Vanderbilt University

NIH Research Projects · FY 2025 · 2023-08

Project Summary Nanosized extracellular vesicles and particles (EVPs) have been identified as an important means for cells to communicate with neighboring and distant cells. EVPs are actively investigated to understand their roles in cancer, non-invasive disease diagnosis, and therapeutics. One of the most significant urgent challenges to overcome in EVP research is understanding the heterogeneity of EVPs. EVPs are heterogeneous in their size and molecular cargo contents. As a result, single EVP analysis has been identified as crucial to deciphering the heterogeneity of individual EVPs and understanding their biological roles in diverse diseases. As an example, an ongoing scientific question concerns whether the newly discovered extracellular particles called exomeres and supermeres are monolithic nanoparticles enriched with multiple makers such as proteins, RNA and lipids or if they are a distribution of different functionally-active nanoparticles (such as proteins, nucleic acid and lipids) co-isolated together. The widely used analysis techniques such as mass spectrometry are incapable of analyzing individual EVPs and hence these assays mask the impact of the heterogeneity of EVPs, which has made it impossible to address this question and other open questions to date. To select individual EVs for analysis in a non-destructive manner, it is imperative to develop methods for trapping them in solution. Optical tweezers recently recognized with a 2018 Nobel Prize in Physics have been demonstrated as effective approaches for trapping single cells and larger EVs. Unfortunately, the diffraction limit of light precludes their use for the trapping of single nanosized EVs, and the recently discovered exomeres, and supermeres that are only 35 nm and 25 nm in diameter, respectively. This MIRA research program is comprised of a collection of projects designed to develop new optical nanotweezer technologies for high throughput parallelized trapping of single nanosized EVPs combined with enhanced Raman analysis to provide unique information on the global biomolecular composition of individual nanosized EVs, exomeres and supermeres. Subsequently, we will investigate the use of these tools to address ongoing controversies in EV research. First, we will develop a novel optical nanotweezer approach based on nanoplasmonic structures that will enable: (i) parallelized trapping of thousands of single EVPs within seconds; (ii) enhancement and acquisition of Raman signals from single trapped EVPs nondestructively while they are trapped in solution near nanoplasmonic cavities; and (iii) biomolecular component analysis to determine the global biomolecular composition of individual trapped EVPs. Secondly, we will utilize the developed technologies to address ongoing questions in EVP research including whether the newly discovered exomeres and supermeres are monolithic or comprise a diverse distribution of functionally active nanoparticles. The pertinent findings to be obtained from the proposed research program will greatly improve our ability to understand the heterogeneity of EVPs, address ongoing controversies and guide the nature of future scientific questions to be investigated in the EVP research field.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ABSTRACT Substance use is a major public health concern that disproportionately affects individuals from lower socioeconomic status (SES) backgrounds. Although part of this association is attributable to downward socioeconomic mobility among individuals who develop problematic patterns of use, emerging evidence suggests that part may also reflect disadvantage-driven changes in brain development that increase risk of use and negative use-related outcomes. However, because much of the research supporting this hypothesized pathway is cross-sectional in nature, characterized by limited measurement of relevant constructs, and conducted in disproportionately White, middle-/upper-class samples, the utility of targeting this pathway with intervention/prevention efforts is unclear. The aims of the proposed career development award are thus twofold: (1) to train the candidate in the use of geospatial tools and related statistical techniques, neuroimaging approaches to characterizing distributed changes in brain structure and function, and theory and principles from health disparities research, and (2) to use this training to test for associations between childhood socioeconomic status, brain structure and connectivity, and substance use trajectories that generalize across racial/ethnic groups using three population-representative, longitudinal cohort studies. The candidate will receive training essential for his development as an independent research scientist under the guidance of an outstanding team of mentors and consultants with extensive experience studying socioeconomic status, brain development, self-regulation, health disparities, and substance use (Drs. Sylia Wilson, Monica Luciana, Damien Fair, Shervin Assari, Martha Farah, and Daniel Berry). The research will be conducted at the Institute of Child Development (ICD) and Minnesota Center for Twin and Family Research (MCTFR), which together offer unparalleled resources to support work identifying the neural and behavioral mediators connecting children’s early-life environments and later substance use. Altogether, the candidate will address four specific aims: (1) test whether children from low SES backgrounds are more likely to develop problematic substance use trajectories and establish whether these associations generalize across different types of substance use; (2) test whether low childhood SES is associated with individual differences in brain structure and resting-state functional connectivity across distributed networks associated with self-regulatory abilities (i.e., cognitive control, reward sensitivity, negative emotionality); (3) test whether these differences in distributed neural networks mediate associations between low childhood SES and substance use trajectories; and (4) test whether the strength of these associations differ across racial/ethnic groups in accordance with the notion of marginalization-related diminished returns. Results of the proposed project have great potential to prompt reconceptualization of brain-based models of substance use and addiction, and, in turn, guide prevention and intervention efforts to reduce an important behavioral health disparity.

NIH Research Projects · FY 2026 · 2023-07

ABSTRACT Alcohol Use disorder (AUD) is a disorder in which alcohol alters a wide range of neural circuits to cause maladaptive behaviors across several behavioral domains. Despite the prevalence and cost of this disorder, treatment strategies are ineffective, especially in preventing relapse. A key feature in some individuals is the induction of negative affective states when alcohol consumption is ceased. In these individuals, alcohol taking and seeking is hypothesized to be motivated by negative reinforcement, where individuals continue consuming alcohol to avoid negative internal states that are triggered by abstinence. While past research has focused on how abstinence produces negative affective states, the question remains as to how affective disturbances motivate behavior (i.e. negative reinforcement). To this end, the goal of this proposal to understand how circuits that control the motivation to avoid aversive stimuli are engaged by alcohol and associated cues to drive alcohol seeking. At the center of reinforcement is the nucleus accumbens (NAc). The NAc is a heterogeneous region primarily composed of two non-overlapping cell types: D1 and D2 medium spiny projection neurons (MSNs) which play complementary roles in controlling motivated behaviors4. While previous work has implicated D1 MSNs in positive reinforcement, our data show that D2 MSNs respond to cues that signal negative reinforcement and causally control the motivation to avoid aversive stimuli. We hypothesize that D2 MSNs are engaged by alcohol-associated cues following withdrawal from chronic intermittent ethanol exposure (CIE; achieved via vapor inhalation), and drive alcohol seeking. To address these questions, will use a variety of cutting-edge optical approaches to record from and manipulate these cells to define the temporal patterns by which they respond to alcohol associated cues and link this to alcohol seeking following CIE exposure. We will determine how the development of D2 responses to alcohol-associated cues is predicted by the negative affective states that develop over withdrawal from CIE. Finally, using patch clamp electrophysiology with channelrhodopsin assisted circuit mapping we will 1) define how CIE changes glutamatergic drive onto D2 MSNs that are specifically activated by negative reinforcement (from the prefrontal cortex, thalamus, and basolateral amygdala) and 2) use drugs acutely restricted by tethering (DARTS) in vivo to prevent glutamatergic drive selectively through AMPA receptors on NAc cells activated by negative reinforcement and prevent alcohol seeking. Together, we will define how this critical cell population that controls negative reinforcement drives operant alcohol seeking. This understanding will be critical to our conceptualization of AUD and why individuals drink following withdrawal.

