University Of South Florida

NIH Research Projects · FY 2026 · 2024-01

Project Summary/Abstract Numerous disease processes impair the regulation of vascular function, leading to abnormally high or low blood pressure. While hypertension is considered as the most important risk factor for global burden of disease, severe hypotension and vasodilatory shock are of major importance in critical illness. Morbidity and mortality of vasodilatory shock remain unacceptably high and treatment strategies have not been improved since decades. Thus, a better understanding of the mechanisms that regulate vascular function is a prerequisite for the development of improved treatment strategies for critically ill patients. Although dysfunction of a1-adrenergic receptors (AR) is thought to constitute the hallmark in the development of vasodilatory shock, the molecular mechanisms leading to impaired α1-AR function have been unknown. Similarly, information on the regulation of other important G protein-coupled receptors (GPCRs) that mediate vasoconstrictor responses, such as arginine vasopressin receptor (AVPR) 1A or angiotensin II receptor 1, during the cardiovascular stress response and the development of vascular dysfunction in critically ill patients has not been available. The main goal of the PI’s research program is to understand the molecular mechanisms that regulate vascular function during the cardiovascular stress response and to identify new therapeutic approaches to stabilize vascular function in critically ill patients. The PI’s laboratory discovered that chemokines and their receptors regulate vascular GPCRs that mediate the effects of key stress hormones, i.e. catecholamines/α1-ARs and arginine vasopressin/AVPR1A, and identified hetero-oligomerization between chemokine receptors (CRs) and vasopressor receptors as a molecular mechanism underlying their cross-talk. In this MIRA application, we propose to build upon our recent progress in the field and to address critical knowledge gaps regarding the prevalence, assembly and molecular signaling properties of hetero-oligomeric complexes between CRs and vasopressor receptors, and to elucidate their relevance in health and disease processes. We will focus primarily on the roles of CRs in the regulation of vasopressor function, but also plan to expand studies on the roles of vasopressor receptors in the regulation of CR function. We propose a multi-faceted and translationally relevant approach, spanning from state-of-the-art molecular biology techniques to analyze receptor heteromer formation and function at the molecular and cellular level to in vivo animal models and analyses of the function of freshly isolated vascular smooth muscle cells and intact resistance arteries from animals and patients. We believe that this project has the potential to establish a new paradigm in the understanding of the regulation of vascular smooth muscle function in health and disease and is likely to identify new molecular targets to modulate vascular function and blood pressure regulation in various disease processes.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY/ABSTRACT Aneurysmal subarachnoid hemorrhage (aSAH) is a type of stroke that usually results from rupture of an intracranial aneurysm. Delayed cerebral ischemia (DCI) is a clinical syndrome of a focal neurological deficit, cognitive deficit, or both is the strongest predictor of poor outcomes after aSAH. One of the most feared complications of aSAH, which occurs in approximately 30-50% of patients, mostly between days 4 and 10 after the initial aneurysm rupture. Cerebral vasospasm is considered the key pathophysiology that causes DCI after aSAH. The central hypothesis of this proposal is that bedside monitoring of cerebrovascular physiology can be used to track and predict functional outcomes after aSAH, including the occurrence of DCI, as well as assess the efficacy of medical and endovascular interventions. Various diagnostic tools have been applied to detect patients with cerebral vasospasm after aSAH such as transcranial doppler, angiography or perfusion imaging. However, many of these tools have significant limitations. If better means existed to identify which patients were at highest risk of developing DCI, or those in very early stages of DCI, clinicians may be able to more effectively intervene and manage this debilitating complication of SAH. Here, we propose to use a novel, noninvasive, bedside optical instrument Pathlength Resolved Diffuse Correlation Spectroscopy (PR-DCS) to continuously measure cerebral tissue hemodynamics in patients with aSAH. The PR-DCS instrument will provide an accurate, depth-sensitive measurement of cerebral blood flow (CBF), overcoming systemic errors prevalent in conventional optical instruments. Further, these CBF measurements enable bedside characterization of cerebrovascular autoregulation, which hold the power/potential to predict complications in the recovery from aSAH. Aim 1 deploys PR-DCS technology to measure cerebral physiology of patients hospitalized with aSAH. We will use PR-DCS to monitor CBF and to estimate an index of cerebrovascular autoregulation throughout the duration of acute hospitalization. Aim 2 uses PR-DCS as a clinical tool for real-time monitoring of CBF changes during pharmaceutical and endovascular interventions aimed at treating DCI. These innovations directly overcome the limitations of current clinical monitoring instruments that have prevented continuous beside monitoring of brain ischemia in aSAH. Our project is highly translational because the knowledge gained around the mechanisms of cerebrovascular autoregulation can be applied to individual patient care situations.

NIH Research Projects · FY 2025 · 2023-09

Primary angle-closure glaucoma (PACG) is a significant cause of irreversible blindness worldwide, affecting c. 17M people. PACG is more prevalent among women; unfortunately, the underlying reasons for this unequal prevalence are unknown. Factors other than sex, such as anatomical deficits in the anterior chamber, race, and age, are associated with PACG, with anatomical deficits being the accepted primary clinical criteria used to assess PACG risk. However, several clinical trials have shown that such anatomical factors are surprisingly poor predictors of PACG development, indicating the involvement of other unaccounted factors in PACG. The pathophysiological mechanisms of PACG are closely related to the biomechanics of the iris. Specifically, in pupillary block (PB), a key feature of PACG, contact between the iris and the lens induces a pressure gradient between the anterior and posterior chambers. Subsequently, PB leads to occlusion of the outflow pathway (i.e., angle closure [AC]) by anterior deformation of the iris, with associated elevation of intraocular pressure and potential glaucomatous vision loss. We and others have shown that by using pupillary reflexes (e.g., triggered by light), one can non-invasively evaluate the biomechanical properties of the iris. Interestingly, in patients with a history of PACG, the iris is stiffer compared to controls. However, the role of iris biomechanics in inducing AC and PACG is unknown. Our central hypothesis is that iris biomechanics plays a crucial, unappreciated role in developing PACG, based on the natural connection between iridial deformations and iridial biomechanical properties. Therefore, this pro- ject objective is to investigate the role of iris biomechanics in PACG through the evaluation of iridial biome- chanical properties and mechanics of AC and PB. In addition, we will investigate sex-dependent differences in iris biomechanics and their potential role in predisposing women to a higher risk of developing PACG. This project s specific aims (SA) are: SA1 - Investigate sex differences in the biomechanical properties of the iris using a hybrid in vivo/ex vivo approach in rabbits (K99 mentored phase). SA2 - Investigate sex differences in biomechanical properties of the human iris using in vivo and ex vivo (cadaver) analyses. SA3 - Investigate bio- mechanical conditions required to induce AC and PB, and their relation to sex and history of AC (R00). These studies will provide an improved understanding of the pathophysiology of PACG and a unique opportunity to combine engineering, basic science, and clinical research to address a significant public health issue. During the mentored phase, the applicant will learn multiple foundational techniques, including in vivo animal studies in rabbits, biomechanical analysis of active (muscular) tissue, histology, OCT imaging, and human sub- ject studies. In addition, he will significantly expand his professional training through various mechanisms. The skills and techniques learned during the mentored phase will build on the applicant s background in tissue bio- mechanics and allow him to pursue a successful and impactful independent academic career.

