University Of Florida

NIH Research Projects · FY 2025 · 2023-08

The proposed fellowship plan is an interdisciplinary research project integrating training in anthropology, medicine, and implementation science. This project is supervised by Drs. Adrienne Strong, Clarence Gravlee (Dept. of Anthropology), Ramzi Salloum (Dept. of Health Outcomes and Bioinformatics), and Grant Harrell (Dept. of Community Health and Family Medicine) with the resources and support of the University of Florida (UF) College of Liberal Arts and Sciences, the UF College of Medicine and the UF Clinical Translational Sciences Institute. The proposal is designed to equip the trainee with the skills necessary to become a physician-scientist who bridges the divide between the social sciences and clinical medicine. Non-medical health factors (e.g. lack of access to transportation, insufficient housing, low socioeconomic status, geographic isolation and rurality) are known to impact a wide array of physical and mental health outcomes. However, little is known about how physicians identify and interpret patient social information, and act upon this information in support of their patient’s health and wellbeing. This proposal will focus on US outpatient primary care clinics for two reasons: primary care prioritizes holistic well-being of patients through the life course and there has been a significant push in recent years to incorporate SDH interventions into primary care clinics. Previous investigations have focused primarily on development of screening tools and limited interventions focused on special populations or specific SDH. Such investigations pay little attention to the informal methods providers and primary care clinics have developed to identify and address clinically relevant non-medical factors in day-to-day practice, activities here collectively termed “social prescribing”. Thus, an investigation grounded in interdisciplinary health services and implementation science literature, with an exploratory component to allow for the description and analysis of these as yet uninvestigated practices and beliefs relating to social prescribing is warranted. The overarching hypothesis is that patient, provider, and clinic level factors affect primary care provider conceptualization and implementation of social prescribing practices. The specific aims will lead to greater understanding of knowledge translation of social science research to clinical medicine in primary care through (1) identification of factors affecting social prescribing conceptualization among primary care providers and (2) identification of factors affecting social prescribing implementation among primary care providers. Successful completion of these studies will enhance understanding of factors affecting development, implementation, and outcomes of social prescribing interventions across care contexts. This research directly targets multiple Clinical and Health Service Research Interest Areas defined by NIMHD.

NIH Research Projects · FY 2024 · 2023-08

ABSTRACT The proposed work outlined in the training plan for this NRSA Individual Predoctoral Fellowships to Promote Diversity in Health-Related Research (F31) Award will support fourth year pre-doctoral candidate Zavier Eure throughout his graduate program at University of Florida. Mr. Eure has displayed phenomenal excellence in his academics and his learning of new lab techniques while being in the UF BMS program and currently supported by the Oral Biology Department R90/T90 Training Grant. The research and training plan for Mr. Eure will provide him a repertoire of skills needed to matriculate to the next level of his pre-doctoral training and also opportunities to further advance his career and professional development. Being awarded this F31 will offer Mr. Eure the ability to achieve his long-term goal of becoming a leader in science and provide a significant contribution to the field of biomedical research. Periodontal disease is a highly prevalent chronic inflammatory disease affecting up to 42% of adults in the US over the age of 30. Periodontal disease is orchestrated by various microorganisms that make up the subgingival microbiota. In susceptible individuals, dysbiosis of the subgingival microbiota promotes a dysregulated inflammation that causes an irreversible destruction of the soft and hard tissues supporting the teeth. Porphyromonas gingivalis (Pg) is an oral bacterium commonly associated with the microbial dysbiosis leading to periodontal disease. Pg is referred to as a pathobiont because its known to manipulate the host inflammatory milieu to facilitate disease states, however Pg can also remain in the oral cavity during healthy states. The various mechanisms behind how Pg contributes to the changing inflammatory milieu is yet to be identified. Our lab has discovered Pg sphingolipids (SLs) limit host inflammation and host cell SL transfer via outer membrane vesicles is a potential transport mechanism for Pg. The mechanism of OMV uptake by host cells is known to impact elicited inflammation and OMV composition (SL+ or SL-) plays an important role in the route for OMV uptake. Myeloid differentiation factor 88 (MyD88) is also known to be involved in the innate immune sensing of Pg OMVs and has been found to be a target for immunomodulation by Pg. I hypothesize Pg SLs facilitates an OMV uptake mechanism that limits inflammation and that SLs promote an immunomodulatory mechanism linked to targeting of MyD88. I plan to use the THP-1 human cell line and primary human macrophages to characterize the uptake mechanism of SL-containing Pg OMVs (Aim 1) and to determine the role of MyD88 in Pg SL-mediated immunomodulation (Aim 2).