NIH Research Projects · FY 2025 · 2023-07

PROGRAM ABSTRACT This proposal requests support for a Vanderbilt Chemistry-Biology Interface (V-CBI) Training Program at Vanderbilt University. Forty faculty preceptors from over eight Ph.D. granting departments and programs in the Colleges of Arts and Sciences, Engineering, School of Medicine Basic Sciences, and Vanderbilt University Medical Center will serve as mentors for the Program. Significant training and education in biology will be provided to students receiving in-depth training in synthetic/mechanistic and analytical chemistry and significant training and education in the chemistry will be provided to students receiving in-depth training in the biological sciences. Highlights of the training program include a common chemical biology 15 week course, elective courses for cross education, a course in experimental design and research reproducibility, student organized research and professional development seminars, a seminar series in chemical biology, an annual research symposium, participation in an annual career development conference and a unique experience in laboratory experimental design and technical training for application in an undergraduate research laboratory. Students will receive in-depth education and training in a core discipline and cross training in complementary fields to allow them to work effectively in multidisciplinary research environment. Support for ten trainees is requested. Trainees will obtain Ph.D. degrees working with preceptors in such the Departments of Biochemistry (11 preceptors), Chemistry (13), Cell and Developmental Biology (2), Pathology and Microbiology (4), Pharmacology (3) and Molecular Physiology & Biophysics, Anesthesiology and Pharmacology (3), Chemical and Biomolecular Engineering (2), Pediatrics (2), Radiology and Radiological Science (1-2 each), among others.

NIH Research Projects · FY 2025 · 2023-07

This training grant is designed to address three critical threats to the field of Communication Sciences and Disorders (CSD): (1) a national shortage of PhD level researchers; (2) increased need for translational, interdisciplinary, and cross-methodological training; and (3) gaps in the implementation of scientific advances to educational, healthcare, and community settings. This proposal requests support for four predoctoral students to participant in a translational and interdisciplinary training program that draws on faculty from the departments of Communication Sciences and Disorders, Psychology and Human Development, Otolaryngology, Biostatistics, and Medicine and leverages the resources and training opportunities available at Vanderbilt (e.g., Vanderbilt Institute for Clinical and Translational Research, The Implementation Science Core). Vanderbilt is uniquely positioned for the proposed research as it is a recognized leader the fields of CSD, and translational and implementation research. The purpose of this training grant is to prepare students for independent scholarship in translational and interdisciplinary research. Trainees take formal coursework across different disciplines, including instruction in quantitative methods, gain hands on experience with multiple methodologies, complete a collaborative-interdisciplinary research rotation (spanning two or more approaches), in addition to working in the lab of their primary advisor. Trainees have access to a number of research programs including in auditory neuroscience, pediatric audiology, cochlear implants, language development and disorders, language neuroscience and rehabilitation, psycholinguistics, developmental disorders and disability, cognitive-communication disorders, genetics, reading and reading disorders, acquired brain injury, hearing and hearing aids, motor speech, voice, and fluency. Experimental methodologies include animal models (e.g., monkey, mice, rabbit), neuroimaging (e.g., MRI/fMRI, DTI, fNIRS, EEG/ERP), physiological measurement (e.g., heart rate, respiration, skin conductance), computational modeling, eye-tracking, neuropsychology, lesion method, various behavioral paradigms, electromagnetic articulography, and advanced statistical approaches. Students participate in expanded training in responsible conduct of research, replication and reproducibility, scientific communication and grant writing, journal clubs, and research colloquia. Key outcomes include submission of peer-reviewed manuscripts, application for external research funding (e.g., NIH NRSA F31), participation in national or international meetings, and eventually, employment at a research university. The overarching goal of the training program is to prepare a number of highly trained and qualified researchers who are well-equipped to address the challenges facing the field of CSD.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Diabetic retinopathy (DR), a microvascular complication of diabetes, is the leading cause of irreversible vision loss in working-age Americans. As the number of Americans with diabetes continues to climb, the prevalence of DR is expected to rise in coordination. Current therapies for DR treat only late stages of disease after irreparable damage to the retina has occurred, highlighting the need for therapeutic interventions to prevent early-stage progression. Since the 1960s, it has been hypothesized that retinal inflammation may drive early DR progression in a cyclooxygenase (COX)-dependent manner. However, trials of various nonsteroidal anti- inflammatory drugs (NSAIDs) to inhibit COX as a DR treatment have failed in large part due to severe cardiovascular or gastrointestinal side effects associated with chronic, broad-spectrum COX inhibition by these drugs. Alternatively, targeting specific prostanoids—the lipid signaling molecules downstream of COX—and/or their receptors could offer a therapeutic approach that isolates anti-inflammatory benefits while avoiding the severe side effects of NSAIDs. Five prostanoids are generated in the COX pathway, signaling through nine prostanoid receptors. The goal of the research proposed here is to determine the therapeutic potential of inhibiting individual prostanoid signaling to slow DR onset and progression. My preliminary studies have identified that two of the five prostanoids—PGE2 and PGF2α—are elevated in primary human retinal cells cultured in conditions of dyslipidemia or inflammation relevant to diabetes. PGE2 is elevated in Müller glia, cells responsible for maintaining homeostasis in the retina, and PGF2α is elevated in retinal microvascular endothelial cells, which form retinal blood vessels. I hypothesize that these two prostanoids are critical drivers of proinflammatory cytokine production and leukostasis, hallmark pathologies associated with DR. This proposal expands upon these findings to define the landscape of retinal prostanoid elevation under conditions relevant to systemic diabetes and to determine the preclinical efficacy of small molecule prostanoid receptor antagonists as targeted therapeutic strategies against DR progression. I propose utilizing primary human cultures of Müller glia and retinal microvascular endothelial cells as well as a diabetic mouse model to interrogate antagonism of prostanoid signaling in both cell- and animal-based disease-relevant experimental models. In completing these studies, I aim to characterize a novel therapeutic strategy to precisely target molecular signaling pathways that may drive retinal vascular inflammation in early-stage DR before irreversible damage occurs. I will carry out my work in the supportive mentoring environment of Dr. John Penn’s laboratory at Vanderbilt University, an institute with rich support of both prostanoid and vision research and with a long history of exemplary graduate training. The training plan outlined in this proposal, paired with my research goals, will aid invaluably in my training to become an independent academic research scientist.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Anhedonia, a core symptom of depression and other forms of psychopathology characterized by loss of interest or pleasure, is associated with a range of negative health outcomes, including suicide risk, and poor treatment response. Low activation of positive valence systems, particularly low reward responsiveness (RR), is a key brain-behavioral process underlying anhedonia. Critically, there is growing evidence that low RR is observable in children at risk for anhedonia due to maternal symptomatology and reflects a vulnerability for the later emergence of anhedonia and depression. Thus, targeting RR in high-risk children is critically needed to prevent the development of anhedonia, alter risk trajectories, and mitigate the tremendous health burden of anhedonia-related psychopathology. Combining principles from adult positive affect treatment and family cognitive behavioral preventive interventions, we developed an innovative, neuroscience-informed dyadic preventive intervention, Family Promoting Positive Emotions (F-PPE), for 8- to 12-year-old children and mothers with a history of major depressive disorder with anhedonia. F-PPE is designed to specifically target RR in children, assessed at the neural level using well-validated electroencephalogram methods. The first phase of the project (R61) will focus on target engagement, testing whether F-PPE increases child neural RR relative to an active comparison. Children (N=60) will complete neural measures of RR pre-intervention, 4- weeks into the intervention to determine dose effects, and post-intervention (8 weeks). Biological mother-child dyads will be randomized to F-PPE or a psychoeducation preventive intervention comparison. Target engagement will be defined as an increase in neural RR in the F-PPE relative to psychoeducation group with at least a medium effect size (d > .40). Change in the target at 4 weeks will be examined to determine dose effects and integrated with participant feedback to refine F-PPE for the R33 phase. The R33 phase will be a replication and extension to clinical outcomes with 100 biological mother-child dyads. Dyads will again be randomized to F-PPE or a comparable number of psychoeducation sessions. In addition to neural RR, ecological momentary assessment of real-world experiences of interest and pleasure and clinical symptoms of anhedonia will be assessed pre- and post-intervention and at a 6-month follow-up assessment. We will examine effects of F-PPE on momentary experiences of interest/pleasure and symptoms of anhedonia across the longitudinal follow-up and test change in RR as a mechanism of clinical effects of F-PPE. These projects will take critical next steps in translating developmental affective neuroscience research to prevention and moving towards precision medicine to reduce the burden of anhedonia and associated psychopathologies.