NIH Research Projects · FY 2025 · 2023-09

The Human Teratogens Course, directed by Sarah G. Običan, MD, is co-sponsored by the University of South Florida (USF) Health, the Society for Birth Defects Research and Prevention (BDRP), and the Organization of Teratology Information Specialists (OTIS). The Human Teratogens Course is a three-day educational meeting held virtually every two years. The next Course will take place February 23-25,2026. The Human Teratogens Course is a unique educational opportunity that aims to enhance the knowledge and skills of healthcare providers in the field of human teratology. The course is targeted toward healthcare professionals and trainees involved in the care of pregnant and breastfeeding women and their infants. These healthcare professionals include but are not limited to, obstetricians, gynecologists, perinatologists, neonatologists, pediatricians, genetic counselors, teratogen information specialists, and pharmacists. The course may also be of interest to researchers and scientists involved in the field of teratology. The course will be led by a team of expert faculty members and consist of lectures that will cover basic principles of teratology, embryology, epidemiology, and teratogen risk assessment and communication. The course will also provide up-to-date information on various exposures that providers are likely to encounter in their practice. The course aims to provide participants with the tools and resources they may to identify, evaluate, and manage exposures in pregnant women and their infants, ultimately leading to improved outcomes for these individuals. The course was previously held in person in Tampa, Florida in 2019 and virtually in 2021 and 2023. The virtual course drew participants from 30 different States as well as 15 different countries. The upcoming course will be a virtual event with live presentations. The virtual format will enable participants from different locations to attend. The virtual format will also include interactive question-and-answer sessions that will facilitate engagement among participants.

NIH Research Projects · FY 2026 · 2023-09

Alzheimer’s dementia (AD) affects over 35 million people worldwide, and this number is expected to nearly triple by 2050. AD is clinically characterized by progressive cognitive decline; pathologic AD is defined by amyloid plaques and neurofibrillary tangles in the brain. Despite substantial effort, our understanding of its pathophysiology remains incomplete. A thorough understanding of the underlying mechanisms is a prerequisite for discovering novel therapeutic targets. Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide backbone attached to one or more carbohydrates (i.e., glycans). GSLs are especially abundant in the brain and play important roles in brain development, brain aging and neurodegeneration. However, due to technical limitations, our knowledge about the global composition and structures of brain GSLs and their associations with AD pathology remain limited. The mechanisms through which altered GSL expression contributes to AD also remain an enigma. Recently, we have developed several novel technologies for identifying and quantifying intact GSLs (both glycan and diverse lipid forms together as a whole), and for synthesis of GSLs and their derivatives. We have also successfully validated these methods in both human and mouse brain tissue samples. Using these innovative techniques, we will test the hypothesis that altered expression of brain GSLs is causally implicated in AD pathology. Our objectives are to generate the first complete map of brain GSLome (i.e., all GSLs in brain), to identify specific GSLs associated with AD neuropathology (e.g., amyloid-β, neurofibrillary tangles), and to elucidate the mechanisms through which altered expression of GSLs causally contributes to AD pathology. To achieve these goals, we leverage a large collection of human postmortem brain tissue samples (dorsolateral prefrontal cortex, DLPFC) in two community-based cohorts of aging and dementia: Religious Orders Study (ROS) and Rush Memory and Aging Project (MAP). Deep clinical and neuropathological phenotypes as well as rich omics data (e.g., GWAS, DNA methylation, RNA-seq, proteomics) are already available in both cohorts. In Aim 1, we will generate the first complete map of brain GSLome and identify specific GSLs associated with AD neuropathology. Using a data-driven and system biology approach, Aim 2 will integrate brain GSLome data with other brain omics data, including genomics (GWAS), epigenomics (DNA methylation, histone acetylation, and miRNA), transcriptomics (RNA-seq) and proteomics in the same brain cortex (DLPFC), to decipher the mechanisms through which altered brain GSLs causally contribute to AD pathology. Aim 3 employs a gene-centric approach to functionally validate the top-ranked genes in Drosophila model of AD. Such results will provide novel mechanistic insight into AD pathology and would offer immense opportunities for targeting the GSL pathways in developing novel therapeutics for AD treatment.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Owing to the aging of populations worldwide, Alzheimer’s disease (AD) is reaching epidemic proportions, with a large social and economic burden. While the most notable symptom of AD is the severe memory loss, patients with AD also suffer from neuropsychiatric symptoms, including impaired sociability and aggression, which represent significant challenges to the care for these patients. Unfortunately, the mechanisms underlying these neuropsychiatric deficits during AD pathogenesis remain to be fully understood and effective treatments are limited. The brain 5-hydroxytryptamine (5-HT, serotonin) regulates multiple physiological functions, including the control of anger, aggression, mood and cognition. Interestingly, numerous studies reported that the brains of AD patients display extensive “5-HT denervation”, as demonstrated by reduced 5-HT neuron numbers or 5-HT bioavailability. These suggest that impaired brain 5-HT signaling contributes to certain AD symptoms. We identified several loss-of-function point mutations in the human HTR2C gene, encoding 5-HT 2C receptor (5-HT2CR), from individuals with cognitive deficits and social incompetence. We generated a knock-in mouse model, Htr2cF327L, to mimic one such mutation and found that these mutant mice recapitulate human symptoms, including impaired memory, decreased sociability and increased aggression. Given the similarity between the Htr2cF327L-induced phenotypes and those seen in AD, we tested effects of lorcaserin (a selective 5-HT2CR agonist) in an amyloid precursor AppNL-G-F knock-in AD mouse model. Interestingly, lorcaserin ameliorates cognitive and neuropsychiatric deficits in AppNL-G-F mice, associated with enhanced neural plasticity in the ventral hippocampal CA1 (vCA1). These findings led to a general hypothesis that the 5-HT/5-HT2CR signaling ameliorates cognitive and social behaviors in AD. To test this hypothesis, we will first combine the retrograde chemogenetics and loss- or gain-of-function mouse models to determine the role of the 5-HT→vCA1 circuit in cognition, sociability and aggression in health and AD pathogenesis. Using site-specific gene manipulation and the humanized genetic mouse models, we will also determine the role of vCA1 5-HT2CRs in cognition, sociability and aggression in health and AD pathogenesis. Finally, we will test lorcaserin effects in two pre-clinical AD models (with distinct pathogenic mechanisms): AppNL-G-F and PS19. Importantly, we will test these mice at various ages along the disease progression to determine the crucial time window for this pharmacological strategy to be most effective. Results obtained from these studies are expected to advance our understanding about the fundamental biology of cognitive/social behaviors and the neurobiology of human AD progression. In addition, these studies carry significant translational values and will provide a framework for novel therapeutic strategies to ameliorate cognitive and neuropsychiatric symptoms in AD.

