately, the arrangement of components within these TNCs remains unclear. This makes it challenging to determine whether disease-causing mutations disrupt functional TNC formation. This proposal employs an innovative, multidisciplinary approach that combines cutting-edge cryogenic electron tomography (cryo-ET), biochemical methods, mass spectrometry, cell imaging and electrophysiological recordings to generate a nanoscale, macromolecular blueprint of synaptic transmission. Our central hypothesis is that synaptic proteins form subsynaptic PSD nanoblocks, receptor nanodomains and cleft adhesion molecule pairs as key building components for TNC alignment and activity dependent re-organization, which is critical for regulating synaptic transmission and plasticity. Toward proving this hypothesis, the authors have already used cryo-ET on cultured primary neurons, induced human neurons, and isolated nerve terminals to directly visualize the nanoscale organization of TNCs in near native state. These efforts have provided the first molecular-resolution information on such TNC assemblies. Advancing from that success, we will utilize two independent yet complementary aims to establish the sub-10 nm resolution structure of TNCs in healthy physiology and diseases: (1) investigate the molecular architecture, composition, and assembly of postsynaptic nanoblocks, (2) determine the in situ structures of synaptic adhesion molecular pairs and glutamate receptors, then investigate their organization within the synaptic cleft and their alignment with PSD nanoblocks. This research will significantly advance scientific understanding of the molecular architecture, dynamics, and functions of synaptic nanostructures, particularly TNCs. This knowledge will enable development of new therapeutics that target nanoscale structures.

NIH Research Projects · FY 2026 · 2023-12

Neural circuits are actively restructured during development as synapses are dismantled in some locations and assembled in others. Despite the importance of synaptic remodeling to circuit function, the underlying mechanisms are largely unknown. To investigate this question, we are exploiting the DD GABAergic motor neurons in C. elegans which undergo synaptic remodeling during larval development. In newly hatched larvae, DD presynaptic boutons are initially positioned on ventral muscles but are then relocated over a ~5 hr period to the dorsal DD neurite for input to dorsal muscles. DD remodeling is transcriptionally regulated by the Iroquois-type homeodomain protein, IRX-1. IRX-1 directs synaptic remodeling by upregulating UNC-8, a sodium epithelial channel (ENaC), which triggers a Ca2+-dependent mechanism of presynaptic disassembly. Additional downstream effectors are likely required, however, because UNC-8 dismantles a subset of presynaptic components (RAB3, v-SNARE, liprin-, endophilin) whereas IRX-1 also acts in a parallel pathway to remove additional presynaptic proteins (UNC13, ELKS, Clarinet). To identify additional IRX-1 targets, we used single-cell RNA-Sequencing (scRNA-Seq) to detect transcripts that are upregulated in remodeling DD neurons. An RNAi screen of this dataset detected a necessary role for the neural cell adhesion protein, NCAM-1, in DD synaptic remodeling. A genetic mutant of NCAM-1 impairs both the removal of ventral synapses as well as their reassembly in dorsal DD neurites, thus confirming that NCAM-1 normally promotes remodeling. Considering the established roles of NCAM in vertebrate neural development, my studies of the C. elegans NCAM homolog could reveal conserved mechanisms for synaptic plasticity that also operate in the brain. I hypothesize that NCAM-1 mediates presynaptic disassembly in remodeling GABAergic neurons in parallel to UNC-8. In Aim 1, I will test the role of NCAM-1 as a regulator of presynaptic remodeling using (A) smFISH to determine if ncam-1 is regulated by IRX-1, (B) live-cell imaging of GFP tagged Clarinet in ncam-1 mutants to determine if NCAM-1 functions in a parallel to UNC-8, and (C) GFP-tagged NCAM-1 to determine its location in remodeling DD neurons. In Aim 2, I will use genome-engineering to identify NCAM-1 structural domains that are required for presynaptic disassembly. Additionally, I will use cell-specific RNAi (csRNAi) to determine if ncam-1 function is required in DD neurons for synaptic disassembly. Finally, in Aim 3, I will determine if NCAM-1 is required for recycling of photoconverted (red) dendra2::RAB-3 from ventral to dorsal synapses in remodeling DD neurons. Because the vertebrate homolog of NCAM-1 mediates the removal of GABAergic inputs in the developing cortex, we suggest that the underlying mechanism may be conserved and thus can be molecularly defined by studies in C. elegans.

NIH Research Projects · FY 2026 · 2023-12

Project Summary Circuit-localized kinase signaling in the brain modulates local circuit activity and consequent behavioral output. An enormous roadblock to understanding this core mechanism has been the inability to image kinase signaling in vivo, but this barrier has finally been overcome with separation of phases-based activity reporter of kinase (SPARK) biosensors, which can visualize rapid, reversible kinase enzymatic function in targeted brain circuits. This proposal images 3 key pathways – protein kinase A (PKA), extracellular signal-regulated kinase (ERK), and calcium/calmodulin-dependent protein kinase II (CaMKII) – both independently and in combination. We will test roles of circuit activity, mapped neuromodulatory input, and kinase-kinase interaction in normal animals, as well as behavioral learning/memory dysfunction and epileptic seizure disease models. The exquisitely mapped Drosophila central brain mushroom body (MB) learning/memory center provides individually-identified MB inputs, core Kenyon cells and MB output neurons for circuit-level dissection of kinase signaling in vivo, within an expansive connectivity network. Learning acquisition and memory consolidation depends on Kenyon cells, which have well-defined input/output connectivity nodes ideal for informed, targeted SPARK imaging studies. In Aim 1, we image bidirectional activity-dependent kinase signaling with PKA- and ERK-SPARK biosensors. To test localized circuit signaling, we use neuron-targeted optogenetics, channel manipulations (e.g. TRPA1), and neurotransmission blockade (e.g. transgenic tetanus toxin) to dissect activity-dependent kinase signaling. We then assay the roles of this local kinase signaling on circuit function (employing GCaMP imaging) and on learning/memory behavioral output. In Aim 2, we image behavioral learning/memory dysfunction and epileptic seizure disease models for changes in circuit-localized kinase signaling. We then test the effects of genetic and pharmaceutical correction of kinase signaling on circuit function, learning/memory and seizure behavior. We assay the roles of defined neuromodulatory neuropeptide, serotonergic and dopaminergic synaptic inputs, using neuron-targeted ligand/receptor RNAi to dissect regulatory mechanisms. In Aim 3, we generate and test an essential new transgenic CaMKII-SPARK biosensor for in vivo circuit signaling studies. We image CamKII- SPARK in activity interaction (as in Aim 1) and behavioral mutant model (as in Aim 2) analyses. We then test kinase signaling, circuit function and behavioral output dependent on PKA/ERK/CaMKII signaling interactions. We image all three SPARK biosensors in combination with loss-of-function and gain-of-function of the other kinase signaling pathways, including Meng-Po kinase (human SBK1), which we propose balances/coordinates local circuit function. In multiply mutant kinase combinations, we will test local circuit activity with targeted GCaMP imaging, and consequent behavioral output in both learning/memory and seizure models. The overall goal of this proposal is the genetic dissection of circuit-localized PKA, ERK and CaMKII interactive signaling as an activity control in brain learning/memory circuitry, to improve both circuit function and behavior performance.