NIH Research Projects · FY 2024 · 2023-07

Project Summary Despite their high prevalence, the underlying neurobiology of substance use disorders is still not understood, limiting effective pharmacological treatment options. Characterized by disruptions in learning processes, substance use disorders are thought to develop with the transition from being motivated by the positive reinforcing effects of the substance, to negative reinforcement where use occurs to avoid or alleviate an aversive state during abstinence. It is hypothesized that substance-induced upregulations in kappa opioid receptor (KOR) signaling play a causal role in the dysphoria that drives negative reinforcement. In support of this idea, KOR antagonists decrease drug intake in models of chronic use or dependence and the nucleus accumbens, a region critical to reinforcement learning and motivated behaviors, is one of the regions in which substance use has been shown to upregulate the KOR system. Despite this evidence of a causal role for KOR signaling in substance use disorders, this system’s function in adaptive reinforcement processes and the sufficiency of its upregulation in driving maladaptive learning remains unknown. Here, we aim to address these issues through direct investigation of the role of the KOR system in positive and negative reinforcement learning through pharmacological manipulation, sub-second measurement of in vivo dynorphin dynamics, and optogenetic activation of the KOR system. In aim 1, I will pair systemic antagonism of the KOR system with positive and negative reinforcement operant conditioning tasks to elucidate the role of KOR signaling in adaptive learning. In aim 2, I will conduct sub-second resolution, in vivo optical imaging of dynorphin release in the nucleus accumbens to uncover the role of endogenous KOR system activity in encoding information during reinforcement learning processes. Finally, in aim 3, I will optogenetically evoke accumbal dynorphin release to determine the sufficiency of augmentation of the KOR system in driving the maladaptive learning that underlies addiction. Through the completion of this proposal, I will receive well-rounded training in conducting independent research and contribute to the field of addiction neuroscience’s understanding of the mechanism by which the KOR system is involved in adaptive and maladaptive reinforcement learning processes.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Cells must synthesize nucleic acids to create genetic information that is used for protein synthesis, an essential process for all life. Nucleic acids are composed of nucleotides containing a nitrogenous base (nucleobase) that dictates base-pairing in the macromolecule and defines the genetic code. Nucleobases must be either synthesized or salvaged from the environment for nucleic acid synthesis, and cellular energy demands often dictate which of these processes is used. Bacterial pathogens must synthesize or salvage nucleic acids for optimal growth and survival during infection, often through pathways that differ from the host, making nucleobase metabolism an attractive target for therapeutic approaches. The vertebrate gastrointestinal tract is colonized by a cooperative group of microorganisms that prevent colonization by invading pathogens by depleting the gut environment of essential nutrients for colonization. The enteric pathogen Clostridioides difficile infects the host gastrointestinal tract upon perturbation of the gut microbiota and is the leading cause of antibiotic-associated infections. Antibiotic perturbation of the gut microbiota alters the nutrient milieu in the gut environment, and C. difficile must compete with the host and microbiota to obtain critical nutrients to colonize and cause disease. Amongst the nutrients altered in the gut following antibiotic treatment are nucleobases, and we hypothesize that C. difficile salvages nucleobases from the antibiotic perturbed gut. Our preliminary data indicate that C. difficile possesses a unique metabolic pathway to salvage a thio-modified uracil nucleobase, 4-thiouracil (4-TU), that is present in the vertebrate gastrointestinal tract. C. difficile can metabolize 4-TU as a uracil source for growth instead of the energetically demanding pyrimidine biosynthetic pathway. Recently, an enzyme capable of metabolizing 4-TU has been described from an Aeromonas species, representing a member of a large family of enzymes containing a DUF523 domain. However, the mechanism by which C. difficile metabolizes 4-TU has not been described. We have identified that two paralogous proteins (CD196_RS03875 and CD196_RS15345) contain a DUF523 domain in C. difficile. Furthermore, our work has uncovered that 4-TU is growth inhibitory to Escherichia coli, which lacks a DUF523 homolog. We discovered that CD196_RS03875 which we named TudS (thiouracil desulfurase), is required for 4-TU metabolism and protects C. difficile from 4-TU mediated toxicity. We hypothesize that 4-TU metabolism enables C. difficile to thrive in the competitive gut environment, and experiments in this proposal will test this hypothesis. In Specific Aim 1, we will define the molecular mechanism by which TudS converts 4-TU to uracil in C. difficile and identify other C. difficile gene products important for 4- TU metabolism through an innovative transposon screen. In Specific Aim 2, we will determine the contribution of 4-TU metabolism to C. difficile pathogenesis using an animal model of infection with mutants defective in 4- TU metabolism. These studies have the potential to define a pathway for salvage and detoxification of an understudied, unconventional nucleobase that may contribute to the pathogenesis of an important gut pathogen.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Indeterminate pulmonary nodules (IPNs) are highly prevalent radiologic findings that represent a substantial burden to patients and the national health care system because of the diagnostic challenge they present. There is a dire need to accurately stratify IPNs into low and high malignancy risk subgroups which are associated with clinical management pathways that are standardized and well validated. Clinical prediction models have the potential to do so in a scalable, cost-efficient, automated, and noninvasive manner, but advances in predictive accuracy must be made before they can make a substantial impact in medical practice. An unexplored direction in this area is integrating repeated measures of computed tomography (CT) studies and clinically-collected information within the same prediction model. This joint learning strategy has advantage of potentially modeling how dynamic radiologic changes like nodule growth rate vary with the trajectory of clinical variables such as smoking patterns and laboratory abnormalities. This perspective motivates the hypothesis that integrating information from longitudinal imaging and longitudinal clinical records will improve personalized IPN risk stratification and lung cancer subclassification From a clinician’s lens, this finding would not be surprising given the many time-varying modalities that are involved in diagnosis and decision making. This project leverages artificial intelligence (AI) and radiomic methods to analyze three retrospective cohorts with the possible addition of a large prospective cohort. The proposed work in Aim 1 will extend upon existing deep learning techniques to train a joint learning model on longitudinal images and clinical records to estimate the malignancy probability across time in patients with IPNs in a combined cohort exceeding 2000 subjects. This novel strategy will be evaluated against single-modality models and convention models that are used in practice. The evaluation will compare the models’ performance in stratifying IPNs into the low and high risk subgroups as a measure of clinical utility. Aim 2 asks if longitudinal change in radiomic features can distinguish between indolent and aggressive lung adenocarcinoma, other lung cancer subtypes, and pulmonary metastases. The proposed study will be the first to comprehensively characterize longitudinal radiomics across lung cancer subtypes and has the potential to identify novel longitudinal radiomic features that will aid early IPN evaluation and noninvasive lung cancer subclassification in patients with repeated imaging. In summary, the proposed research asks if clever integration of longitudinal information across different modalities can be leveraged to advance IPN risk stratification and lung cancer subclassification. This fellowship will be conducted at Vanderbilt University in a highly collaborative training environment with mentors in medical imaging AI, pulmonary oncology, biomedical informatics, radiology, and biostatistics. The proposed research and training plans are synergistically designed to ultimately prepare the candidate for a physician scientist career at the intersection of engineering innovation and precision oncology.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY/ABSTRACT Epilepsy is a debilitating neurological disorder that affects 1% of the population worldwide and temporal lobe epilepsy (TLE) is the most common form. Seizure onset in TLE is typically localized to the mesial temporal lobe, however, patients can suffer from distant effects such as loss of consciousness during seizures (ictally) and neurocognitive deficits between seizures (interictally), both of which impair one’s activities of daily living, ability to work, and quality of life. Prior work investigating these global effects have resulted in the Network Inhibition Hypothesis, which states that focal seizure activity observed with stereotactic electroencephalography (SEEG) spreads to subcortical structures responsible for neocortical activation, resulting in ictal loss of consciousness in focal impaired awareness seizures (FIAS). Studies with functional MRI (fMRI) have provided evidence that the same anatomical areas have abnormal functional connectivity (FC). It thought that recurrent FIAS lead to chronic interictal decreases in subcortical to neocortical FC, but this knowledge gap but this knowledge gap has not been addressed. We aim to relate FC of ictal and interictal states using SEEG (Aim 1). I specifically hypothesize during FIAS ictal FC will decrease in the same anatomical regions as those implicated in interictal FC abnormalities. In addition to recurrent seizures, patients can also have devastating interictal neurocognitive deficits. These widespread neurocognitive deficits suggest that there is a common factor, which is thought to be the subcortical arousal structures. It has been shown that fMRI FC abnormalities of subcortical to neocortical structures are associated with neurocognitive deficits, seizure frequency, and can recover after surgery. While general subcortical to neocortical abnormalities have been outlined, there is a gap in understanding of specific brain networks associated with neurocognitive deficits. This could be due in part to not adequately controlling for arousal state. The high vigilance or “sustained attention” state, is a state of cognitive engagement mediated by subcortical arousal structures which fluctuates at rest. It is associated with subcortical to neocortical FC changes, associated with extratemporal neurocognitive deficits, the state active during neurocognitive testing, and thought to be a confounder for resting-state fMRI by some. We aim to link specific subcortical to neocortical network abnormalities with neurocognitive deficits by controlling for the high vigilance state with fMRI-EEG (Aim 2). I specifically hypothesize that during high vigilance states, patients will have significantly decreased FC within subcortical to neocortical networks compared to controls, that the decrease will be associated with extratemporal neurocognitive deficits, and that the network will recover after successful surgery. This proposed fellowship will provide research training in a collaborative research atmosphere with expert mentors. Research training will be conducted in an environment that combines an academic medical center with a level 4 epilepsy center, world class imaging institute, and engineering all on one campus. Studying multiple modalities to characterize epileptic networks may lead to improved neuromodulation targets for TLE.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY SF3B1 is the most commonly mutated splicing factor in cancer, occurring in thousands of cancer patients annually. Mutations in SF3B1 result in a neomorphic protein that causes aberrant splicing of hundreds of transcripts, including known cancer associated genes. While the mechanisms by which these alterations promote tumorigenesis are incompletely understood, our lab has previously shown SF3B1 mutations are attractive therapeutic targets. SF3B1 mutations are prevalent in many cancers (breast, melanoma, bladder, pancreatic, leukemias), so improving our ability to target these mutations could have major public health implications. To do this, there is a fundamental need to better understand how SF3B1 mutations drive tumorigenesis. Recent work in acute myeloid leukemia shows differences in missplicing, oncogenic effects and prognosis among various SF3B1 hotspot mutations, yet there are no studies to date investigating these in solid malignancies. To determine potential therapeutic strategies, novel model systems are required. An innovative genome editing approach will allow us to study the mutations at the most common hotspots from breast cancer and melanoma, K700 and R625, respectively in several representative cell line models. Changes in the transcriptome and phenotypic differences in proliferation, migration, and invasion will determine whether there are specific alterations in SF3B1 that lead to distinct oncogenic phenotypes. Additionally, preliminary systematic analysis of online cancer databases shows SF3B1 mutations and TP53 alterations are mutually exclusive in cancer. This often suggests either synthetic lethality or a lack of selection for co-occurrence due to shared roles in tumorigenesis. Successful generation of dual SF3B1 mutant and TP53 mutant or TP53 knock out cell lines demonstrates that the mutations are unlikely to be synthetic lethal. Instead, this relationship likely demonstrates a shared role and will allow us to determine novel mechanisms of SF3B1-mediated tumorigenesis. Previous findings in SF3B1 mutants demonstrate dysfunctional cellular respiration due to missplicing and degradation of a UQCC1, a component of mitochondrial complex III. There is a resultant increase in glucose, similar to p53’s well known role in promoting the Warburg effect. Further studying the relationship between mutant SF3B1 and TP53 may identify therapeutic vulnerabilities that can be additionally leveraged against the large subset of cancers with TP53 mutations. The sponsor’s robust history of utilizing genome editing strategies to study individual mutations in breast cancer in conjunction with the abundant resources and core facilities at Vanderbilt University make these Aims achievable. Completion of these aims provide an excellent foundation in cancer molecular genetics. This will allow the PI to acquire the technical skills to build toward an independent investigational career in oncology, specifically studying novel pathologic features of cancers that lead to uniquely targetable vulnerabilities.