NIH Research Projects · FY 2025 · 2023-09

Hispanics/Latinos, the largest and fastest-growing minority group in the US, are disproportionately affected by type 2 diabetes (T2D). Sociocultural, psychosocial, and behavioral factors [collectively called “socioenvironmental factors” hereafter] are believed to contribute to T2D disparity in Hispanics/Latinos, but the mechanisms through which they get under the skin are unclear. DNA methylation (DNAm), one of the most studied epigenetic modifications, is responsive to various socioenvironmental exposures across an individual’s life course, and aberrant DNAm has been associated with aging and age-related diseases including T2D. To date, little is known about the genomewide DNAm patterns associated with socioenvironmental exposures [in totality named “socioenvironmental exposome”]. The mechanisms through which socioenvironmental exposome becomes biologically embedded into aging and risk of T2D have not been well studied in a nationally representative sample of Hispanics/Latinos. Building on our prior work, we hypothesize that altered DNAm evoked by socioenvironmental risk and protective factors contributes to risk for T2D in Hispanics/Latinos. Our objectives here are to characterize the socioenvironmental exposome in relation to risk of T2D, and identify key biological pathways through which socioenvironmental exposome affects risk of diabetes, independent of known risk factors. To achieve this, we leverage a wealth of deep clinical phenotypes and socioenvironmental factors collected by the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) and its ancillary study - the Sociocultural Ancillary Study (SCAS). Using peripheral blood genomic DNA collected at baseline (2008-2011) from 3,323 non-diabetic Hispanics/Latinos (aged 18-74) followed through 2024, we will first identify unique latent constructs using a unified theoretical framework and then conduct epigenomewide association studies (EWAS) to identify methylated genes/regions in response to each unique socioenvironmental construct (Aim 1). Findings from Hispanics/Latinos will be replicated in non- Hispanic Whites, African Americans, and American Indians (total N=7,184). In Aim 2, we will prospectively determine whether socioenvironment-induced DNAm predict the onset and progression of T2D, independent of standard clinical factors. In Aim 3, we will perform integrated genetic and epigenetic analyses to identify causal epigenetic mediators and molecular pathways through which socioenvironmental exposome become biologically embedded into diabetes risk in Hispanics/Latinos. Successful completion of this project will identify modifiable genes and causal pathways through which socioenvironmental factors become biologically embedded in Hispanic cardiometabolic health. Such results may provide novel mechanistic insights into disease pathology, and are likely to lead to culturally tailored precision strategies for diabetes prevention and intervention, thereby reducing diabetes disparity in this minority population.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Research over the past decade has revealed the importance of long non-coding RNAs (lncRNAs) in the control of cardiac remodeling and heart failure. We have characterized a cardiac specific lncRNA in the control of calcium handling in human and mouse cardiomyocytes. Knockout of this lncRNA causes dysregulation of calcium handling in cardiomyocytes and impairs cardiac function. Knockout mice are susceptible to cardiac arrhythmias caused by catecholaminergic challenging and premature death. In this project, we propose to (1) determine mechanisms by which lncRNA regulates gene expression at the transcription level, (2) determine how lncRNA regulates gene expression at the alternative splicing level, and (3) determine cardiac protection conferred by lncRNA in response to pathological stresses. The results from this project have the potential to facilitate the development of novel therapeutics for heart disease.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Regular physical activity is a powerful intervention that reduces obesity and confers protection against obesity-associated metabolic diseases. The mechanisms responsible are incompletely understood but are likely to extend beyond activity-associated increases in energy expenditure alone. We recently identified a lactate-derived metabolite called N-lactoyl-phenylalanine (“Lac-Phe”) as the most significantly elevated metabolite in blood plasma after an intense exercise bout. We further demonstrated that pharmacological elevation of plasma Lac-Phe to mimic exercise training can robustly suppress feeding in obese mice, and repeated Lac-Phe regimen results in chronic hypophagia, weight loss, and reduced adiposity, associated with improved glucose tolerance. While these findings raise the possibility that Lac-Phe could be used as an anti-obesity agent, the neurobiological mechanisms underlying Lac-Phe hypophagia remains unknown. Our preliminary studies identified Agouti-related peptide (AgRP)-expressing neurons in the arcuate nucleus of the hypothalamus (ARH) as one direct target of Lac-Phe action and mediate its hypophagic response. One objective is to examine effects of Lac-Phe and exercise on afferent synaptic inputs to AgRP neurons, and efferent outputs from AgRP neurons to their synaptic targets. Our data also suggest that Lac-Phe inhibits orexigenic AgRP neurons via increasing an outward potassium current, namely KATP current. Thus, the second objective is to use the CRISPR-Cas9 approach to genetically disrupt the expression of KATP channel subunits in AgRP neurons, and use these models to determine the functional relevance of KATP channel in Lac-Phe-induced AgRP inhibition and hypophagia. Finally, we also observed that Lac-Phe activates neurons in four other brain regions, the lateral septum (LS), the paraventricular nucleus of the hypothalamus (PVH), the parabrachial nucleus (PBN), and the nucleus of solitary tract (NTS). Thus, we will combine the Targeted Recombination in Active Populations (TRAP) approach with electrophysiology, chemogenetics and scRNA-Seq to determine whether Lac-Phe stimulates these neurons directly or indirectly, whether these neurons functionally participate in the Lac-Phe-induced hypophagia, and what are neurochemical identities of these Lac-Phe-activated neurons. These proposed experiments will reveal the neurobiological basis for Lac-Phe hypophagia, which may identify Lac-Phe or the associated pathways as targets for weight management.

NIH Research Projects · FY 2024 · 2023-08

Abstract Apicomplexa parasites contribute significantly to human disease burden, including ~1/3 of human populations permanently infected with Toxoplasma gondii. Existing treatments are limited and often toxic to the most affected population of immunocompromised patients. We need a profound knowledge of parasite biology to develop efficient anti-parasitic drugs. Our group focuses on the studies of cell cycle mechanisms that are central to parasite survival and offer a wealth of druggable targets. The cell cycle program orchestrates cell division and ensures the inheritance of the genetic material. Apicomplexan cell cycles are strikingly different from the cell cycles of their hosts. Although T. gondii tachyzoite divides by endodyogeny that resembles a binary division of the conventional eukaryotes, there are substantial differences in cell cycle organization and regulation. It includes the atypical S-phase of Toxoplasma endodyogeny, which is a primary focus of our study. The need for appropriate tools to examine the intricacy of the apicomplexan cell cycle and unconventional regulators significantly impedes the related studies. To fill a major gap in our knowledge of the essential biology of apicomplexan parasites and boost the Toxoplasma cell cycle studies, we engineered a new Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) probe. In the Aim 1 experiments, we will test the hypothesis that Toxoplasma endodyogenic cell cycle includes a composite S/M/C phase that runs for nearly half of the division cycle. Using our new ToxoFUCCI probes, we will determine how the intertwined S/M/C phase is organized. In Aim 2, we will determine the mechanism of the S-phase regulation. Designed experiments will test the hypothesis that, contrary to conventional S-phase cyclin dependent kinase (Cdk), controlled release of the sequestrated Cdk-related kinase TgCrk5 regulates DNA replication in the tachyzoites. Using the conditional expression model of TgCrk5, we will determine the functions of the sequestered and the nuclear TgCrk5 and identify the TgCrk5 substrates. Given that T. gondii lacks conventional S-Cdk substrates, we expect to discover a novel TgCrk5 network. The project will advance our knowledge of the fundamental process of parasite survival and have a high potential to discover future efficient drug targets.