NIH Research Projects · FY 2025 · 2023-08

In the past two to three decades, there has been a surge in the use, abuse, and misuse of opioids. Among opioid users, Illicit fentanyl use has become widespread in the US. Moreover, synthetic fentanyls rapidly flood the illicit drug market due to their cheap manufacturing, enhanced potency, and chemical composition that evades toxicology screening. Furthermore, black-market drug makers create new fentanyl analogs to avoid classification as illegal, to get around policy restrictions. Although drugs of abuse and HIV are entwined epidemics, it is becoming clear that Fentanyl plays an alarming role in new outbreaks. Recent reports suggest that there's an elevated risk of acquiring HIV-1 infection in those who inject fentanyl. Additionally, fentanyl is commonly detected in urine drug-screen tests in people living with HIV (PLWH). Advances in antiretroviral therapy has extended the lives of PLWH. However, as people live longer, the risk of altered neurological manifestations increases. It is also well known that drugs of abuse compound these effects of HIV. Even though it is known that opioids and their receptors are implicated in the pathogenesis of NeuroHIVs, there's essentially no information available about whether fentanyl has effects on the status of HIV infection in the CNS. In this study, we will examine the overall hypothesis that: fentanyl and novel synthetic fentanyl analogs facilitates HIV-1 leukocyte transendothelial migration, microglial HIV infection/replication and impairs the integrity and function of the BBB. We propose three specific aims to explore the hypothesis. In Aim 1, using our latest tissue-engineered microfluidic model of the human BBB, we will perform analyses of the kinetic changes in BBB permeability, transporter status and immune-endothelial interaction in response to fentanyl and other synthetic fentanyls. Since microglial is the primary refuge for HIV in the brain, Aim 2 will determine the effects of synthetic fentanyls on dynamic HIV-1 infection in the resident immune cell of the brain, the microglial. Comparisons will be made to monocyte derived macrophages. Aim 3 will examine the molecular mechanism responsible for a dysfunctional BBB by fentanyls. This proposal comprehensively addresses the key tenets of RFA-DA-23-012 and features multiple synthetic fentanyls, a translational model of the BBB, and experiments to define the underlying molecular mechanism involved. Our studies will provide important insights on how the interplay of two major pathologic factors (HIV and fentanyl) in the CNS impair the BBB and compromise the intracellular anti-HIV immunity of microglia, the key mechanisms that could significantly accelerate the development NeuroHIV.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Patient-centered communication improves health outcomes, yet is under-utilized in the pediatric emergency department (PED). The PED is a high-stress environment in which physicians and families quickly establish rapport and exchange information in brief, fragmented interactions despite never having met previously, while PED physicians engage in complex decision making with high levels of uncertainty for multiple patients concurrently. This creates barriers to patient-centered communication, which may negatively impact the quality of care provided to acutely ill and injured children. Dr. Colleen Gutman is an Assistant Professor of Pediatric Emergency Medicine at the University of Florida. Her long-term goal is to become an independently funded physician-scientist with a focus on investigating and implementing strategies to promote patient-centered pediatric emergency care. Through the support of a KL2 award, Dr. Gutman gained introductory skills in multi-center research, qualitative analysis, and health communication science. To achieve a successful transition to research independence, Dr. Gutman and her multidisciplinary mentorship team devised a career development plan that builds from that foundation. With this K23 award, Dr. Gutman will develop advanced skills in 1) the conduct of parent-engaged research, 2) the science of patient-centered communication, 3) advanced mixed methods, 4) academic leadership, and 5) scientific writing. The career development plan will be complemented by mentored research experiences. Using the NIMHD Research Framework, the proposed research seeks to define elements of the physician-parent interaction that contribute to child health outcomes. The objective is to define factors that influence the use of patient-centered communication behaviors in the PED. This is a necessary first step that will inform targeted interventions aimed at improving the health of all acutely ill and injured children. In Aim 1, Dr. Gutman will use a parent-engaged modified Delphi approach to establish physician behaviors (e.g., assumptions about parent characteristics and access to resources; communication with parents) that are critical for patient-centered communication in PED care. In Aim 2, she will collect paired questionnaires from PED physicians and parents. She will analyze physician-parent response concordance on items evaluating parent characteristics and access to resources. Through this analysis, she will assess for clinical and patient-level factors that are associated with the accuracy of physician assumptions. In Aim 3, she will video-record PED encounters to analyze the relationship between clinical and patient-level factors and physician communication. The findings will inform a multicenter R01 proposal that is fully powered to conduct a mediation analysis assessing the relative importance of the defined factors and behaviors on patient-centered health outcomes. Long term, this research will inform targeted interventions to promote patient-centered communication and improve the health of acutely ill and injured children.