NIH Research Projects · FY 2025 · 2023-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The long-term goal of this training program is to develop members of a workforce with rigorous training in an area of biomedical science and to foster a deep understanding of the principles of pharmacology to create scientists who will be well positioned to participate in the process of moving basic science discoveries to much needed therapies. This training will be accomplished in an interdisciplinary environment that brings together trainees from multiple institutions who represent a broad range of scientific interests and a shared desire to understand the principles that underlie the development of safe and effective therapies. A cohort of 3 trainees will be appointed for the second and third year of their graduate training, a stage in their training where pharmacology-specific training is best delivered and solidified. We aim to train scientists equipped with the principles of the discipline of pharmacology who are uniquely prepared to identify and solve research questions in the biomedical sciences. Mastery of the principles of pharmacology will allow trainees to serve as leaders in transitioning discoveries into therapies. To this end we have developed a predoctoral training program that will: provide each trainee with the foundational tenants of the discipline of pharmacology such as receptor theory, drug metabolism, and pharmacokinetics; develop the professional skills necessary for success; support individual research mentoring that provides directed research in an area of biomedical science of interest to the trainee alongside discipline-specific training; and evaluate the attainment of individual trainee and program goals. Timely and actionable feedback on the progress of trainees is provided by a competency-based assessment plan developed in collaboration with a select group of like-minded predoctoral training programs. All facets of the proposed program are supported by a comprehensive evaluation plan designed to both assure the development of individual trainees and provide feedback to improve the overall training program. These experiences are supplemented to build competence in research ethics, scientific rigor, presentation of scientific data, and assessment of data. We convey to trainees the expectation and the tools to pursue life-long learning as an essential component for success. Professionalism will be intentionally addressed to impart skills in areas such as communication, teamwork, mentoring, and self-assessment in order to aid in the development of resilience, integrity, and leadership. These program aims are supported by longstanding strengths that include the history of collaboration amongst the partner institutions, the past success of trainees, and a rigorous research environment that supports pharmacological investigation. Our goal is to produce trainees who will become leaders in careers found in academic and industrial research, government and regulatory affairs, and education.