NIH Research Projects · FY 2026 · 2023-11

PROJECT SUMMARY Postpartum depression (PPD) is prevalent and associated with negative outcomes for both mothers and infants. PPD emerges during a period of psychosocial and neurobiological change, and there is a critical need for integrated research on trajectories of risk processes. Low activation of RDoC’s positive valence systems (PVS) prospectively predicts depressive symptoms outside of the peripartum period and may be particularly relevant for driving social motivation in mother–infant relationships. PVS function is reliably and objectively measured at the neural level, but very little research has examined brain function across pregnancy. Critically, psychosocial and neurobiological changes occurring across the peripartum period, including acute stress and elevated cortisol, are also known to impact PVS function. These disparate lines of research support the scientific premise of low PVS function as a key risk process in PPD and highlight the need to consider multimethod trajectories of change in PVS function as predictors of PPD, rather than relying on single measures and time points. We developed safe, feasible, and robust methods for repeatedly assessing reward- related brain function across the peripartum period, which we will combine with ecological momentary assessment of positive affect to chart trajectories of PVS function across the peripartum. For comparison, similar measures of negative valence systems (NVS) function will also be collected. We will recruit 300 pregnant women in their 2nd trimester (at least 50% classified as high risk for PPD) for an intensive longitudinal study with follow-up assessments conducted every 10 weeks through 25 weeks postpartum (4 lab and 2 virtual assessments). PVS function will be assessed from 15 weeks gestation to 5 weeks postpartum. Hair samples will allow for retrospective assessment of cortisol and other hormones to be estimated from conception through birth, and acute stress and birth trauma will be measured through gold-standard interviews. At 15 weeks postpartum, mothers and infants will participate in an observed free play interaction to assess mutual enjoyment. Depressive symptoms and diagnoses will be assessed from 15 weeks gestation through 25 weeks postpartum. This innovative study will advance understanding of the role of PVS function in PPD, including examining PVS function as a prospective predictor of PPD, comparing both baseline functioning vs. trajectories of change and PVS vs. NVS as predictors of depression (Specific Aim 1). We will also test PVS function across peripartum as a predictor of new mothers’ experiences of enjoyment in interactions with infants and examine low mutual enjoyment as a mechanism of the effects of low PVS function on PPD (Specific Aim 2). Finally, we will test longitudinal associations between acute stressors, cortisol levels, and subsequent PVS function, providing unique insight into the psychosocial and neurobiological processes shaping PVS function, and ultimately, depression risk (Specific Aim 3). The study will advance understanding of the dynamic interplay of processes impacting PVS and inform effective timing and targets for intervention to reduce the burden of PPD.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Our world is highly multisensory, and we acquire information about it via a number of distinct sensory systems. Typically, objects or events are specified by more than a single sense, and the integration of this multisensory information confers powerful and adaptive perceptual and behavioral advantages such as faster and more accurate responses. These advantages become pivotal when navigating complex environments where motion is ubiquitous, as is the case in the real world. However, multisensory processing also presents a computational challenge for the brain: to carry it out efficiently, the brain needs to not only decide which pieces of sensory information belong to the same event (and thus should be integrated or bound), but also which information needs to be segregated. Although great strides have been made in recent years to further our understanding of multisensory perception and its neural correlates, there are still significant gaps in our knowledge with regards to processing more ecologically-valid stimuli, such as those containing motion. One of these gaps revolves around how motion information is transformed in the presence of modulatory, cross-modal input as it makes its way through successive stages of the cortical processing hierarchy, and how these transformations map on to behavior/perception. The experiments outlined in the current proposal begin to address this issue using behavioral paradigms that we have developed in which macaques signal the direction of an auditory, visual, or audiovisual motion stimulus. During performance of the task, we will record neural activity in two cortical domains reflecting successive levels in the processing hierarchy: the medial temporal (MT) and medial superior temporal (MST) areas. The first aim will examine how modality and motion strength within audiovisual stimuli impact discrimination behavior and contribute towards causal inference. The second aim seeks to characterize responses to auditory, visual, and audiovisual motion information in these areas with the overarching hypothesis that as motion information ascends from MT to MST, there will be an increase in the role of modulatory auditory input, reflective of a gradual shift from encoding low-level stimulus features such as signal strength toward the encoding of features relevant to goal-oriented behavior such as stimulus direction and task demands. Collectively, the work will shed great light on the mechanistic underpinnings of multisensory perception in nodes critical to motion processing. Additionally, success in these experiments would challenge how we think about the modularity of the sensory cortical processing hierarchy. Such knowledge is of increasing importance given the growing recognition of altered multisensory function in those with neurodevelopmental conditions and/or sensory function loss, as well as the value of brain-informed algorithms for naturalistic virtual and augmented reality technology.