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Endogenous metabolism and environmental exposure can result in a battery of DNA lesions. Failure to repair these DNA lesions gives rise to genome instability and mutation accrual, which lead to accelerated aging, cancer, and neurodegenerative diseases. Abasic site (AP site) is one of the most prevalent forms of DNA damage. Although AP sites are known to be repaired by AP endonuclease in human cells, the dynamics of AP site induction and repair across the genome and the cellular factors modulating these processes remain elusive. In this application, we aim to explore how site-specific induction and repair of AP sites are affected by toxicant exposure and epigenetic alterations. We propose three specific aims to achieve our objective: 1) we will develop a DNA-protein cross-linking followed by next-generation sequencing (DPC-Seq) method for mapping, at single- base resolution, AP sites in the human genome after toxicant exposure; 2) we will explore how the formation and repair of AP sites are influenced by epigenetic state of chromatin; and 3) we will employ DPC-seq and whole- genome mutation analysis to examine how AP sites contribute to mutational signatures observed in cancer genomes. To accomplish this proposal and enable my transition to an independent academic career, I assembled a mentoring committee with expertise in animal models, bioinformatics, mutagenesis, and toxicology. The proposed research is built upon my research experience/skill sets, strong preliminary data, and the proposed mentoring plan. The outcome of the proposed research will establish a new method for mapping AP sites at single-base resolution and provide important insights into the occurrence and repair of AP sites and how they are influenced by toxicant exposure and genomic contexts. The proposed research will also reveal the roles of AP sites in cancer etiology, which will ultimately lead to better strategies for the prevention and therapeutic interventions of human cancer. Moreover, this proposal will fill gaps in my training and collect the data for my independent publications and research grant applications, thereby enabling me to transition to become an independent scientist in the field of environmental toxicology.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Nearly one-million people in North America are now living with Parkinson’s disease (PD), and that number is projected to rise to nearly 1.2 million by 2030. With advancements in neuromodulatory technologies, increasingly more of these individuals elect to undergo deep brain stimulation (DBS) surgery in order to control symptoms of the disease, including refractory tremor, medication-induced dyskinesias, and PD-associated dystonia. The two most common DBS neural targets for controlling these symptoms are the globus pallidus internal segment (GPi) and the subthalamic nucleus (STN). Recent meta-analyses have shown relative equivalence between these two sites at controlling core PD symptoms. To date, there is not conclusive evidence regarding the potential impact of DBS to GPi or STN on laryngeal-mediated functions of voice, swallowing, and cough, and consequently no guidance on whether these outcomes should be considered when selecting DBS target. Therefore, the goal of this application is to determine the impact of DBS neural target (STN versus GPi), lead location within the target, laterality, and stimulation settings on voice, swallow and cough function in people with PD. The larynx is an important player in each of these functions, and our central hypothesis is that spread of stimulation to corticobulbar fibers in the genu of the internal capsule have deleterious effects on laryngeal motor control, resulting in voice, swallow, and cough dysfunction. We have identified three specific aims for this application: 1.) To compare laryngeal function during volitional voice tasks pre-post DBS, and when DBS placement is bilateral versus unilateral for STN and GPi targets. 2.) To compare laryngeal function during volitional and induced cough tasks pre-post DBS, and when DBS placement is bilateral versus unilateral for STN and GPi targets. 