NIH Research Projects · FY 2026 · 2023-06

Approximately 33-50% of intensive care unit (ICU) survivors develop long-term cognitive impairment – a well-established form of Alzheimer’s Disease Related Dementia (ADRD). ICU-acquired ADRD prevalence is especially high in acute respiratory distress syndrome and sepsis survivors, affecting up to 80%. This loss of cognition leads to loss of independence, employment, and quality of life and persists for months to years. ICU-acquired ADRD is part of a broader syndrome known as Post-Intensive Care Syndrome (PICS), including physical, mental, and socioeconomic impairments. ICU Recovery Clinics (ICU-RC) are a feasible and promising intervention to collaboratively address ICU-acquired ADRD and other PICS impairments, but in-person access is limited. There is a need to study efficacy of ICU-RCs with large cohorts using alternative delivery strategies to expand availability and reach. Older (age >=45) septic shock and/or acute respiratory failure patients are at a combined risk for ICU-acquired ADRD among other impairments, and the ideal population to first address this knowledge gap. The Vanderbilt ICU Recovery Center team has nearly 10 years of experience providing interdisciplinary, collaborative care to identify and treat ICU-acquired ADRD and other PICS impairments experienced by ICU survivors. We hypothesize that a collaborative telemedicine-delivered interdisciplinary ICU-RC intervention effectively identifies and improves long-term cognitive function, and as secondary outcomes, physical and mental health function, social integration, and self-management behaviors vs. a control condition with follow-up chosen by the discharge team. Therefore, in a sample of older septic shock and acute respiratory failure survivors, we aim to examine the efficacy of telemedicine ICU-RC services vs. control follow-up chosen by the discharge team in identifying and managing ICU-acquired ADRD and other PICS impairments (i.e., physical, mental health, social, self-management function) at 6 months after hospital discharge. We will address our hypothesis and aims by conducting a controlled trial of 202 patients randomized 1:1 with age stratification to telemedicine ICU-RC or control (101 per group). Telemedicine recipients will receive a minimum of 2 ICU-RC visits within 3 months of hospital discharge or return to home if discharged to another institution, with additional follow-up determined by the severity of PICS impairment. Our primary outcome is cognitive function (Aim 1) at 6 months using the MoCA-Blind and PROMIS Cognitive Function from the Long-term Core Outcome Measurement Set for ICU survivors. Our secondary outcomes are physical and mental health functioning (Aim 2) and social integration and self-management behaviors (Aim 3). In addition to 6-month measurements, we will assess pre-hospital function and 1-week post-discharge to assess discharge functional trajectories. This research will provide scientific justification for the continued development, implementation, and scaling of ICU recovery programs. Ultimately, such knowledge can improve the quality of life for millions of ICU survivors and family members by reducing ADRD burden.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Mating, host-seeking and blood-meal-acquisition behaviors in disease-carrying mosquitoes are facilitated by an array of chemosensory and feeding appendages that are critical for transmission cycles. In order to find a mating partner or distinguish host odor cues (kairomones) from numerous other environmental stimuli, Anopheles coluzzii mosquitoes use a keen sense of smell/taste that relies on at least three large families of chemosensory receptors: these are the gustatory (AcGrs), odorant (AcOrs) and variant ionotropic (AcIrs) receptors that are expressed in peripheral chemosensory tissues and convey information about the chemical environment to the brain. In contrast to the highly divergent and relatively well-studied AcOr family, the AcIr family of chemoreceptors is largely conserved and relatively poorly understood. Preliminary studies from our group utilizing state-of-the-art transgenic reporters and gene-targeting approaches have begun to explore the expression and function of AcIr76b, one of the triad of obligate IR co-receptors. These studies confirm broad expression profiles and, more importantly, have begun to elucidate the essential role that IR signaling plays in mediating responses to many odor cues that underlie important mosquito behaviors, such as mating and host seeking/blood feeding. This has led us to hypothesize that AcIrs play a critical role in neuronal sensitivity to as-yet undefined mating and blood-feeding cues as well as several well-established human skin odorants in sensilla that reside on the adult antennae, maxillary palps, labella, and tarsi. For example, grooved- peg sensilla are responsive to important human-derived kairomones—such as ammonia, lactic acid and butylamine—yet the neurons housed within them express AcIr76b and other AcIrs but not AcOrs. In this proposal, we seek to comprehensively elucidate the molecular functionality of AcIrs and their potential roles as host kairomone, mating and blood-feeding receptors and thus clarify key components of the chemosensory processes in the malaria vector mosquito An. coluzzii. Broadening our understanding of the host-seeking, blood- feeding, and mating biology of An. coluzzii and indeed other disease-transmitting mosquitoes in which Irs are extremely well conserved may have important future implications for human health by providing new ways to interfere with disease transmission.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY/ABSTRACT RyR2 is an intracellular calcium (Ca) release channel expressed in the sarcoplasmic reticulum (SR) of cardiomyocytes. In the normal heart, RyR2 Ca release from the SR is tightly regulated and only occurs during systole to facilitate heart contraction. In heart disease, RyR2 Ca release can occur during diastole and is considered pathologic. Pathologic Ca release can be caused by RyR2 mutations or RyR2 post-translational modifications. Pathologic Ca release during diastole reduces cardiac contractility due to depletion of SR Ca stores and is pro-arrhythmogenic due to delayed after-depolarizations resulting from sodium (Na) flux into the cell via the Na-Ca exchanger. RyR2 mutations cause catecholaminergic polymorphic ventricular tachycardia (CPVT), a genetic arrhythmia syndrome, while post-translational modifications have been widely documented in congestive heart failure (CHF) caused by myocardial infarction. Both conditions are associated with a high risk of sudden cardiac death (SCD). My mentor discovered that an old antiarrhythmic drug – flecainide – prevents pathologic rather than physiologic Ca release and is strikingly effective in preventing ventricular arrhythmias in CPVT patients. Importantly, he recently discovered that flecainide’s efficacy depends not on Na channel block but rather RyR2 block. Unfortunately, due to its Na channel blocking properties, flecainide increases mortality in patients with CHF and cannot be used in this patient population. To address these patients’ risk for SCD – currently unmitigated by available drugs – I aim to develop a flecainide analogue that maintains RyR2 block but not Na channel block. In doing so, I will investigate the mechanism of action of flecainide and test the hypothesis that its efficacy depends on a change in the membrane potential across the SR. My research background and the established use of patch clamp electrophysiology and calcium imaging in my mentor’s lab will enable me to test flecainide analogues generated by our collaborators in synthetic chemistry. To probe the mechanism of action underlying flecainide’s voltage-dependent RyR2 block, I will employ a variety of tools including genetically encoded voltage indicators and voltage-sensitive dyes to capture the theoretical membrane potential change that occurs at the SR. The results from this aim will not only clarify flecainide’s mechanism of action but also yield a novel therapeutic principle – that voltage-dependent block of RyR2 channels is a key feature for the development of future RyR2 inhibitors as antiarrhythmic drugs.