NIH Research Projects · FY 2025 · 2023-09

The overarching goal of this proposal is to create a four-state Mid-South REACH Hub to accelerate real-world impact of biomedical innovations through education, mentorship, and financial support for aspiring entrepreneurs. This Hub is critically needed, as much of the region is not supported by any REACH Hub, and our consortium dramatically extends REACH geographically to a network of hundreds of colleges and universities who currently encounter challenges in accessing federal support for innovation and entrepreneurship. The significance of this endeavor is twofold: we will turn many more academic discoveries into real-world products that save lives and improve human health, and we will catalyze a medical innovation economy in states that have a robust innovation pipeline but limited resources to translate these discoveries into a knowledge- based biomedical technology industry. Leveraging more than $2M per year in committed state and institutional matching funds, Mid-South REACH will exponentially expand the impact of the REACH program. Innovation in our Hub comes from an approach to ensure high quality decision making, the ability to reach a large network of colleges and universities across four states, and the new flexible, scalable, and sustainable Hub model we propose in which many states and universities contribute matching funds that are then used to fund innovators within their own constituencies. This model makes it possible to add new partners; each can be confident that their money will stay in their local area, enhancing sustainability. Our approach is to extend excellent education, proactive mentoring, and financial support throughout a coalition of states and universities across the Mid-South, combining Vanderbilt’s expertise in entrepreneurial education with the collective experience of our partner institutions in supporting biomedical innovation. Our Hub will be led by an experienced Multi-PI leadership team from each state who bring extensive networks, and outstanding experience in innovation, entrepreneurship, and medical research to the team. Together, we will recruit an exceptional External Review Board, with funding decisions made by the board and the PIs. We will experientially educate our teams via proactive mentoring that begins from the earliest moment of the pre-proposal stage and continues throughout their projects, encouraging them to focus on product-market fit and to “fail fast” by identifying and working toward the critical six-month technical and educational milestones that would justify subsequent funding tranches. Ultimately, the payoff of this process in terms of startups founded, job creation, and economic impact will motivate institutions and state governments to continue providing funding and in-kind administrative support, making the Mid-South Hub self-sustaining and multiply NIH’s initial seed investment manyfold into the future.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract The re-allocation of metabolic resources towards cell processes that promote somatic maintenance is a long- held hypothesis to explain how diverse longevity paradigms promote healthier aging. One of the best models of this hypothesis is dietary restriction (DR), which optimizes metabolic efficiency while maintaining animal fitness and longevity. However, we possess surprisingly little insight into how specific nutrient resources are differentially utilized at the cell level during DR. In addition to altering metabolic processes, DR promotes a dramatic remodeling of organelle structures and functions. Increasingly we understand that not only is the structure of a discrete organelle critical for its functional state, but also the spatial relationships and contact sites formed between different organelle networks. The restructuring of the overall subcellular architecture thus plays a critical role in determining metabolic performance. However, our preliminary data suggest that age-dependent accumulation of molecular damage causes remodeling of the endoplasmic reticulum (ER), a hub of inter- organellar communication. Our overarching hypothesis is thus that remodeling of inter-organelle interactions is is both a key route by which aging cells lose functional resilience and an essential mechanism of DR-mediated reprogramming of metabolism. To advance this hypothesis we propose to exploit new correlative electron microscopy (EM) and stable isotope imaging technology in a combination of mouse and C. elegans models to establish a framework for how DR restructures the organelle interactome and re-allocates nutrient flux between organelles. Altogether this proposal aims to establish a framework for how DR remodels the organelle interactome to promote healthy aging with a focus on the role of the ER.

NIH Research Projects · FY 2025 · 2023-09

Understanding how learning occurs in early childhood has the potential to transform our understanding of human learning and our approach to building intelligent machines, yet critical windows in early childhood remain under-sampled and consequently provide little insight concerning learning. One fundamental and long-standing question in human learning is the process by which neural specialization for visual letter and digit processing emerges in the first grade. This knowledge is critical for addressing public health concerns related to reading and math literacy because first-grade letter and digit knowledge are the strongest predictors of future reading and math abilities, and children who fall behind in reading and math in elementary school will likely experience medical and financial instability as adults. This project employs a multi-level approach to understanding learning in childhood that will support critical advancements in several disciplines, including human and artificial learning, developmental and cognitive neuroscience, educational neuroscience, neuroimaging methods, computer vision, and learning sciences broadly. The first aim is to create and distribute a large corpus of images from Sesame Street episodes annotated for educational content, such as letters and digits, as well as for other common object categories. The image corpus will be the first to capture the visual statistics of child learners and can be used to train different artificial learning architectures to better understand human learning. The second aim is to collect, preprocess, and distribute a dense longitudinal MRI dataset of brain structure and function sampled at multiple time points throughout the first grade year. The dense longitudinal MRI dataset will provide experimentally measured brain responses to images from the Sesame Street corpus that will be of benefit for understanding human learning and of appropriate scale for constraining artificial learning architectures. The third aim is to evaluate the emergence of selective neural processing for letters and digits as learning occurs throughout the first year of schooling. This aim will address an open question in human learning concerning the process by which neural specialization for letters and digits emerges, namely the role of the motor system in emerging specialization. Understanding the time course of changes in brain function and structure during early learning is critical for developing accurate predictors of long-term life outcomes and for identifying sensitive windows of great plasticity to optimize intervention timelines.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Social determinants of health (SDoH) are associated with poor health outcomes and health disparities. The National Academies of Sciences, Engineering, and Medicine’s Future of Nursing Report encourages clinicians to address SDoH, which are the environmental conditions in which people live, learn, work, play, and worship, that can affect health, functioning, and quality-of-life. While there is a large corpus of knowledge associating SDoH with childhood asthma, a gap exists on screening of SDoH in school-based health centers (SBHCs). SBHCs care for children pre-kindergarten to 18 years of age, working collaboratively with the school nurse to advance the health and well-being of all youth. Although SBHCs have access to individual-level health information about the child, SBHC providers do not have easy access to SDoH information at the neighborhood and community level. Screening pediatric families for SDoH in a systematic way can facilitate connection to pertinent resources. However, there is a dearth of information regarding SDoH screening and the role of SBHCs in pediatric SDoH interventions. This project focuses on the critical need to identify the facilitators and barriers of SDoH information gathering behaviors in SBHCs. Through an exploratory sequential design, the project will include the development of an informatics resource providing SDoH information to SBHC providers. Asthma will serve as the first clinical condition for this novel work. The investigators will address three specific aims: Aim 1. Describe the information gathering behaviors for SDoH and asthma performed by school-based health providers (i.e., nurse practitioners, physicians, physician assistants) for preventative asthma care, and what barriers exist for information gathering. Aim 2. Develop and evaluate an informatics resource that provides actionable SDoH information related to the preventative management of asthma. Aim 3: Evaluate the feasibility of an informatics resource comprising SDoH among school-based health providers for use in the preventative management of asthma. The PI will receive research training through the rich scientific environment of Vanderbilt University. Training goals will focus on acquiring knowledge and skills in biomedical informatics, research methodology, qualitative focus groups, SDoH, and responsible conduct of research. The highly experienced mentorship team will provide support to make continued progress toward independence in a program of research focused on SDoH in school-based health centers, of which this project is a key component. Upon successful completion of the proposed study, contributions are expected to fill a critical knowledge gap with evidence that supports screening for SDoH by school-based health providers. These contributions will be significant because they are expected to provide a way to operationalize SDoH, to prevent asthma exacerbations, and address social needs to improve the lives of school-age children. The proposed study supports the mission of NINR through inquiry that addresses both the SDoH and Health Equity research lenses in the unique setting of school-based health centers.