3.) To compare airway safety associated with laryngeal onset, degree, and duration of maximum closure during swallowing, pre-post DBS, and when DBS placement is bilateral versus unilateral for STN and GPi targets. These hypotheses were developed based on compelling published and unpublished preliminary data. We will accomplish these aims by enrolling people with PD who are being considered for DBS surgery, and a control cohort with similar age, disease duration and severity, who are not currently being considered for DBS surgery. We will measure physiologic, functional, and quality of life parameters of voice, swallow and cough pre- and post-surgically (and at equivalently spaced time-points for the control group.) The realization of the proposed aims is significant because it will address a substantial gap in our understanding of DBS outcomes related to communication and airway protection, which are important in terms of morbidity, mortality, and quality of life for patients with PD. The translational potential to provide additional guidance to DBS surgical teams regarding whether voice, swallow or cough functions should be considered with selecting DBS target and/or laterality is high. Ultimately, the project fits squarely within the overarching goal of the research team to deliver the best possible care to people with PD.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Spatial cellular heterogeneity contributes to the complexity of diseases, therapeutic treatment, and drug response, which commonly involve the interplay between different molecular levels including genetic, epigenetic, and cellular levels. Recent technological advances of spatial technologies have enabled the elucidation of single cell heterogeneity with rich information and spatial locations that offer remarkable opportunities to understand biological processes and molecular interplays involved in disease and therapeutics. Moreover, traditional approaches mostly focus on a single type of data that cannot fully address this complexity and heterogeneity. Therefore, there is a lack of integrative approaches that leverage the strengths of data from multiple sources (e.g., genomics, epigenomics, clinical data) to achieve full insights into the pathobiology of complex disease and drug response. Given these challenges and my unique multi-disciplinary training, the overall goals of my research program are to develop a novel class of machine learning, statistical and deep learning approaches for the enhancement, prioritization and interpretation of spatially organized cells in complex tissue, to better understand the molecular mechanisms underpinning diseases and drug response, which will empower precision medicine by identifying individualized biomarkers for disease prevention, diagnosis and treatment. Specifically, in the next five years, my team will (i) develop a novel transfer learning approach to impute the transcriptomics and epigenomics profiles in spatial slices; (ii) develop a computational framework to reveal disease-associated phenotypes in spatially distributed cells, through leveraging Genome-Wide Association Studies (GWAS) studies; (iii) develop a novel domain adaptation method to predict drug responses of spatial cells, using pharmacogenomics knowledge base; (iv) develop a novel class of statistical methods for the joint analysis of spatial transcriptomics and single-cell multi-omics data, thus unveil the underlying regulatory mechanisms in diseases and drug response. In the meantime, supported by Wake Forest Comprehensive Cancer Center, we will apply the methodologies to different studies such as Brain Metastasis and Alzheimer’s Disease for novel scientific findings. We will work closely with collaborating biostatisticians and biologists to interpret the biological discoveries. Importantly, we will work with experimental labs to validate the findings. In line with our previous work, we will continue to make all developed methods into open-source software tools that are accessible and useful to the biomedical research community.

NIH Research Projects · FY 2026 · 2023-08

Summary Brain-derived neurotrophic factor (BDNF) binds to full-length tropomyosin receptor kinase (TrkB.FL), inducing its dimerization and activation. This activates many signaling cascades that cooperatively promote neuronal survival and regulate neuronal development and synaptic transmission in many brain regions. This signaling pathway is also critical for the control of energy balance, as mutations in the TrkB.FL kinase domain or BDNF lead to hyperphagia and severe obesity in mice and humans. In addition to TrkB.FL, the Ntrk2 gene produces two truncated receptors, a predominant TrkB.T1 and a minor TrkB.T2 (we use TrkB.T to refer both isoforms). TrkB.T has a short intracellular sequence and lacks the tyrosine kinase domain. Neurons mainly express TrkB.FL, whereas astrocytes only express TrkB.T. While binding of BDNF to TrkB.T1 induces Ca2+ signals and activates Rho GTPase in cultured astrocytes, it remains unclear if astrocytic TrkB.T plays a role in the control of energy balance. We generated astrocyte specific Ntrk2 conditional knockout (aNtrk2 cKO) mice where Ntrk2 deletion starts at 5 weeks of age and abolishes TrkB.T expression in astrocytes. We found that the Ntrk2 deletion in mature astrocytes blocked astrocytic reactivity in the arcuate nucleus of the hypothalamus (ARH) and gave mice total resistance to diet-induced obesity (DIO) by reducing energy intake and increasing energy expenditure. In addition to nutritional and trophic