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY/ABSTRACT Alcohol use disorder (AUD) is characterized by problematic alcohol consumption that evolves over an individual’s drinking history. While initial alcohol use is thought to be driven by its positive reinforcing effects, following long-term drinking, negative affective states emerge during withdrawal. These states are associated with increases in alcohol seeking, tolerance, and levels of consumption that follow repeated bouts of abstinence. This has led to the hypothesis that negative reinforcement becomes the primary motivational drive – where individuals consume alcohol to alleviate these negative states. However, the circuit-based mechanisms that control this switch to negative reinforcement are unclear. The goal of this proposal is to outline how alcohol-associated cues recruit circuits that control negative reinforcement before and after chronic alcohol exposure. Our preliminary data show that D2 medium spiny neurons (MSNs) are a critical a negative reinforcement circuit and control the motivational drive to avoid aversive stimuli in an environment. This proposal will test the hypothesis, that alcohol-associated cues recruit D2 MSNs activation in vivo and drive drinking after chronic exposure to alcohol. By combining optical approaches to record from and bidirectionally modulate D2 MSNs in the NAc during alcohol drinking I will define exactly when and how their activity is recruited over a history of drinking and how it controls drinking behavior. In Aim 1& 2, I will optically inhibit or activate D2 MSN activity at the time of an alcohol-predictive cue to determine how this controls operant drinking before and after chronic intermittent ethanol (CIE) exposure. In Aim 3, I will determine if the dynamics of D2 MSNs during operant alcohol drinking are predictive of specific drinking patterns by combining fiber photometry with operant behavior and doing deep-phenotyping analysis. I hypothesize that D2 MSNs – which function as a negative reinforcement signal – are recruited by alcohol associated cues only after a history of CIE exposure, when alcohol use is driven by negative reinforcement. Taken together, the experiments in this proposal will determine the negative reinforcement circuits that are recruited in the later phases of alcohol drinking and whether the dynamics of these neuronal circuits could be predictive of alcohol drinking phenotypes. This proposal encompasses technical and theoretical training that will provide the foundational expertise and conceptual thinking needed to address larger questions regarding how long-term exposure of alcohol changes the brain and drives continued alcohol use. Additionally, these findings can ultimately inform our understanding of underlying reward and learning process and lead to more efficacious treatment interventions for AUD.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Many aggressive cancers have a robust tumor microenvironment composed of heterogenous stromal and immune cells. Although the advent of immune checkpoint inhibitors has shifted therapeutic targets of cancer research to include tumor-cell extrinsic targets, the therapeutic potential of targeting the tumor stroma remains underexploited. Recently, cancer-associated fibroblasts (CAFs) have been implicated as drivers of disease progression. From preliminary attempts to harness the therapeutic potential of targeting CAFs, it has become clear that targeting CAFs as a bulk population of cells will not be sufficient. As such, there have been efforts in breast and pancreatic cancer to define the heterogeneity of CAFs. These efforts have yielded diverse subtypes commonly described by two overarching groups: myofibroblast CAFs (myCAFs) and inflammatory CAFs (iCAFs). In pancreatic cancer, myCAFs are observed to be tumor-adjacent and iCAFs are more distant from tumor cells. While defining subtypes of CAFs is a necessary first step, the development of novel therapeutic approaches will likely require the identification of specific functions of CAF subtypes. To this end, Wnt2 has been identified as upregulated in CAFs from pancreatic, breast, and colorectal cancer, yet the role of CAF-driven Wnt signaling on tumor progression remains largely unknown. Anaplastic thyroid carcinoma (ATC) is a lethal disease (~3-5 month median survival) with an abundant tumor stroma and no efficacious treatment options. The composition of the tumor stroma in ATC has been largely unexplored. My preliminary work identifies a prominent fibroblast population in ATC that expresses WNT2. As ATC is known to have upregulated Wnt signaling relative to other thyroid neoplasms, this provides a unique opportunity to study the dynamics of CAF-driven Wnt signaling. The goal of this proposal is to define the CAF subtypes present in thyroid carcinoma and determine the functional role of CAF-derived Wnt2 on tumor growth. I hypothesize that distinct CAF populations promote tumor growth and invasion in thyroid carcinoma through Wnt signaling and have unique spatial relationships. To test my hypothesis, I will perform experiments in ATC models and papillary thyroid carcinoma (PTC) models. PTC is a predominantly indolent thyroid carcinoma that can transform to ATC in vivo, making it ideal for examining the ability of CAFs to promote disease progression. In aim 1, I will elucidate subtypes of CAFs present in ATC and PTC and probe Wnt ligand-receptor interactions. Further, I will determine spatial resolution of myCAF and iCAF fibroblast populations in thyroid carcinoma. In aim 2, I will utilize >40 primary patient thyroid carcinoma CAF cultures that our lab has collected to demonstrate the role of CAF-derived Wnt2 signaling both paracrine on PTC and ATC tumor cells and autocrine to shape the phenotype of CAFs. In completing these studies, I will for the first time define the heterogeneity of CAFs in thyroid carcinoma and characterize a potential novel therapeutic target applicable to CAFs in multiple cancer types.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Alcohol consumption and related behaviors can account for the third-highest cause of preventable death in the United States. The ongoing COVID-19 pandemic has altered how individuals consume alcohol, which creates a need for new strategies to help and protect the health of individuals with alcohol use disorder (AUD). Studies in animal models suggest that exercise can help improve negative affective behaviors associated with abstinence from alcohol exposure. The insular cortex (IC) is a key component in brain circuitry, contributing to synaptic changes in the extended amygdala during abstinence. We have demonstrated that the IC is critical for expressing negative affective behaviors that emerge from forced alcohol abstinence in mice. The primary motor cortex (MOp) regulates essential information between the IC and extended amygdala during affective behaviors. Voluntary exercise, a behavior dependent on the MOp, causes rapid changes in IC activity in mice. Mice exposed to chronic alcohol intake display negative affective and aversion-resistant behaviors following forced abstinence which is mitigated by exposure to intermittent voluntary wheel running access. There is a need to gain insight into the interconnection of the MOp-IC to determine how exercise modulates forced abstinence-associated behavior following alcohol consumption. The inhibitory neurotransmitter GABA is associated with negative affect in humans. GABAergic somatostatin interneurons (SST-INs) are rich in the neocortex and have been critical in regulating negative affective behaviors in rodent models of substance use disorders. Our data suggest that GABA in the IC plays an important role in stress responsiveness. My preliminary data indicates that MOp neuronal projections innervate IC SST-INs. We need further investigation of MOp-IC microcircuitry to understand how activity within it might be altered by chronic alcohol intake. This information has led me to hypothesize that exercise can decrease negative affect and aversion resistance during abstinence from alcohol via the MOp-insula microcircuit connectivity. Within this fellowship, I will first determine the MOp-mid-IC microcircuit inter-connectivity in the mouse brain via electrophysiological methods. I follow up on these results by investigating how alcohol mediates IC microcircuitry. I will then observe the behavioral implications of this circuit by testing if exercise during alcohol abstinence promotes alterations in the IC SST-IN activity. Completion of this F31 grant will provide me with new techniques and skills to help me advance in my career as an independent academic researcher.