NIH Research Projects · FY 2024 · 2023-08

Abstract Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by neuronal loss driven by deposits of pathological tau. Therefore, identifying and targeting potent regulators of tau is crucial in developing effective therapeutic strategies. My exciting preliminary data show DnaJB6 overexpression decreases tau levels in HEK293T cells more than 50%, and in parallel, knockdown of DnaJB6 results in a 2-fold increase of tau. This suggests that DnaJB6 may regulate tau levels, however, the nature of this relationship has not yet been investigated. In this proposal, I will test the hypothesis that DnaJB6 can prevent tau accumulation through direct and indirect mechanisms. In Aim 1, I will test this through assessing protein-protein interaction dynamics between DnaJB6 and tau with recombinant in vitro assays, including Thioflavin T fluorescence and Surface Plasmon Resonance. A targeted proteomic approach will be used to identify direct and proximal connections between DnaJB6 and tau in primary neurons. In Aim 2, we will use PS19 and non-transgenic mice to assess the associated behavioral and molecular changes that result from DnaJB6 overexpression in the brain. Successful completion of these studies will provide crucial information regarding DnaJB6 biology, direct and indirect effects of DnaJB6 on tau, and how regulation of DnaJB6 affects behavioral and molecular changes in the tauopathic brain.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no definitive treatment that reverses the course of the disease, and we still lack a firm grasp on how older people develop AD. Bridging Integrator 1 (BIN1) is the second-largest genetic risk factor for late-onset AD. At least 12 different alternatively spliced BIN1 isoforms are expressed in the brain, including the neuron-specific BIN1 isoform 1 (BIN1iso1) and the ubiquitously expressed BIN1 isoform 9 (BIN1iso9). In the brain gray matter of patients with AD, there is a decrease in neuronal BIN1iso1 and an increase in BIN1iso9 compared to healthy controls. Thus far, evidence has shown that neuronal BIN1 isoforms participate in clathrin-mediated endocytosis, endocytic recycling, and synaptic vesicle release and retrieval. However, the mechanisms by which BIN1 contributes to these functions and the neighborhood of proteins that BIN1 interacts with to accomplish these cellular tasks remain largely undefined. Therefore, a fundamental gap in the field is an unbiased characterization of BIN1iso1 interacting proteins and proximal neighbors. Closing this gap will help define BIN1’s biological functions in healthy and diseased brain neurons. Utilizing the highly innovative proximity biotin ligase, TurboID, fused to BIN1iso1, will allow the identification of all proteins within a 10-nm radius. TurboID-based proximity labeling coupled with the most recent advanced quantitative mass spectrometry and data analysis methods represents a powerful strategy for discovery research. My preliminary in vitro studies using this approach in mouse N2a neuroblastoma cells resulted in the discovery of 234 proteins as BIN1-associated (proximal) or interacting proteins. These results identified several known BIN1 interactors such as tau, dynamin, synaptojanin, and many previously unknown proximal proteins. The following Specific Aims will translate these findings in vivo and dramatically advance the field. Aim 1. Identify neuronal BIN1iso1 interacting proteins in vivo using wild-type mice under homeostatic conditions. Aim 2. Establish neuronal BIN1iso interactome in mouse models of AD (5XFAD and PS19) before and after the onset of pathology. This project will not only advance the field by providing those studying AD and BIN1 with a list of BIN1iso1 proximal proteins to generate novel hypotheses but will also support the applicant’s pre-doctoral research training in AD pathophysiology, advanced methods such as in vivo AAV transduction, proximity-based labeling in the context of AD pathology, large-scale proteomics data analysis, and bioinformatics analysis of BIN1 functional pathways. The dynamic and highly collaborative research environment at the USF Health Byrd Alzheimer’s Institute will enhance the learning opportunities in cell biology and molecular pathology within an established AD laboratory led by a committed mentor. Furthermore, research training in proteomics approaches offered by the collaborator’s lab and the Proteomics Core facilities will complement the applicant’s interdisciplinary research training in age-related neurodegenerative disease.

NIH Research Projects · FY 2024 · 2023-08

Project Summary / Abstract………………………………………………………………………………..…. Metastatic prostate cancer (PC) typically manifests in the skeleton. The lesions arise from disseminated tumor cells (DTCs) that often persist in a dormant state for months to decades after initial primary tumor treatment. Despite our knowledge that DTCs give rise to incurable bone metastatic PC, there remains gaps in our understanding as to the molecular mechanisms underpinning entry and reawakening from the dormancy program. Insight into those mechanisms could yield new therapeutic approaches that would prevent the metastatic relapse and ultimately death of almost 34,500 American men each year. To address this, we have developed a novel model of PC dormancy in agreement with published markers in the literature in vitro and optimized a surgical technique to study dormancy in vivo. We next performed transcriptomic sequencing on PC cells growing under normal, dormant, or reawakening conditions. Bioinformatic analysis of transcript expression and transcription factor (TF) activity revealed positive regulatory domain zinc finger region protein 16 (PRDM16) to significantly increase during entry into dormancy and decrease during exit across mouse (RM1) and human sensitive / castrate resistance cell lines (LAPC4, 22Rv1). Further, we validated PRDM16 upregulation in dormant cells on the protein level both in vitro and in vivo. Genetic silencing of PRDM16 led to a significant reduction in the ability of PC cells to enter dormancy concomitant with decreased expression of anti-apoptotic proteins including BCL-2 and increased pro-apoptotic proteins such as BIM & Noxa. Preliminary data shows that PRDM16 expression in PC cells can be induced by BMP-7 and conversely decreased by the BMP signaling antagonist, noggin, factors with known critical roles in regulating bone homeostasis. Based on our findings, we hypothesize that BMP7 induces PRDM16 expression in early skeletal DTCs, which is critical for the initiation of the dormancy program. In the F99 phase, we will identify PRDM16 transcriptome in PC dormancy and confirm BMP signaling control on its expression using genetic approaches and validation of findings in pre-clinical models. In K00 phase, we will study how aging contribute to the reawakening of dormant cancer cells in the bone microenvironment. We will identify biological and molecular determinants of cancer dormancy using Cherry-niche technology and comprehensive fluorescence-activated cell sorting (FACS), high dimensional mass cytometry, and single cell RNASeq experiments. We will then validate our findings in pre-clinical models and patient tissue sections. Findings from this work will advance our understanding of the intrinsic and extrinsic factors regulating the cancer dormancy program in skeletal metastasis in the context of aging, which is relevant to most prostate cancer patients.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Hazardous alcohol use (HAU) leads to tremendous morbidity and mortality in millions of individuals in the United States and worldwide annually. The cognitive, neuronal, and psycho-social aspects of alcohol use have been well established. More recently, clinical, and basic research has begun to understand the role in which HAU contributes to gut dysbiosis, which plays a significant role in one’s overall health status. Furthermore, a large portion of deaths associated with alcohol use are related to digestive diseases. HAU predisposes and contributes to the manifestation of several comorbidities, like hypertension, metabolic syndrome, and diabetes mellitus which drive and exacerbate vascular dysfunction and cardiovascular disease (CVD). Previous research has demonstrated that the gut microbiome too plays a critical role in the diagnosis and prognosis of individuals with established CVD. Increased levels of circulating trimethylamine-N-oxide (TMAO), a gut derived metabolite, has been shown to drive the development of atherosclerotic heart disease. However, the relationship between HAU- induced gut dysbiosis and its’ metabolites towards vascular and CV function is not well-defined. We hypothesize that HAU-induced gut dysbiosis leads to endothelial dysfunction and increased risk of CVD via gut- derived metabolites (i.e., TMAO). Furthermore, we believe that HAU-induced gut dysbiosis exacerbates the progression of heart failure and that gut microbiota-targeted therapies (MBTT) will restore vascular function thereby improving CV health in the setting of HAU. Through utilization of mouse models of HAU and microbiota adoptive transfer we plan to execute a series of studies that demonstrate HAU-induced dysbiosis and CV-related pathology are related; moreover, that the dysbiotic microbiome is sufficient to cause the vascular dysfunction and increased risk of CVD. We plan to utilize metagenomics, metabolomics, and cardiovascular function assessment to demonstrate the causal relationship between HAU, the gut microbiome and CVD. We will then investigate the effects of prior HAU gut dysbiosis on the progression of heart failure in a murine model of myocardial ischemia-reperfusion (MI/R). This will answer questions regarding the predisposition of individuals who participate in HAU and their risk for worsening CV outcomes after MI/R-induced heart failure. Concurrently, we will examine continuous HAU prior to and after MI/R-induced heart failure to understand if HAU leads to increase morbidity or mortality in the presence of CVD. Successful completion of these studies will significantly advance our understanding of the pathology of alcohol-induced gut dysbiosis and its’ effects on vascular and cardiac function.