NIH Research Projects · FY 2024 · 2023-09

Project Summary In this MOSAIC K99/R00 Pathway to Independence application, Dr. Brian O’Grady proposes training in models of subarachnoid hemorrhage (SAH) and development of therapeutics that will strategically compliment his expertise in the development of arteriole-specific growth of ex vivo brain tissue in a biomimetic hydrogel and 3D printed microfluidic fabrication. The training plan is paired with scientific studies that will develop and apply a novel microfluidic device for modeling subarachnoid hemorrhage stroke events and for use as a screening platform for a dual-targeted nanoparticle as a potential therapeutic for the damage caused by SAH and delayed cerebral ischemia. Dr. O’Grady’s primary goal is to become an independent researcher focused on creating biomimetic in vitro models of the brain vasculature and developing novel therapeutics for neurological diseases. The rigorous training described and the outstanding team of mentors in vascular biology (Dr. Lippmann), neurological disease pathology (Dr. Jefferson), and nanoparticle development and therapeutics (Dr. Duvall) will ensure his success in transitioning to independence. Through his training plan, Dr. O’Grady will gain 1) deeper knowledge of blood-brain barrier physiology and the neurovascular unit; 2) experience synthesizing and characterizing nanoparticles; 3) knowledge of modeling SAH and neurological disorders in vitro; and 4) strategies for running a successful interdisciplinary and collaborative research lab. SAH is defined as a cerebrovascular disease with the initial event of a ruptured brain aneurysm and accounts for 5% of all types of strokes. Despite this small percentage, SAH accounts for one third of all stroke-related years of potential life lost before the age of 65. While a new era of neurocritical care management has contributed to improved outcomes for SAH, the secondary consequences result in delayed cerebral ischemia (DCI). DCI has varying degrees of patient functional outcome and has no known interventions to improve quality of life. This lack of effective treatments is largely attributed to the high failure rate of translating brain-targeting drugs from animals to humans. Recently, there has been a global effort to produce a tissue engineered, in vitro model system that can represent the complex vascular anatomy and microenvironment of the neurovascular unit. Dr. O’Grady’s preliminary work demonstrates that a novel biomimetic hydrogel supports induced pluripotent stem cell-derived neural, mural, and glial cells and induces arteriole-specific growth of ex vivo human brain vasculature. This new vasculature consists of anatomically correct, concentric layered structures that were previously unobtainable. When supported by a microfluidic device, the arterioles anastomose and can be lumen- perfused and photoablated. Based on his preliminary data, Dr. O’Grady hypothesizes that the dynamic neurovascular microenvironment of a stroke-like event can be accurately modeled by this new in vitro system. In addition to developing a new in vitro model of SAH, this project will test and validate the neural protective efficacy of a dual-targeted therapeutic for SAH and DCI in the human in vitro model.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Long QT syndrome (LQTS) is a cardiac disorder characterized by the prolongation of the latter portion of the electrocardiogram trace (the QT interval) that increases risk of cardiac arrythmia, cardiac arrest, and sudden unexpected death. Approximately 1 in 2500 individuals suffer from the congenital form of LQTS, with 30-50% of cases being caused by mutations in the voltage gated potassium channel protein KCNQ1 (type 1 LQTS, or LQT1). Over 250 LQT1-associated mutations in KCNQ1 have been identified, but the impact of these mutations on the channel’s structure and function, and whether there are common mechanisms through which these mutations lead to KCNQ1 dysfunction in LQT1, is still unknown. The primary goal of this proposed project is to explore mistrafficking as a potential mechanism of KCNQ1 loss of function in long QT syndrome. Previous studies of LQT1-associated mutations in the KCNQ1 voltage sensing domain (VSD) found that the majority decreased KCNQ1 trafficking to the plasma membrane and destabilized the VSD. Additional studies have shown that mutations in KCNQ1 can lead to retention in the endoplasmic reticulum (ER) and increased proteasomal degradation. This has led to the hypothesis that mistrafficking is a common mechanism of protein dysfunction in LQT1. Mistrafficking has been identified as a disease mechanism in several other membrane-protein associated diseases, such as cystic fibrosis and retinitis pigmentosa, and in the case of cystic fibrosis, drugs have been developed to rescue mistrafficking and alleviate disease symptoms. This leads to the additional hypothesis that drugs that bind nascent KCNQ1 channels and increase their stability can increase the trafficking of KCNQ1. In line with these hypotheses, I propose two aims: 1) to classify mutations across KCNQ1 based on their impact on KCNQ1 trafficking, and 2) to develop a high throughput screening method to identify small molecules that increase cell surface trafficking. In Aim 1, I will classify the trafficking of all possible KCNQ1 variants with a fluorescence-activated cell sorting (FACS)-based deep mutational scanning method. This will allow me to determine whether the majority of LQT1-associated mutations cause mistrafficking and also provide information on variants of unknown significance (VUS) and other KCNQ1 variants. In Aim 2, I will utilize immunofluorescence and high content imaging to screen for small molecules that increase WT or mutant KCNQ1 trafficking. This will test the hypothesis that KCNQ1 mistrafficking is rescuable with small molecules. Together, the results of these aims will provide further insight into the molecular mechanisms behind KCNQ1 dysfunction in LQT1 and explore a possible route for developing novel treatments for LQT1.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY In response to damage from injury or disease, lost neurons in the adult mammalian retina are irreplaceable which can lead to visual impairment including blindness. Some non-mammalian vertebrates, such as teleost fish, can regenerate the retina in response to damage via the Müller glia. In teleost fish, Müller glia will respond to signals from damaged neurons by asymmetrically dividing to produce retinal progenitor cells. These progenitor cells then differentiate into neuronal cell types and functionally restore tissue. Mammalian Müller glia are quiescent and do not display regenerative capacity in response to damage. However, mammalian Müller glia demonstrate some cell state plasticity and can be reprogrammed into induced pluripotent stem cells (iPSC) in vitro. Mammalian Müller glia likely have the capacity for reprogramming but lack the cell intrinsic- or extrinsic-signaling necessary to do so. I have found that overexpression of L-Myc, a robust reprogramming factor used to reprogram many somatic cell types