NIH Research Projects · FY 2024 · 2023-06

PROJECT SUMMARY At the excitatory synapse, a heterogenous molecular layout creates a precise pre- and post-synaptic structure that facilitates different modes of neurotransmission. The spatial segregation of neurotransmission leads to the autonomous function of spontaneous glutamate release. The investigation into the structure/function relationship at the excitatory synapse has facilitated the discovery of disease pathways and targets for psychiatric disease intervention. However, aberrant inhibitory neurotransmission is also implicated in numerous psychiatric illnesses including schizophrenia, depression, and anxiety. Despite the fundamental importance of inhibitory neurotransmission in disease, few studies have investigated the relationship between nanostructure and function at the inhibitory synapse. My preliminary data suggests a segregation of action potential dependent and spontaneous neurotransmission at central inhibitory GABAergic synapses, but how this segregation is achieved is unknown. Few studies have investigated the inhibitory structure/function relationship due to the limited tools that allow for the selective manipulation of different modes of inhibitory neurotransmission. I have generated preliminary data that a novel small molecule drug, Artemisinin, selectively dysregulates inhibitory spontaneous release as it competitively binds in the GABAAR-gephyrin binding pocket; providing a new pharmacological tool that will be utilized throughout this proposal. This project will investigate the GABAergic synapse in two-fold by elucidating the post-synaptic structure that segregates neurotransmission and how this structure leads to an autonomous function of spontaneous GABAergic transmission in homeostatic plasticity. In primary hippocampal culture, Artemisinin will be used as a tool to decrease GABAergic spontaneous release to investigate how this corresponds to nanostructure and signaling pathways. The central hypothesis is GABAergic synapses have a specific post- synaptic gephyrin scaffold and GABAAR nanostructure that facilitates an autonomous role of spontaneous neurotransmission. Aim#1 will investigate how the post-synaptic structure segregates neurotransmission using super resolution and electron microscopy to assess 1a. gephyrin clustering dynamics and 1b. GABAAR subunit localization and clustering. Aim#2 will investigate the functional pathway this nanostructure facilitates by delineating the role of spontaneous GABAergic neurotransmission in homeostatic plasticity. 2a. First downstream gene expression pathways will be assessed, specifically BDNF expression. 2b. Then how spontaneous GABAergic signaling alters calcium dynamics to trigger gene transcription pathways will be delineated. This research will elucidate the relationship between structure and function at the inhibitory synapse, ultimately providing novel insight into the regulation of synaptic strength underlying excitatory/inhibitory balance in health and disease.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Obstructive sleep apnea (OSA) is a sleep-related breathing disorder associated with major co-morbidities and is estimated to affect nearly one billion people worldwide. Moreover, there are differences in prevalence, diagnosis rates, and co-morbid outcomes for OSA based on the demographics of a patient, such as age, race, and gender. The diversity of the clinical manifestations, objective measurements, and outcomes – the phenotype – of OSA underscores the opportunity for predictive models to improve care of patients with OSA. Predicting future (i.e. 5- year post-diagnosis) risks of OSA co-morbid outcomes and predicting how different treatments for OSA affect these risks can help clinicians and patients choose the best treatment strategies. Current OSA outcomes research has key limitations. Prior studies have characterized groups of OSA patients that exhibit similar characteristics, referred to as sub-phenotypes of OSA. However, these studies have been limited by analyzing relatively few variables obtainable from questionnaires. To address this limitation, we will use rich longitudinal electronic medical records (EMR) data to characterize OSA sub-phenotypes and to predict OSA outcome risks for individual patients. To extract insights from EMR data, we will leverage modern computational methods based in machine learning (ML). A second major limitation of existing OSA research is worse predictive model performance for some groups. Model biases have real-world negative implications. The ubiquitous STOP-BANG questionnaire used to screen patients for further OSA testing performs worse for women and Asian individuals, leading to potential delayed, under-, or misdiagnosis of OSA in these groups. To address this limitation, this proposed project will assess and mitigate biases present in our predictive models. To better understand patient factors associated with OSA outcomes, this project has two aims. In Aim 1 clustering methods will be applied to identify groups of OSA patients who share similar sub-phenotypes according to combinations of clinical features and objective measurements present in EMR data. Then, sub- phenotypes will be compared by the rates at which they exhibit different OSA outcomes, providing intuition into potential underlying pathophysiologic differences. In Aim 2, ML classifiers will be applied to build and validate algorithmically fair predictive models for future OSA outcome risks as well as effects of OSA treatments. Patient- specific factors that are consistently associated with differences in OSA outcome risks through Aims 1 and 2 will provide both personalized insights into treatment options and stronger evidence of underlying pathophysiology worthy of further investigation.

NIH Research Projects · FY 2025 · 2023-06

Project Summary Insulin secretion from pancreatic β cells is crucial to maintaining blood glucose homeostasis, allowing cells throughout the body to take up glucose from the blood and obtain nutrients for normal cellular processes. In type 2 diabetes, however, peripheral tissues become insulin resistant, β cells over-secrete insulin to compensate, and eventually β cells become dysfunctional. It is therefore necessary to thoroughly understand the mechanisms of insulin secretion in order to identify potential therapeutic targets for type 2 diabetes treatment. Insulin secretion is known to predominantly occur at the vascular face of the β cell, at regions termed “hot spots.” Hot spots are characterized by proteins common to the active zone of neurons as well as proteins of cortical microtubule-stabilizing complexes and focal adhesions. Our laboratory has also found that microtubule destabilization promotes insulin secretion specifically at hot spots. Others have shown that the actin cytoskeleton also plays a critical regulatory role in insulin secretion. Given the known connections of microtubules, focal adhesions, and actin with known hot spot proteins, it is likely that the interplay of these cytoskeletal elements is essential for the hot spot organization and function. I therefore hypothesize that insulin secretion hot spots are “secreting adhesions”: mechanosensitive subcellular domains which use cytoskeletal regulation to accomplish directed and clustered secretion. My specific aims are to 1) characterize the cytoskeleton networks, their interplay, and other hot spot components in β cells; and to 2) test if MT- regulated RhoA-dependent contractility promotes clustered secretion via secreting adhesion assembly. To address these aims, I will use high- and super-resolution fixed and live-cell imaging techniques combined with computational analyses. Improving the understanding of hot spot identity and establishment via the cytoskeleton will contribute to overall knowledge of insulin secretion, and potentially uncover novel targets for diabetes therapeutics.