NIH Research Projects · FY 2026 · 2023-08

Project Summary. A fundamental characteristic of alcohol use disorders is the loss of control over alcohol consumption that results in progressively escalating levels of alcohol use and facilitates the progression to alcohol-dependence. Given the comorbidity of alcohol dependence and disorders of affect such as depression is extremely high, it has been posited that self-medication of negative affective states contributes to continued excessive alcohol use and relapse. Furthermore, negative affective states produced by chronic alcohol exposure can influence the neurocircuitry of cognitive control systems to perpetuate further excessive alcohol use. Once that degree of dysregulation is reached, components of the dependence cycle serve to facilitate each other in a manner that is extremely deleterious to personal, familial and societal welfare. The principal investigator’s long-term goal is to identify effective therapeutic targets and strategies for the treatment of alcohol use disorder (AUD). The objective of this application, which is the next step in pursuit of that goal, is to understand the neuroadaptations in Oprk1 (kappa-opioid receptor gene)-regulated systems that occur in response to chronic alcohol exposure and contribute to maladaptive behavioral regulation. The central hypothesis is that the Oprk1-regulated neurocircuitry becomes progressively dysregulated in a manner that promotes the continued excessive consumption of alcohol and perpetuates the cycle of alcohol dependence. The rationale for the proposed studies is that understanding the contribution of dysregulated Oprk1 expression in AUD will lay the foundation for the development of effective therapies designed to alleviate maladaptive behavioral regulation produced by alcohol dependence. This hypothesis will be tested by utilizing inducible and conditional CRISPR/CAS9 gene editing and chemogenetic approaches to recapitulate or ameliorate symptoms of alcohol dependence in non-selected and transgenic rats. Animal models of operant alcohol self-administration, negative affective-like behavior and executive function including working memory will serve as functional end-points to systematically investigate the role of Oprk1 expression in maladaptive behavioral regulation related to alcohol dependence. In addition, Oprk1 gene expression will be assessed as a complement to the behavioral approaches. The proposed research will help to identify the functional importance of neuroadaptations in Oprk1-regulated systems that result from chronic alcohol exposure and will provide much needed information regarding the influence of Oprk1 on the neurocircuitry of AUD-related phenotypes. Such a contribution is significant because it will help identify and develop therapeutic targets to treat AUD that focus on the removal of maladaptive phenotypes; a strategy that should greatly increase treatment compliance and decrease rates of relapse.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Opioid therapy is commonly prescribed for patients with chronic widespread musculoskeletal pain, but offers questionable benefit for long-term pain management and is associated with arrhythmias, overdose, and death. Individuals with chronic pain experience high rates of comorbid chronic insomnia, arousal, and abnormal brain activation in response to painful stimuli. Research shows individuals with chronic pain exhibit increased brain activation in regions associated with pain modulation in response to painful stimuli compared to healthy controls. Withdrawal from opioids is difficult; and inadequately managed pain contributes to that difficulty. The Cognitive Activation Theory of Stress (CATS) tests the hypothesis that poor sleep and arousal lead to critical changes in brain activation that increase pain severity and lead to opioid use. Research shows cognitive behavioral treatment for insomnia (CBT-I, an evidence based intervention for chronic insomnia) improves sleep, arousal, abnormal brain activation, and pain in individuals with comorbid chronic pain and insomnia, but does not reduce opioid use. However, because CBT-I improves each of the mediators hypothesized to contribute to opioid use, it warrants examination as a neoadjuvant to gradual tapering of opioid medication. The proposed trial tests the novel hypothesis that improving sleep and decreasing arousal will lead to normalized brain activation and decreased pain prior to gradual tapering, which will facilitate reduced opioid use. This hypothesis is supported by theory (CATS) and empirical findings. It also reflects federal pain research priorities. Trial Design. 165 adults who use prescription opioid users (18+ years of age) and have chronic pain and insomnia will be randomized to CBT-I or Sleep Hygiene and Related Education (SHARE). They will then undergo a gradual tapered withdrawal protocol for opioids. Outcomes (sleep, arousal, brain activation, pain, opioid use, opioid related problems) will be examined at baseline (BL), post intervention (P1), post withdrawal (P2), and 6-month follow-up. Specific Aims 1 and 2 test the impact of CBT-I on sleep, arousal, brain activation, pain, opioid use, and opioid related problems compared to the active SHARE control. Specific Aims 3 and 4 test the impact of tapering opioid use following CBT-I on sleep, arousal, brain activation, pain, opioid use, and opioid related problems compared to the combined SHARE and tapered withdrawal control. An Exploratory Aim examines the relationships between changes in the mechanistic outcomes and changes in the opioid outcomes, and their potential moderators (e.g., craving, withdrawal symptoms, sex, age, race, ethnicity). Public Health Implications: Demonstration that a relatively brief behavioral sleep intervention facilitates tapered withdrawal from opioid medication and protects against relapse through improvements in sleep, arousal, abnormal brain activation, and pain has important implications for the millions of individuals living with chronic pain, their families, communities, and healthcare.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract The long-term objective of this application is to develop innovative therapies for intracerebral hemorrhage (ICH), which causes high rates of death and disability worldwide. This is consistent with the mission of NINDS. This proposal aims to investigate the biological functions of fibroblast-derived laminin in blood-brain barrier (BBB) repair and fibroblast biology after ICH and explore the underlying molecular mechanisms. In Aim 1, the function of fibroblast-derived laminin in ICH pathogenesis will be investigated in two clinically relevant ICH models using middle-aged transgenic mice with laminin deficiency in fibroblasts. In Aim 2, the function of fibroblast-derived laminin in BBB repair after ICH will be investigated both in vitro and in vitro. First, how loss of fibroblast-derived laminin affects BBB permeability and inflammatory cell infiltration after ICH will be examined (Aim 2A). Next, whether loss of fibroblast-derived laminin exacerbates BBB disruption via paracellular and/or transcellular mechanisms will be investigated (Aim 2B). Third, the receptors that mediate fibroblast-derived laminin’s “BBB-repairing” effect on endothelial cells will be identified using both pharmacological and genetic approaches (Aim 2C). In Aim 3, the function of fibroblast-derived laminin in fibroblast biology and fibrotic scar composition will be investigated. First, fibroblast biology (proliferation/apoptosis/migration), fibroblast morphology, and fibrotic scar components will be examined in vitro and in vivo (Aim 3A). Next, how exactly fibroblast-derived laminin regulates fibroblast biology and fibrotic scar composition will be explored by bulk and/or single-cell RNAseq analysis (Aim 3B). Third, the receptors that mediate these changes in fibroblasts will be identified using both pharmacological and genetic approaches (Aim 3C). Successful completion of this proposal will elucidate novel functions of fibroblast-derived laminin in BBB repair and fibroblast biology after ICH, identify the receptors that mediate these effects on both endothelial cells and fibroblasts, provide new molecular targets with therapeutic potential, and promote the development of innovative and effective treatments for ICH.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Nearly half of the US adult population has hypertension, which puts them at increased risk for stroke, vascular damage, heart attack, heart failure, and kidney disease. Recent genome-wide association studies have linked a number of mutations in the gene, CLCN6, to reduced hypertension and stroke risk. CLCN6 encodes the voltage-sensitive chloride channel 6 (ClC-6). To date, very little is known of about the function ClC-6, or its role in blood pressure homeostasis. The overall goal of this proposal is to establish the physiological and molecular roles of ClC-6 on vascular smooth muscle cell (VSMC) function and blood pressure. Mentored Phase: Preliminary data have demonstrated that ClC-6 is expressed in the Golgi apparatus of VSMCs. I hypothesize that ClC-6 activity in VSMCs regulates luminal Golgi Ca2+ stores by providing a charge balance for Ca2+ uptake, thereby maintaining membrane electroneutrality. This occurs through an association with the Golgi-specific Ca2+-ATPase, SPCA1, and loss of ClC-6 reduces Golgi Ca2+ stores and signaling, which alters VSMC contractility. Specific Aim 1. Establish the role of ClC-6 on Golgi Ca2+ handling in VSMCs. I will utilize planar lipid bilayer electrophysiology and sophisticated Ca2+ imaging to determine ClC-6 chloride channel properties and their impact on the Ca2+ storage capacity of the Golgi. Furthermore, these experiments will provide the first evidence of the role of Golgi-specific Ca2+ release in VSMC Ca2+ handling in response to vasocontraction and dilation stimuli, which has never before been examined. Independent Phase: This phase of the project will develop an independent line of investigation into the molecular effects of ClC-6 on VSMC function. Insight gained from these experiments will further explain the physiological mechanism underlying ClC-6 regulation of blood pressure control. My preliminary data have established that ClC-6 prevents or slows large artery vessel stiffening during development of hypertension. I will further examine the effect of ClC-6 on cellular proliferation, migration, apoptosis, and extracellular matrix protein deposition in normotensive and hypertensive vessels. I hypothesize that loss of ClC-6 will reduce extracellular matrix protein secretion and slow cell proliferation, thereby reducing hypertension induced fibrosis and media thickening. These changes will result in abrogated arterial stiffening and vessel remodeling, and reduce peripheral vascular resistance, providing a rationale for how ClC-6 moderates blood pressure. Specific Aim 2. Elucidate the influence of ClC-6 on vascular stiffness and vessel remodeling during hypertension. Experiments to address this hypothesis will include a diverse range of experiments, including pulse-wave velocity measurements, histological analyses, migration assays, myography, TUNNEL and BrdU assays, and transcriptomic analysis. These studies will inform our understanding of the protein's function by defining the role of this channel in vasculature at the molecular, cellular, and systemic levels.