support to neurons, astrocytes contact synapses through their processes to regulate synaptic transmission by taking up neurotransmitters from the synaptic cleft and releasing gliotransmitters into the synaptic cleft. Thus, we hypothesize that TrkB.T-mediated Ca2+ signals promote astrocytic reactivity in response to high-fat diet (HFD) feeding, which in turn disrupts astrocytic support to neurons and astrocytic regulation of synapses and shifts energy homeostasis to energy surplus. Our studies will focus on astrocytes, AgRP/NPY neurons, and POMC neurons in the ARH. We propose to examine the impact of Ntrk2 gene deletion on astrocytes in the ARH (Aim 1), to determine the impact of astrocytic Ntrk2 deletion on neurons and synaptic transmission (Aim 2), to identify the site where astrocytic TrkB.T ablation blocks diet-induced obesity (Aim 3), and to determine if attenuating astrocytic Ca2+ signals can prevent obesity in HFD-fed mice (Aim 4). In conclusion, this research project will test several novel concepts, including a crucial role for TrkB.T- mediated Ca2+ signals in induction of astrocytic reactivity, an active role for TrkB.T in the regulation of energy balance, and altered astrocytic regulation of synaptic transmission in HFD-fed mice. Our studies will likely show that attenuating TrkB.T-mediated Ca2+ signals in astrocytes can be a novel and effective strategy for therapeutic interventions of DIO, the most common form of obesity in humans.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Improved methods for assessing pregnancy well-being, and assessing possible molecular and genetic correlates of poor pregnancy outcomes are currently in development. This knowledge is of limited value, however, without the ability to intervene in troubled pregnancies. The obvious difficult step is to translate knowledge to be gained into placental therapeutics to improve fetal health. Treating the placenta within the pregnant mother has several significant concerns. Placental therapeutics will need to selectively target the placenta to avoid unwanted off-target effects on maternal physiology and to ensure that treating the placenta does not result in inadvertent negative consequences for the fetus. The long-term goal is to establish safe, efficacious and placenta-specific gene therapy in a nonhuman primate in order to establish potential treatment strategies for fetal growth restriction and other pregnancy pathologies in which the placenta plays a role. Furthermore, the development of an organ-specific targeting approach in a nonhuman primate will allow the development of other disease-specific models of pregnancy in an animal model highly translatable to human. The overall objective of this application is to assess the feasibility and safety of a polymer-based nanoparticle that can be taken up by the placental syncytium and result in transgene expression and allow tracking and targeting developments for organ-specific delivery of nanocarriers. The rationale behind this proposal is that it is expected to make significant steps toward a specific, translatable delivery mechanism for therapy in the placenta and to broaden the capabilities of disease modelling in the nonhuman primate. These forward-looking and innovative studies will position University of Florida and University of Wisconsin- Madison investigators to be poised to work towards the ultimate goal, to be able to “treat the placenta”, an ephemeral organ fundamental to our existence, yet exceedingly difficult to address with therapeutic intervention, in the interest of improving pregnancy outcomes, women’s health and lifelong health. This study will also open possibilities for other organ-specific disease models to be developed in the nonhuman primate.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT The first 1,000 days (conception to age 2) have been deemed a critical period for obesity prevention yet, effective, sustainable efforts are lacking. Current home visitation programs (HVP) targeting at-risk families for other child development related issues are a potential innovative opportunity for early childhood obesity prevention. The overall goal of the project is to reduce the prevalence of overweight and obesity in children under the age of 1 year thereby reducing obesity rates of older children and adults in the long term. Prior pilot work by members of the research team demonstrated feasibility of embedding an early childhood obesity prevention (ECHO) program in an existing home visitation program (HVP+) during an infant’s first year of life. Five obesity-associated behaviors (i.e., breastfeeding, introduction to solids, limiting juice, sleep routines, screen time) were emphasized through brief interactive lessons utilizing behavior change strategies and an ecological approach by providing linkages to community resources that support healthy behaviors. The pilot program was well received by families, mothers breastfed longer, infants had fewer nocturnal awakenings, were less likely to receive juice, and had a lower weight-for-length (WFL) z-score at 12 months. However, there is a critical need for alternative and innovative, consistent, and sustainable digital delivery methods especially