NIH Research Projects · FY 2025 · 2023-06

ABSTRACT / SUMMARY The goals of this research are to extend our discoveries of how blood oxygenation level dependent (BOLD) signals in white matter (WM), detected using functional magnetic resonance imaging (fMRI), are related to neural activity in gray matter (GM), and to implement new analyses that properly incorporate WM signals into models of brain function derived from imaging data. For the past three decades, nearly all analyses of brain fMRI data have ignored WM signals and usually have removed them as nuisance regressors. However, that view has changed in light of more recent evidence that WM BOLD signals represent potentially important and heretofore overlooked indicators of neural activity that are intimately related to how cortical regions communicate, and so should be incorporated into complete assessments of functional connectivity. We have recently shown that BOLD signals are robustly detectable in WM when appropriate analyses are used, that the hemodynamic response function in WM is different from GM, and that WM tracts show reproducible patterns of apparent connectivity which may be summarized in Functional Connectivity Matrices (FCMs), obtained by analyzing resting state correlations between segmented WM and GM parcellations. Furthermore, distinct, reproducible networks of WM emerge from data-driven analyses in similar manner to cortical circuits. In this proposal we aim to develop new analyses and apply them to large numbers of publicly available data. We aim (1) to quantify the functional relationships between WM fibers and GM circuits at a finer scale and in greater detail. We will extend the concept of FCMs to three dimensions to derive those WM tracts that show synchronous time courses with pairs of GM regions that themselves are identified from a matrix of GM-GM connectivity; (2) to use data-driven, model-free independent component analyses to identify WM and GM functional networks and quantify the correlations between them; and (3) to construct a suite of detailed and quantitative atlases characterizing functional connectivity and network topology in WM, and establish their relationships with behavioral and cognitive measures. Templates and digital atlases provide a way to spatially normalize data to common spaces, and measure normal and abnormal variations quantitatively. Extending and applying the methodology from structural and diffusion MRI fields to create atlases of WM functional data will enable reproducible quantification, normalization, and interpretation of our results. Each analysis will also examine the influence of gender and age on WM functional metrics. Impact: BOLD signals in WM reflect neural activity that is related to cortical brain function, so analyses of the functional engagement of WM are essential to properly model brain networks. This research would demonstrate how WM and GM activities are related, and how to integrate them to obtain a more complete model of brain organization. The results will lay a firm foundation for exploiting functional connectivity in white matter associated with pathological or other changes across a spectrum of disorders and conditions.

NIH Research Projects · FY 2025 · 2023-05

K.220.7: PROJECT SUMMARY/ABSTRACT Training: The major objectives of the proposed K99/R00 Pathway to Independence Award are Dr. Ashley Watts’ training in alcohol research and the launching of her career as an independent scientist. As a postdoctoral research fellow in the Department of Psychological Sciences at the University of Missouri, Dr. Watts is well along the path to becoming a leading psychopathology researcher. Dr. Watts seeks training and mentoring during the K99 phase to narrow her focus to alcohol use and alcohol use disorder and gain more training in quantitative genetics and modeling. Training in AUD will emphasize on AUD nosology; modern models of addiction, including their key neurobehavioral mechanisms; and individual differences in alcohol consumption. She will attend conferences related to alcohol use/misuse, behavior genetics, and psychopathology. She will also attend behavioral and molecular genetics workshops that span from human to animal models, as well as a neuroscience workshop; each of these workshops provides hands-on training in quantitative genetic techniques. This training will greatly extend Dr. Watts’ foundational knowledge and skill set. Dr. Watts will dedicate a great deal of her time to the dissemination of findings from the proposed research through manuscript preparation and submission. The proposed mentor and co-mentor (Drs. Kenneth Sher and Andrew Heath), collaborators (Drs. Wendy Slutske, Douglas Steinley, and Phillip Wood), and consultants (Drs. John Crabbe, Arpana Agrawal, Kathleen Bucholz, Kristina Jackson, and David Watson) collectively provide expertise that is ideally suited to facilitate the successful completion of the proposed training and research activities. Furthermore, the University of Missouri is a world-class research institution with the resources necessary to facilitate successful completion of the training (K99) phase of the proposed project. K99 Phase: With supervision, Dr. Watts will conduct secondary data analysis on phenotypic and genetic heterogeneity within AUD. This work will be achieved using a combination of large-scale cross-sectional and longitudinal phenotypic and genetically informative data. She will receive training via mentorship, coursework, workshops, readings, and consultation. R00 Phase: Dr. Watts will ascertain features of AUD that are specific to alcohol use, rather than general to substance use, externalizing psychopathology, or psychopathology more broadly using phenotypic, behavior genetic, and molecular genetic analysis. Significance: Her work will ultimately target sources of heterogeneity within AUD, characterize specific AUD vulnerabilities, and inform future precision-medicine efforts, each of which are emphasized by NIAAA’s emphases on the improvement of AUD diagnosis and understanding its relations with other forms of psychopathology (NIAAA, 2017).

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Prescription opioid therapy plays a critical role in the clinical management of pain in multiple acute and chronic settings. The challenges of effective pain management have led to over 2 million adults in the US, and over 12 million globally, with an opioid use disorder (OUD). OUD accounts for over 120,000 deaths annually worldwide. The dominant target of therapeutic opioids is the µ-opioid receptor (MOR). The analgesic effects of MOR agonists are due to Gα,i/o/z-protein signaling, and it has been proposed that undesirable side-effects of MOR agonists, such as respiratory depression and tolerance, can be mitigated through partial recruitment of Gi/o/z- protein subtypes. Thus, it is of clinical interest to determine the relationship between MOR signaling and analgesia versus side-effects to guide the design of therapeutic agonists that selectively activate the desired signaling pathway. G-protein coupled receptors (GPCRs), including MOR, are known to adopt a range of different functionally distinct configurations upon engaging orthosteric modulators and/or intracellular effector proteins. These induced-fit structural rearrangements cannot be modeled with existing computer-aided drug discovery algorithms during docking or design due to the time and resources required. It is the objective of this proposal to develop a customizable, multi-purpose computer-aided drug design (CADD) platform that can efficiently model largescale induced-fit conformational changes during small molecule and/or receptor sequence design. Completion of the proposal will enable structure- based design of biased agonists and DREADDs (Designer Receptors Exclusively Activated by Designer Drugs). This proposal will include innovative algorithms that leverage deep learning protein structure prediction methods and ultra-large make-on-demand chemical libraries to rapidly screen synthetically accessible molecules for those that can induce conformational changes required to activate G¬i¬-protein signaling in MOR. In collaboration, I will synthesize (Dr. Craig Lindsley), functionally validate (Drs. Craig Lindsley, Heidi Hamm, and Vsevolod Gurevich), and structurally characterize (Drs. Beili Wu and Matthias Elgeti) designed molecules and DREADDs. Experimentally validated partial and biased agonists and DREADDs will be fed back into the computational platform to be used as starting points for subsequent rounds of optimization. In this way, we will establish a computational-experimental iterative feedback loop.