to iPSCs, leads to cell cycle re-entry in adult mammalian Müller glia in ex vivo retinal tissue. Using murine retinal explant tissue as an injury model, I will characterize the effects of L-Myc overexpression on proliferation and dedifferentiation in mammalian Müller glia. I hypothesize that L-Myc overexpression in Müller glia leads to direct and indirect changes in gene expression and chromatin accessibility that reprogram Müller glia toward a progenitor-like state. To test this hypothesis, I will first characterize the effects of L-Myc expression on proliferation. I will do so by defining the interval of proliferation as it relates to L-Myc expression and determining if Müller glia or their descendants are able to divide repeatedly following L-Myc overexpression. Next, I will characterize the effect of L-Myc overexpression in Müller glia on cell identity and gene regulation. Using data integration of RNA-sequencing, ATAC-sequencing, and CUT&Tag of FACS sorted L-Myc overexpressing Müller glia, I will identify gene regulatory networks affected directly or indirectly by L-Myc overexpression and identify changes in cell state. Combining Müller glia, a highly promising population within the mammalian retina for reprogramming, and L-Myc, a potent reprogramming factor to promote proliferation and dedifferentiation, creates an ideal scenario to reveal targets to promote regeneration in the mammalian retina through the analysis of gene regulatory networks activated or inhibited by L-Myc overexpression. Regardless of the cell state changes that occur in response to L-Myc overexpression in Müller glia, this project will reveal what signaling barriers exist that prevent tissue regeneration via Müller glia in the mammalian retina. This study is designed to identify future targets for genetic therapies to promote restoration of tissue and visual function after injury in the mammalian retina, which one day could lead to treatments for currently untreatable visual diseases.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Sepsis, or life-threatening organ dysfunction due to a dysregulated host response to infection, is a critical public health issue. Affecting nearly 50 million people annually, sepsis is a leading cause of death worldwide, and significantly impacts the global economy. A major reason for the substantial burden of sepsis is an insufficient understanding of the biologic mechanisms that potentiate its pathogenesis. One of the hallmarks of sepsis is endothelial injury, which manifests as endothelial barrier hyperpermeability and results in organ dysfunction including acute respiratory distress syndrome (ARDS). A known contributor to the disruption of endothelial barrier integrity in sepsis is cell-free hemoglobin (CFH), hemoglobin released into the circulation from lysed red blood cells. CFH is elevated in the majority of patients with sepsis and is associated with higher rates of organ dysfunction, such as ARDS, and death. This proposal seeks to define the pathophysiologic role of CFH in endothelial hyperpermeability in sepsis. A primary regulator of endothelial permeability is the endothelial glycocalyx, a matrix of glycoproteins and proteoglycans that lines the vascular lumen. In sepsis, this function is impaired due to increased activity of heparanase, an enzyme that degrades the endothelial glycocalyx. Importantly, greater glycocalyx breakdown correlates with worse sepsis outcomes. Given that heparanase expression is, in part, modulated by transcription factors that are stimulated by reactive oxygen species (ROS), and that CFH undergoes oxidation in the inflammatory environment of sepsis, producing ROS including superoxide in the process, I hypothesize that CFH-generated superoxide triggers glycocalyx cleavage via induction of heparanase expression, thereby serving as a critical mediator of endothelial hyperpermeability and consequent organ injury in sepsis. I will test the effect of CFH on the pulmonary endothelial glycocalyx using mechanistic approaches in both cultured primary human lung microvascular endothelial cells and murine polymicrobial sepsis. Both models will be used to accomplish each Aim. In Aim 1, I will determine the impact of superoxide and CFH on glycocalyx degradation, endothelial barrier function, and sepsis-associated lung injury, severity, and mortality. Aim 2 will define the role of CFH in the modulation of heparanase expression and activity. I will also interrogate whether alterations in heparanase expression and activity affect endothelial barrier permeability and sepsis outcomes. Finally, I will delineate the impact of CFH- generated superoxide on heparanase expression and activity to complete my investigation of this proposed pathway. In resolving the role of CFH in glycocalyx degradation and endothelial dysfunction, I will deliver unprecedented insights into the consequences of elevated circulating CFH during sepsis, with potential to unveil new approaches to the development of therapeutics for the treatment of sepsis-associated lung injury. Furthermore, the completion of this project will facilitate the development of my technical, critical thinking, and communication skills that will be crucial to my success as an independent physician-scientist.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Calcium in cardiomyocytes is released via an intracellular calcium channel, ryanodine receptor 2 (RyR2). In hearts where there are mutations in RyR2, spontaneous Ca2+ leakages can occur resulting in cardiac arrhythmias. Small molecule therapeutics such as flecainide, tetracaine, and dantrolene have low specificity, low membrane permeability, low solubility, poor selectivity, low potency, or toxicity. Therapeutic selectivity between RyR isoforms (RyR1, RyR2, RyR3) is lacking, heightening our interest in developing ent-verticilide as an anti- arrhythmic agent. This proposal focuses on the discovery and development of new therapeutics as antiarrhythmic agents. The proposed work is founded on our discovery of potent and selective inhibition of RyR2-mediated calcium flux by ent-verticilide. Through a cross-disciplinary collaboration, it was discovered that ent-verticilide is a selective inhibitor of RyR2-mediated calcium release, including a preliminary study of efficacy in vivo. As a 24 membered cyclic depsipeptide with a molecular weight of 853 Da, ent-verticilide falls outside of the category of a traditional small molecule drug. While “Beyond Rule of 5” compounds with in vivo activity are growing in number, an understanding of their pharmacokinetics (PK) has lagged, thereby requiring new chemical tools and creative tactics to advance the field. We will investigate the permeability of this unnatural product and its analogues by development of a structure-activity relationship profile focused on both permeability and increased efficacy. We aim to systematically design and synthesize structural analogues of ent-verticilide with varying degrees of N-methylation. Following the synthesis, we will study passive permeability and collect structural data to inform SAR, providing additional perspective for feedback to Specific Aim 1. By methodicalstructural change to ent-verticilide, we will create an SAR-based feedback loop between permeability, activity, structure, and conformation. A strength of this approach is the combination of rigorous tools to study passive membrane permeability, and cardiomyocyte-based functional studies using both permeabilized and non-permeabilized cells to achieve an overall hypothesis-driven approach to discover how analogues of ent-verticilide travel through cellular membranes and ultimately target RyR2. Another strength is our positioning to prepare diverse analogues that include ring-chain variants likely to exhibit contrasting permeability. With increased understanding of the mechanism of action, we hypothesize that we can design analogues of ent-verticilide with improved potency and selectivity, thereby providing a potential therapeutic against fatal ventricular arrhythmias.