during times when face-to-face home visits are not feasible. Qualitative interviews were conducted with home visitors (n=27) from Florida’s (FL) Maternal, Infant Early Childhood Home Visitation (MIECHV) Program and revealed that they were highly receptive to using digital learning with at-risk families. The specific aims of the proposed research are to a) Develop, refine, and conduct usability testing of early childhood obesity prevention digital learning modules with mothers (n=30) participating in FL MIECHV; and b) Conduct a pilot RCT of a 12 month digitally-enhanced early childhood obesity prevention intervention (HVP+E), with 50 mother-infant pairs (25 HVP+E/25 standard HVP) participating in FL MIECHV to determine feasibility and acceptability of the HVP+E intervention and study recruitment, implementation and evaluation protocols; and obtain data on preliminary efficacy of the intervention on children’s WFL z-scores (primary outcome), maternal feeding practices, child sleep and screen time (secondary outcomes). Mother-infant dyads (n=50) enrolled in the Maternal, Infant Early Childhood Home Visitation (MIECHV) Program parentally or within one month of giving birth will be randomized to receive either the standard home visitation program (HVP) or the digitally delivered obesity prevention-enhanced home visitation program (HVP+E) for 1 year. HVP+E content will also be expanded to support mothers in engaging fathers and other family members in target behaviors. Mother-infant dyads will be assessed at study entry and at 6 and 12 months. If efficacious, the intervention has potential for public health impact through early childhood obesity prevention in an underserved population, and dissemination through home visitation programs nationwide.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/ Abstract Channelopathies are diseases or physiological disorders caused by the dysfunctional ion channel proteins. For example, the essential mechanosensitive channels PIEZO1 and PIEZO2 have been tightly linked to multiple diseases, such as distal arthrogryposis, dehydrated hereditary stomatocytosis, and Gordon Syndrome. There are ~100 disease alleles that have been identified in PIEZO1/2, most of which caused severe physiological disorders in cardiovascular, vestibular, neuronal, and connective tissues. Despite the electrophysiological studies in the patients’ cells indicated that these symptoms are likely due to a mechanotransduction defect, the underlying mechanisms or molecular determinants of PIEZO diseases remain largely unknown. Here, I introduce a facile and powerful in vivo system for the functional study of PIEZO; the stretch sensitive and responsive C. elegans reproductive tract. I have discovered that the dysfunctional PEZO-1 (the sole ortholog of PIEZO in C. elegans) causes severely reduced brood sizes due to the crushing oocytes in the spermatheca and poor sperm motility (3). This proposed study aims to discover the nature of the pathways and genetic interactors that enable PIEZO to respond to mechanical stimuli and coordinate mechanotransductive tissue function in vivo. Furthermore, I will identify new genetic suppressors and associated pathways in the C. elegans reproductive tract. This basic research will shed light on the understanding of channelopathy diseases caused by PIEZO dysfunction and the potential therapeutical drug target design. To achieve these goals, I will pursue three specific aims: The first aim is to identify novel genetic interactors of PEZO-1 in C. elegans. A combination of genetic screens and biochemical assays will be used to achieve this aim. I expect that completing the proposed aims will establish the C. elegans reproductive system as a simple and genetically tractable model to elucidate PIEZO biological functions and to better understand the molecular mechanisms of PEZO-1 activity. The second aim is to determine whether inter-tissue signaling pathways (such as the sex hormone prostaglandin) is affected in pezo-1 mutants. To achieve this aim, I will perform genetic and biochemical assays to determine whether PEZO-1 contributes to prostaglandin synthesis and secretion, which are essential for sperm attraction. The final aim is to identify target tissues and relative contribution of PIEZO disease alleles to intracellular Ca2+ homeostasis and signaling. To achieve this aim, I will generate a set of the tissue-specific Ca2+ indicators to quantify the calcium influx in each mutant. These studies should lead to a comprehensive delineation of genes that interact with pezo-1, and new pathways that involve mechanotransduction. This research will also shed light on the molecular mechanisms of the genetic diseases caused by PIEZO dysfunction. Overall, this K99/R00 award will strengthen my research skillset and facilitate my transition into an independent researcher in the field of genetics, development, mechanobiology, and translational science of human rare diseases.