NIH Research Projects · FY 2026 · 2023-08

Alcohol use disorder (AUD) remains a major issue in the United States (US) despite the various laws, campaigns, and preventative efforts. Alcohol is by far the most commonly-abused drug across the lifespan. According to 2015 data from The National Survey on Drug Use and Health, there are currently 138 million alcohol users in the US, with almost half of drinkers reporting problem drinking (binge or heavy alcohol consumption), and 15.7 million reporting an AUD. A host of pharmaceutical targets have been identified, but treatment efficacy and abstinence rates both remain low due to factors such as cost, negative physiological effects, and requirements of long-term commitment to treatment. Thus, new directions for addiction treatment are needed. In one such direction, recent advances in ultrasound technology have enabled the non-invasive modulation of deep brain systems such as the reward circuit. We propose a novel combination of low intensity focused ultrasound (LIFU) with photoacoustic tomography (PAT) localization to modify the activity of the two primary components of the reward system, the ventral tegmental area and nucleus accumbens. Our preliminary work has demonstrated that this approach reduces alcohol intake and preference when administered once daily in either region. Thus, in the proposed studies, we will examine the mechanisms by which LIFU reduces alcohol intake, the longevity of this effect, and potential side effects of this treatment on the brain. We will first optimize our protocol for maximum efficacy in the deep brain regions of the reward circuit. Next, we will assess a variety of behaviors related to affect and to alcohol-seeking in order to identify the neurological processes on which LIFU is exerting its effects. Finally, we will assess the molecular effects in the brain following LIFU treatment, in order to identify those neuronal changes that may explain the behavioral shifts, and to determine whether there are any adverse effects on neuronal function following chronic LIFU treatment. For these studies, we will use male and female crossed high-alcohol-preferring mice, in order to identify whether there are sex-dependent differences in the effects of LIFU on alcohol-seeking, or in the neuronal effects of LIFU. These studies will provide an essential foundation of knowledge for the use of LIFU to reduce AUD, and will open the door to a wide spectrum of further studies examining other substance use disorders, and use of LIFU in traditionally difficult to treat populations, such as adolescents, or for disorders commonly comorbid with AUD, such as sleep disorders.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract The urgent need to refill the physician-scientist pipeline by training the next generation of physicians in basic and translational laboratory and applied clinical research has been widely recognized. A declining number of surgeon-scientists, however, threatens future advancement of surgical practice. The overall goal of this training program is to train the next generation of surgeon-scientists, who have the knowledge, skillsets and motivation to perform translationally relevant and meaningful research that will lead to significant improvements in patient outcomes. Our training program seeks applications from surgery residents interested in a 2-year full-time research training centered on injury pathobiology and outcomes in critical illness, a broad research arena essential for all surgical specialties. Each year, 1 trainee, who has typically completed 3 years of surgery residency, will be recruited primarily from a large pool of diverse and highly talented applicants for the general surgery and the integrated vascular surgery residency programs in the Department of Surgery, Morsani College of Medicine at University of South Florida (USF). The central hub of our training program is the Division of Surgical Research (DSR) in the DOS. Experienced investigators in the DSR, alongside carefully selected investigators from 5 other USF departments will form the training faculty. The training faculty is composed of 14 individuals who are committed to the research training of young surgeons, have an establish track record of extramural funding related to various aspects of injury pathobiology and outcomes in critical illness, and currently hold more than 30 NIH/VA awards as principal investigators. Many of our training faculty are physician scientists, which provides our trainees with critical experience and knowledge in both medical and basic sciences. Based on the experience of the MPI/PDs as PIs of previous T32 training programs at other institutions, we propose an optimized training plan to provide trainees with intensive state-of-the-art training in responsible conduct of rigorous and reproducible research that is guided by individual development plans. While trainees will select a scientific mentor of their choice, all trainees will be required to develop written research proposals and progress reports, to give formal research presentations, to participate in seminars, journal clubs and in didactic course work in biomedical data analysis and responsible conduct of research. In addition, trainees will have the opportunity to obtain additional graduate degrees/certifications in health informatics or health care analytics. We have the infrastructure, resources, expertise and motivation to provide our trainees with advanced and individualized research training in a vibrant scientific environment. We believe that our training program will ultimately provide the society with much needed surgeon-scientists, who will be at the forefront of clinical practice in the future.