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Islet glucose-stimulated insulin and somatostatin (SST) secretion are perturbed in patients with type-2 diabetes (T2D) and in animal models of the disease, which contributes to disrupted glucose homeostasis. It is generally accepted that secretagogues stimulate hormone secretion from -cells and -cells in response to elevated intra- cellular Ca2. However, the mechanisms that control inhibition of islet Ca2+ handling via Gi/o-coupled receptors (Gi/o-GPCRs) and how they are altered in T2D are largely unknown. Data from our lab finds that Gi/o-GPCRs reduce islet Ca2+ entry via Src tyrosine kinase-mediated activation of Na+/K+-ATPase (NKA), which hyperpolarizes membrane potential (Δψp) and limits insulin secretion. Further data show that Protein kinase A (PKA) activation by Gs-coupled receptors inhibits islet NKA activity and stimulates Ca2+ entry. Moreover, we find that islet SST provides paracrine signaling that slows glucose-stimulated -cell Ca2+ oscillations via oscillations in NKA activity, which depends on the action of Src tyrosine kinase and PKA. Finally, our preliminary data provide the first evidence that diabetic conditions diminish islet NKA activity, which contributes to perturbations in glucose and GPCR control of Ca2+ handling. Based on these exciting preliminary data, the overall objective of this pro- posal is to elucidate how islet NKA is controlled and becomes disrupted during the pathogenesis of diabetes. This project will test the central hypothesis that that islet NKA activation by tyrosine kinases limits Ca2+ entry and hormone secretion through Δψp hyperpolarization; whereas, PKA inhibition of islet NKAs enhances Ca2+ entry and hormone secretion by depolarizing Δψp. The rationale that underlies this project is that understanding sig- naling that integrates NKA modulation of islet cell Ca2+ handling and hormone secretion will expose novel thera- peutic targets for restoring glucose-stimulated hormone secretion in T2D. This project will be accomplished with the following two specific aims: 1) Determine the mechanisms regulating NKA control of β-cell function in health and diabetes; and 2) Determine how NKA modulates -cell function and dysfunction. Under the first aim, trans- genic mice with -cell ablation of the - - and -subunits of the NKA complex subunits as well as human -cells with knockdown of NKA - - and -subunits will be utilized to assess the roles of NKA during secretagogue and Gi/o-GPCR modulation of -cell Ca2+ handling and insulin secretion. Aim1 will also determine how diabetic con- ditions impact NKA signaling and insulin secretion. Under the second aim, NKA control of -cell Ca2+ handling and function will be determined in mice with -cell specific ablation of NKA - - and -subunits or in human pseudoislets with -cell specific knockdown of NKA - - and -subunits. Furthermore, Aim2 will determine how reduced NKA function in -cells under the stressful conditions associated with diabetes contributes to -cell dysfunction. This project is significant because it is expected to illuminate mechanisms that alter -cell and -cell Ca2+ handling and disrupt islet hormone secretion in T2D. Moreover, this project plans to identify potential phar- macological strategies for normalizing islet hormone secretion and reducing islet dysfunction in T2D.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY A fundamental question in neurobiology is how environmental signals – both developmental and ongoing– induce plasticity in neural circuits and networks to shape behavior. Circadian photoperiod, the proportion of daylight in a solar day, is a pervasive environmental signal that varies substantially with latitude and season, and drives acute and long-term effects on mood regulation in humans and in animal models. The associations of the molecular circadian clock and photoperiod with mood disorders are clear, but the neurobiological mechanisms remain incompletely understood. The serotonergic dorsal raphe nuclei (DRN) are a critical nexus for integrating circadian photoperiodic input with mood and reward. They receive light input from the circadian visual system and polysynaptic input from the biological clock nuclei and make widespread outputs, including to midbrain nuclei mediating motivation and reward through dopaminergic transmission. Seasonal photoperiods (winter–like “short days” vs. summer-like “long days”) induce enduring changes in mouse DRN serotonin neurons - programming their excitability and intrinsic electrical properties, their serotonin content, as well as anxiety and depressive-like behaviors. We have previously shown that the TREK-1 K+ channel mRNA expression is photoperiodically regulated in DRN 5- HT neurons, and therefore may play a key role in photoperiodic programming of serotonin excitability. A number of independent lines of evidence indicate that TREK-1 in DRN neurons impact mood regulation and mood disorders. We now also report intriguing sex-dependent photoperiodic regulation of dopamine uptake and release downstream of the DRN in the NAc of female mice, indicating photoperiodic impact on circuitry for motivation and reward that mirrors the reported female bias of Seasonal Affective Disorder (SAD) in humans. We propose as our overall hypothesis that DRN 5-HT neurons are a primary site of photoperiodic programing – in which transcriptional regulation of TREK-1 plays a key role in regulating neuronal excitability. In congruence with the NIMH RDOC paradigm, we envision photoperiod programing as an extended circuit for positive valence system behaviors in which the output of the programmed serotonergic DRN induces convergent drive by the DRN and VTA inputs to alter NAc function, driving changes in the output of reinforcement/motivated behaviors. We will further elucidate a mechanistic basis of photoperiodic programming of 5-HT neurons involving TREK-1, and downstream effects of this programming on positive valence systems, including NAc dopamine release and uptake, NAc synaptic plasticity, and NAc-driven motivation behavior. Completion of these Aims will enhance understanding of key neurobiological mechanisms underlying photoperiodic regulation of mood, motivation, and reinforcement.

NIH Research Projects · FY 2024 · 2023-08

Project Abstract There are over 20 million sexual and gender minority (SGM) adults in the United States. SGM adults experience significant health inequities such as higher risk for Alzheimer’s Disease and related dementias (ADRD), higher prevalence and severity of chronic health conditions, and higher risk for multiple cancers. SGM discrimination likely accelerates aging, disrupts use of preventive care, and creates barriers to health systems when they are sick. The overall objective is to understand the relationship between provider- and policy-level factors to improve aging and health outcomes of midlife and older SGM adults. In Specific Aim 1, we will estimate population-level prevalence of subjective cognitive decline (SCD), severity of SCD, and receipt of informal care for SCD-related impairments using data from the 2015-2019 Behavioral Risk Factor Surveillance System (BRFSS). This is the largest probability sample of sexual minority (SM) adults available and all analyses will be stratified by sex and sexual orientation for the first time in public health research. We hypothesize that SM adults will report higher SCD prevalence and ADRD risk, but lower access to informal care provided by a spouse or family member. In Specific Aim 2, we will examine the relationship between 7 provider-level attitudes and practices towards SGM patients and preventive healthcare use (e.g., receiving colorectal cancer screenings, flu vaccinations) and level of cognitive impairment. This aim leverages novel panel data on midlife and older SGM adults from the NIA- funded Vanderbilt University Social Networks, Aging, and Policy Study (VUSNAPS). In Specific Aim 3, we will estimate the association of state policies legally permitting denial of health services based on sexual orientation and rates of preventive healthcare use, health status, and health behaviors among SM adults. In this aim, we will use BRFSS data and a difference-in-differences design to compare SM adults in states with and without legal denial policies. Results have significant implications for midlife and older SGM adults — an NIA priority population. By using the largest probability sample of SM adults available, we are able to stratify by sex and sexual orientation. These nuanced findings may inform policies regarding long-term supports and services to meet the needs of sub-populations with both SCD-related impairments and elevated risk for ADRD. Additionally, we use novel panel data on SGM midlife and older adults to identify provider-level predictors of preventive care. Research insights may inform larger scale interventions to improve attitudes and practices with SGM patients at the health system-level.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY In nature and human microbiomes, microbes regularly face challenges due to fluctuations in the availability of resources and nutrients - a lifestyle termed feast/famine. Previous studies investigating microbial adaptation to feast/famine have focused on the specific adaptations that allow microbes to survive extreme starvation, often overlooking how the eventual replenishment of resources affects evolution. However, due to evolutionary tradeoffs between growth and survival, the molecular, cellular, and behavioral phenotypes that evolve in response to feast/famine may vary based on the duration and severity of starvation. Common adaptations to resource limitation include expanding metabolic capability through nutritional competence and increasing efficiency by diversification into cross-feeding ecotypes. As microbial metabolism can be constrained by many biologically relevant factors, including the presence of oxygen, this can complicate evolution and limit potential adaptive trajectories. Research in my lab focuses on how microbes adapt and diversify in novel complex environments by applying multi-omic, systems microbiology approaches to experimental evolution. We plan to investigate how oxygen availability shapes microbial evolution to feast/famine by conducting an adaptive laboratory evolution experiment with two bacterial species, the facultative anaerobe Escherichia coli, and the fastidious aerotolerant anaerobe Lactobacillus crispatus. We will characterize populations for fitness outcomes, common adaptive mutations, and patterns of diversification to determine how oxygen influences adaptation to feast/famine conditions. We will follow up by characterizing the effects of common adaptive mutations on microbial physiology using transcriptomics and high-throughput phenotyping. Further, as oxygen can shift the topography of the adaptive landscape by affecting the rate and spectra of mutations, we will also perform mutation accumulation experiments on facultatively anaerobic, aerotolerant anaerobic, and obligately anaerobic bacterial species in the presence and absence of oxygen. Studies of microbial evolution have historically neglected fastidious microorganisms and anaerobic environments due to the challenges associated with their culture. Our research will provide fundamental knowledge about evolutionary processes in a neglected fraction of the microbial tree of life that accounts for a significant proportion of the human microbiome.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Urinary tract infections (UTIs) are one of the most common bacterial infections worldwide and the bacteria that cause them are becoming increasing resistant to frontline antibiotics. As a result, last resort antibiotics, like fosfomycin, are beginning to be more frequently prescribed. Uropathogenic Escherichia coli (UPEC), which is the primary cause of UTIs, can become resistant to fosfomycin, through mutations that impair the function or production of the UhpT transporter, which imports fosfomycin into the bacterial cell. The current paradigm is that such mutations come at a fitness tradeoff because impairment of UhpT limits the import of the glycolysis intermediate glucose-6-phosphate. However, my preliminary data indicate that mutations that lead to increased resistance to fosfomycin by abrogating uhpT expression, do not impede colonization of the host urinary tract. In fact, loss of fosfomycin import demonstrates increased persistence during long-term infection. Additionally, I have shown that 77% of screened UPEC clinical isolates harbor fosfomycin resistant subpopulations. This raises the alarming hypothesis that fosfomycin resistant subpopulations arise during UTI, possibly in response to a host-imposed stress and may provide additional fitness advantages for the pathogen. I will test this hypothesis through two specific aims which will: determine the contribution of fosfomycin resistant subpopulations to UPEC pathogenesis (Aim 1) and elucidate the basis of prolonged bacteriuria during UPEC pathogenesis following loss of uhpBA (Aim 2). Together my studies will provide insights that will ultimately help us curb the onset and propagation of resistance to one of the last- resort antibiotic agents, by thoroughly elucidating novel mechanisms that lead to fosfomycin resistance during infection and gauging their fitness advantages and disadvantages in comparison to their parental strains. Through the execution of these aims, I will cultivate valuable skills in genomic analysis, advanced microscopy techniques, eukaryotic cell culture, and comprehensive analysis of host-pathogen interactions.