NIH Research Projects · FY 2025 · 2023-07

Tuberous Sclerosis Complex (TSC) is a chronic disease that is caused by mutations in the genes TSC1 or TSC2. The disease affects 2 million people worldwide and there is no cure for TSC disease. Symptoms include formation of large tumors called hamartomas in various organs of the body, including the brain, the kidney, and the lung. One of the severe complications associated with these tumors is chylothorax, which is the accumulation of chyle in the space between the pleura and chest cavity. Chyle is a milky fluid that originates from lymphatic vessels draining dietary fats. Chylothorax can cause difficulty breathing, tachypnea, chest pain, respiratory failure, and death. Although chylothorax indicates malfunctioning lymphatic vessels in TSC patients, the current dogma is that tumors compress/obstruct the lymphatic vessels near the lung resulting in leakage, and it remains unknown whether TSC mutations in lymphatic endothelial cells are a causative factor of chylothorax. Thus, a significant unmet need is to determine whether lymphatic vessels are involved in TSC disease and delineate the pathological changes of the lymphatic vasculature due to TSC loss-of-function, which will identify new signaling pathways and molecular targets for this disease. Our preliminary data show that lymphatic-specific deletion of the Tsc genes in mice leads to chylothorax and is accompanied by a severe loss of lymphatic valves. These data suggest that the lymphatic vasculature is involved in TSC disease and regressing lymphatic valves allow lymph to flow backwards into the paravertebral and intercostal lymphatic capillaries, which causes lymph leakage from the thoracic duct into the chest cavity. Aim 1 will determine pathological changes in the lymphatic vasculature upon genetic deletion of Tsc genes, Aim 2 will identify the molecular mechanisms by which loss of Tsc1 or Tsc2 causes the loss of lymphatic valves, and Aim 3 will investigate novel approach to reverse lymphatic valve loss in the Tsc knockouts. It is highly anticipated that these aims will identify new pathogenic features of TSC disease and new signaling pathways affected by loss of TSC signaling.

NIH Research Projects · FY 2025 · 2023-07

Project Summary: Growing evidence suggests the link between right ventricular (RV) fibrosis, poor function of the pressure-overloaded RV, and mortality in pulmonary arterial hypertension (PAH). PAH patients with decompensated RV failure (RVF) have persistent RV fibrosis even when treated with the conventional therapies for PAH. RVF is the main cause of death in PAH and maintaining RV function in PAH is associated with improved patient survival. However, there are currently no available therapies that specifically target RV fibrosis. Therefore, identifying the molecular mechanisms underlying RV fibrosis in PAH is urgently needed to develop novel therapeutic approaches targeting RVF in PAH. We recently reported the significant roles of o xidative stress-sensitive protein kinase D (PKD) at outer mitochondrial membrane (OMM) and its substrate dynamin-related protein 1 (DRP1), a mitochondrial fission protein, in dysregulating CM functions. We also showed that DRP1-mediated mitochondrial fission limits the size of the matrix cavity, thus causing elevated and sustained mitochondrial Ca2+ (mtCa2+) transient in response to cytosolic Ca2+ elevation. Using a preclinical rat PAH model with RV hypertrophy, failure, and fibrosis that significant , we found PKD activation and DRP1 phosphorylation occurs specifically in cardiac fibroblasts (CFs) in the RV (RV-CFs), but not in CMs under PAH, which subsequently causes an PKD-dependent increase in mitochondrial fission, mitochondrial reactive oxygen species (mROS), and CF proliferation. Moreover, we found that PKD activation is associated with increased phosphorylation of a pro-apoptotic protein Bax, which inhibits apoptotic pore formation in the OMM and potentially contributes to the anti-apoptotic phenotype of RV-CFs in PAH. Lastly, we also found that mtCa2+ uptake via mtCa2+ uniporter (MCU) is required for mROS elevation and subsequent activation of proliferative signaling in CFs. Based on these findings, we hypothesize that 1) PKD-dependent Bax phosphorylation allows RV-CFs to be resistant to apoptosis under PAH; 2) PKD-dependent mitochondrial fission limits mtCa2+ and antioxidant capacity by decreasing the size of the matrix cavity and causing increased mtCa2+ and mROS levels, thus acting as a molecular “switch” for proliferative signaling for RV-CFs in PAH; and 3) CF-specific inhibition of PKD at the OMM in vivo can be leveraged as a novel therapy to attenuate cardiac fibrosis in response to stress/injury such as PAH. In Aim 1, we will establish Bax as a novel PKD substrate in the mitochondria and assess the impact of PKD-dependent Bax phosphorylation on OMM permeability. To specifically inhibit PKD activity only at the OMM, we will use an OMM-targeted dominant-negative PKD1 (mt-PKD- DN) that we have newly validated. In Aim 2, we will test whether PKD-dependent enhancement of mitochondrial fission facilitates RV-CF proliferation via increased mtCa2+ and mROS levels. In Aim 3, we will test the therapeutic potential of mitochondrial PKD inhibition by mt-PKD-DN in the quiescent CFs before they transform into myofibroblasts by CF- specifically expressing mt-PKD-DN in a preclinical rat PAH model. The proposed project is designed to determine the role of mitochondrial fission, Ca2+, and mROS in RV-CF hyperproliferation and RV fibrosis in PAH, which will lead to develop a novel strategy (i.e., PKD inhibition) for the management of RV fibrosis and failure in the setting of PAH.