NIH Research Projects · FY 2025 · 2023-08

Cocaine use disorder (CUD) imposes a large burden on public health, particularly because there are no FDA- approved pharmacotherapies for the disorder. The onset and maintenance of CUD is driven by physiological and molecular changes within the brain that lead to maladaptive behavior associated with cocaine taking and seeking. A key neuronal population in this dysregulation is dopamine 1 receptor expressing medium spiny neurons (D1 MSNs) in the nucleus accumbens (NAc). These cells are activated by acute cocaine, undergo physiological and transcriptional plasticity following repeated cocaine exposure, and are recruited by cocaine- associated cues to drive drug seeking. While their causal role in drug-induced behavior has been identified, the molecular mechanisms underlying cocaine-induced dysregulation remains poorly understood. The goal of this proposal is to define how chromatin regulation – through a recently identified cocaine-induced chromatin modifying enzyme [lysine acetyltransferase 2a (KAT2a)] – is a key substrate involved in the motivation to take and seek cocaine. We present in our preliminary data a detailed series of proteomic bioinformatic studies through which we identified KAT2a as an upstream regulator of the wide-scale transcriptional dysregulation associated with cocaine exposure in the NAc of both males and females. We also show that mutations to KAT2a that impair its function only in D1 MSNs greatly impair cocaine self-administration and disrupt physiological responses in D1 MSNs. I hypothesize that KAT2a acts within NAc D1 MSNs to control cocaine self-administration and cue-induced seeking via regulating D1 MSN activity at baseline and in response to drug-associated stimuli. To address this question, I will combine cocaine self-administration in mice with viral-mediated gene transfer and optical imaging in awake and behaving animals. In Aim 1, I will define the role that KAT2a in D1 MSNs plays in motivation to consume cocaine, cocaine reward sensitivity, and cue-induced seeking. In Aim 2, I will use optical imaging to define how KAT2a alters D1 MSN activity at baseline and in response to cocaine. In Aim 3, I will define the role of KAT2a in D1 MSN responses to cocaine-associated cues in awake and behaving animals and determine how these neural dynamics relate to drug-seeking behavior. The training goals in this proposal will provide the technical and conceptual expertise necessary to investigate the transcriptional and epigenetic mechanisms underlying substance use disorder. Finally, the experimental findings will define a cell type-specific neuroepigenetic mechanism of CUD in males and females.

NIH Research Projects · FY 2025 · 2023-08

Deep neural networks (DNNs) for object classification have been argued to provide the most promising state- of-the-art models of the visual system, accompanied by claims that they have attained or even surpassed human-level performance. However, mounting evidence has revealed that DNNs fail catastrophically when faced with more noisy or degraded viewing conditions. By contrast, the human visual system is far more robust. To better understand and model human vision, one must determine whether the brittle nature of DNN performance arises from flaws in their architectural design, imperfections in their learning protocols, or inadequate sampling of relevant training experiences. This project will investigate the neurocomputational bases of robust object recognition, focusing on challenge conditions of visual noise and blur, to develop new DNN models that can provide a better account of human behavioral and neural responses to object images that will vary from clear to severely degraded. Both feedforward and recurrent DNN architectures will be evaluated, and the critical sets of training experiences needed for DNNs to attain robustness will be determined. In Aim 1, we will evaluate what types of DNNs can adequately predict human behavioral and neural responses to objects embedded in noise on an image-by-image basis. Correspondences between fMRI responses at multiple levels of the human visual pathway will be compared with layer-wise DNN representations to evaluate the goodness of fit for DNN model predictions. In Aim 2, we will determine what types of DNNs can better account for human behavioral and neural responses to blurry object images. We will further explore how training with blurry images modifies the visual representations learned by DNNs, leading to greater robustness to other types of image degradation and greater sensitivity to shape information. In Aim 3, we will investigate whether perceptual training with noisy or blurry objects can allow humans to acquire even greater robustness. We will then determine whether human improvements in behavioral and neural performance can be effectively modeled by DNNs that undergo comparable regimens in visual training. As a whole, this project will lead to the development of powerful new DNN models that provide a better account of human behavioral and neural responses across a wide range of challenging viewing conditions. By developing a better neurocomputational model of the intact human visual system, we will be better positioned to eventually develop models of central visual disorders, which can arise from neurodevelopmental or neurological disorders, stroke, head injury, brain tumors or other diseases. The advancement of more robust, human-like DNNs is also highly relevant to AI applications in computer vision and medical image processing.

NIH Research Projects · FY 2024 · 2023-08

Project Summary and Abstract Alcohol Use Disorder (AUD) impacts upwards of 15 million Americans in the United States and has many consequences on overall health and quality of life for people affected by this disorder. Despite this, only around seven percent of people with AUD seek treatment and options for care remain limited, creating an urgent need for more research into the development and treatment of AUD. A major source of interest lies in what factors predispose certain individuals who consume alcohol over others to develop AUD, which can provide insight into specific preventative and treatment measures for this population. Deficits in cognitive domains of response inhibition and cognitive flexibility have been implicated as risk factors for later development of AUD and are predictors of more severe alcohol induced cognitive dysfunction. The medial prefrontal cortex (mPFC) is at the center of the brain circuitry that mediates aspects of cognitive function known to be impaired in AUD and studying the underlying neural pathways impacted by alcohol exposure has been a major area of study in the field. Human neuroimaging studies have identified mPFC projecting neurons to ventral tegmental area (VTA) as an important circuit in reward processing and initiation of goal directed behavior as well as alcohol craving in AUD models. The aim of this proposal is to utilize cutting edge techniques in single cell in vivo calcium imaging and behavioral tasks in mice to understand the contribution of this important but understudied pathway to preexisting and alcohol-induced deficits in cognitive function. Using complex operant positive reinforcement behavioral paradigms to test domains of cognitive flexibility and response inhibition in mice combined with single cell in vivo calcium imaging dynamics of mPFC→VTA projections as mice complete the task, I will study how this pathway responds to and changes as mice learn before and after alcohol exposure. I will first study whether there are individual differences in cognitive function in response inhibition and cognitive flexibility in mice at baseline with an operant behavioral task, after which I will characterize the activity pattern of mPFC→VTA projecting neurons using head mounted microendoscopes to capture real time neuronal activity of these projections in the mPFC in freely behaving mice as they learn the tasks. Further, using longitudinal tracking analysis, I will determine if there are individual differences in activity patterns between mice with variations in baseline cognitive function and how these changes evolve after binge drinking alcohol exposure in a two-bottle choice task. Completion of this proposal will provide valuable insight into the circuit level changes that underlie individual differences in vulnerability to alcohol induced cognitive deficits and will provide invaluable training for me as a future independent academic researcher.