NIH Research Projects · FY 2026 · 2023-05

Modified Project Summary/Abstract Section Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease (CKD). Diabetic complications manifest themselves differently between men and women. Understanding the molecular mechanisms of these sex-related differences is critical to designing tailored therapeutic approaches. This proposal is focused on sex differences in the progression of DKD with specific emphasis on the contribution of pro-inflammatory signaling pathways in diabetic females and males. To explore the sexual dimorphisms in the development of DKD, we propose to use here non-obese type 2 diabetic nephropathy (T2DN) rats. We have demonstrated previously that T2DN rats develop renal and physiological abnormalities similar to clinical observations in humans with DKD, implying these rats are an excellent model for studying the progression of renal injury in type 2 DKD. Furthermore, our recent studies revealed sexual dimorphism in this model, indicating that while both female and male T2DN rats developed non-obese DKD phenotype, females had significant protection from the development of severe forms of glomerular and tubular damage. The main goals of this project are to determine potential molecular mechanisms causing renal, and specifically glomerular, injury and evaluate sexual dimorphism of these pathways in type 2 DKD. Aim 1 will explore the functional and structural differences between male and female T2DN rats and further identify critical sex dependent pathways in DKD. Furthermore, our pilot studies have revealed that the cyclic GMP-AMP Synthase (cGAS) / STimulator of INterferon Genes (STING) signaling pathway is upregulated in male T2DN rats and can contribute to the observed sex difference. The role of the cGAS-STING pathway in multiple regulatory mechanisms, specifically in cancer and inflammatory diseases, was recently reported. Since then, the development of drugs targeting the cGAS-STING pathway has been within the major interests of pharmaceutical companies, which provides high translational potential due to the possibility of repurposing already approved drugs. The potential role of the STING pathway in renal inflammation and fibrosis was also uncovered. Furthermore, it was proposed that the cGAS-STING pathway plays a key role in podocytopathy and modulating DKD. However, the contribution of this pathway towards the development of DKD and its role in observed differences in female and male diabetic subjects have not been established. Based on our pilot studies, we hypothesize that the cGAS-STING pathway contributes to the progression of DKD and sexual dimorphism, which will be explored in Aim 2. A combination of in vivo and ex vivo studies will be used to address the following Specific Aims: 1) To characterize the sexual dimorphisms in the progression of type 2 DKD; and 2) To define the contribution of the cGAS-STING signaling pathway in the progression of DKD and its contribution to sex difference.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY Atherosclerotic cardiovascular diseases (ASCVDs) are the leading cause of death in the United States and worldwide. Atherosclerosis of the arteries underlies these conditions and is characterized by chronic inflammation and inappropriate proliferation of disease contributing cells. Current medications available for the treatment of atherosclerosis merely target risk factors, not the disease-causing cells themselves. Additionally, invasive procedures such as percutaneous coronary intervention (PCI) treat arterial stenosis but can have adverse effects such as thrombosis. Therefore, despite progress in the field, site-specific and cell-selective therapies that target the cells that form atherosclerotic plaques while sparing the vascular endothelium are not available. Thus, the main objective of this research proposal is to test the feasibility of a novel messenger RNA (mRNA) therapeutic that is site-specific in targeting regions of atherosclerotic plaques only, while specifically targeting disease causing cells, reducing inflammation, and sparing the vascular endothelium. This research is highly significant to public health as ASCVDs represent a major public health issue and place an enormous burden on our Nation. I hypothesize that a combination of microRNA (miRNA) switch and small interfering RNA (siRNA) technology into a single mRNA construct will allow for a first of its kind RNA therapeutic strategy to cause atherosclerotic plaque regression and resolution. In order to test the efficacy of this new therapeutic, I will employ a series of rigorous experiments using in vitro, in vivo, and ex vivo models. Importantly, my ex vivo model uses freshly isolated coronary arteries from human hearts, giving more clinical relevance to my work and allowing me to test my therapeutic on human arteries burdened with atherosclerotic plaques. We aim to make a positive impact on the field by not only designing a novel mRNA therapeutic to treat atherosclerosis, but also, by providing evidence for the effectiveness of this unique therapeutic strategy that can be modified in order to treat other diseases. I will work towards this goal under the mentorship of my sponsor, Dr. Hana Totary-Jain, an expert in the field of CVD as well as RNA therapeutic research, at the supportive research environment of USF Health’s Morsani College of Medicine. My mentor, committee, additional advisors, and department will provide me with all the necessary training and resources needed to complete this very impactful research while also preparing me for my next career stage as a post-doctoral scholar and eventually a principal investigator.

NIH Research Projects · FY 2025 · 2023-05

Project Summary Alzheimer’s dementia (AD) affects over 35 million people worldwide, and this number is expected to triple by 2050. As early as a century ago, Alois Alzheimer noted three significant neuropathological features in the brain of AD patients: senile plaques, neurofibrillary tangles, and lipid granule accumulation. While senile plaques and neurofibrillary tangles are now widely accepted as hallmarks of AD pathology, and thus have been extensively studied, the role of lipid accumulation in AD pathogenesis has been less studied. Lipidomics is a new omics technique that can identify and accurately quantify hundreds to thousands of lipids in biospecimens in large- scale population studies. Using this technology, many lipid species have been reported to be associated with cognitive phenotypes and AD neuropathologies (e.g., amyloid-beta, tau tangles). However, several key knowledge gaps exist in this field. First, previous studies have largely focused on blood, but the brain lipidomic profile is likely different from that of blood. To date, little is known about the global lipid composition and individual lipid species that trigger neuropathologies in human AD brains. Second, of the few existing lipidomic studies in human AD brains, sample size was mostly small and results were inconsistent. Importantly, the coverage of brain lipidome (i.e., collection of all lipid species in brain) in previous studies was low, and thus many disease-related lipids have not been investigated. To date, a full spectrum of brain lipidome in relation to AD pathology is lacking, especially in large-scale epidemiological studies. Finally, the potential causal role of lipid regulation in brain aging and AD neuropathology remains largely unknown and unexplored. To address these important questions, we leverage the large-collection of postmortem brain tissue samples, the deep clinical and neuropathological phenotypes, and the rich brain omics data (e.g., genomics, epigenomics, and transcriptomics) in two community-based longitudinal cohorts of aging and dementia – the Religious Orders Study and Rush Memory and Aging Project (ROSMAP). Specifically, we will conduct the first comprehensive lipidomic profiling in 1,450 frozen dorsolateral prefrontal cortex (DLPFC) using the Metabolon’s Complex Lipid Panel (CLP), a mass spectrometry based platform that can identify and accurately quantify the absolute concentrations of up to 1,100 individual lipid species and 14 lipid classes in large-scale epidemiological studies. Our goals here are to 1) generate the first comprehensive reference map of brain lipidome in relation to Alzheimer’s dementia-phenotypes (Aim 1); 2) identify individual brain lipid species associated with AD neuropathologies and cognitive phenotypes (Aims 1 and 2); and 3) elucidate the potential causal role of altered brain lipid regulation in AD pathology (Aim 3). Such results will shed light on the mechanisms through which lipid accumulation in the aging brain affects AD pathology, and provide evidence for targeting lipid metabolism in developing novel therapeutics for AD prevention and treatment.