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The global epidemic of obesity poses formidable challenges to human health associated with risks of chronic diseases such as type 2 diabetes and cardiovascular disease. Obesity is driven by systemic energy surplus with excessive fat deposition in white adipocytes (WAs). Brown adipocytes (BAs) that are specialized in dissipating energy through non-shivering thermogenesis and increasing energy expenditure represent a potential therapeutic target in obesity treatments. However, there is a critical knowledge gap in fully understanding the molecular mechanisms underlying BA function. Our long-term goal is therefore to explore the novel molecular regulation of BAs in order to facilitate the development of therapeutic strategies to combat obesity. By performing quantitative mitochondrial proteomics, we recently identified Family With Sequence Similarity 210, Member A (FAM210A), an uncharacterized protein, as a critical regulator of thermogenesis in brown adipose tissue (BAT). Emerging studies reported a potential role of FAM210A in regulating skeletal muscle growth and pathological cardiac remodeling, however the function of FAM210A in thermogenic BAs is completely unknown. Using newly developed Fam210a floxed mice, we provided strong preliminary data supporting an essential physiological role of FAM210A in BAT thermogenesis. We showed that 1) FAM210A is highly induced by cold and coupled with cold-induced mitochondrial cristae remodeling; 2) Adipocyte-specific knockout (KO) of Fam210a in mice leads to the whitening of BAT and cold intolerance; 3) Loss of Fam210a causes metabolic dysfunction of BAT; 4) Fam210a KO induces the depletion of mitochondria and disruption of cristae architecture. Based on this exciting discovery, the overall goal of this proposed study is to elucidate the cellular and molecular mechanisms by which FAM210A functions in BAs to regulate thermogenesis, and investigate the physiological role of adipose FAM210A in systemic metabolism. To achieve this goal, we propose three specific aims. In Aim 1, using mice and cells with inducible deletion of Fam210a in adipocytes, we will evaluate the regulatory role of FAM210A in mitochondrial metabolism, synthesis, and degradation in BAs in vivo and in vitro. Employing high-resolution three-dimensional imaging systems, we will dissect the function of FAM210A in controlling cold-induced cristae membrane remodeling. In Aim 2, we will define the molecular mechanisms through which FAM210A regulates mitochondrial homeostasis and cristae remodeling in BAs via the identification and characterization of interacting protein partners that enable FAM210A’s regulation of cristae-shaping protein. In Aim 3, we will utilize our unique loss- and gain-of-function mouse models to test whether FAM210A is required and sufficient to increase energy expenditure and systemic metabolism so as to ameliorate diet-induced obesity and metabolic dysfunction. Upon completion of the proposed studies, we expect to establish the functional role of FAM210A as a novel regulator of mitochondrial dynamics and BA thermogenesis, thus identifying a new potential therapeutic target for the obesity epidemic.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Maternal immune activation (MIA) during prenatal or postnatal development significantly increases the risk for offspring neurodevelopmental disorders (NDDs) later in life. Growing evidence suggest that regardless of the MIA stimuli (infectious or environmental), offspring exhibit an enhanced risk for lifelong neuropathology defects ranging from reduced brain volume to alterations in neurocircuit organization. The brain extracellular matrix- containing chondroitin and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs) are key regulators of brain development and can be biochemically altered by neuroimmune responses. Defects in CS/DS-GAG abundance and/or sulfation patterning (4S (CS-A), 2S4S (CS-B/DS), 6S (CS-C), 2S6S (CS-D), 4S6S (CS-E), 0S (CS-O)) result in the manifestation of similar neuropsychiatric behaviors as reported in offspring affected by MIA, but whether and how MIA affects offspring brain matrix is unknown. By employing a novel laser capture microdissection coupled mass spectrometry methodology (LMD-LC-MS/MS), our Preliminary Data provide the first evidence for inter- and intra-regional differences in CS/DS-GAG sulfation pattern differences throughout the developing mouse and non-human primate (NHP) brain. Specifically, the hippocampus exhibits a significant increase in both developmental 6S (CS-C) and 2S6S (CS-D) isomers compared to the cortex, implying that the hippocampus remains developmentally plastic long after the maturation of adjacent regions. Moreover, we show that infectious Zika virus MIA during gestation in NHPs decreases the abundance of the developmental 2S6S (CS-D) axonal growth factor attractant isomer in the hippocampus, suggesting stunted neurocircuit formation after infectious MIA, while the non-infectious maternal high fat diet (mHFD) MIA during lactation in mice decreases the abundance of the developmental 6S (CS-C) plasticity isomer in the hippocampus, suggested accelerated early maturation of hippocampal neurocircuits in response to non-infectious MIA. The implication that both infectious and non-infectious MIA insults influence the spatiotemporal regulation of brain CS/DS-GAG sulfation patterns fits a global interconnecting theory linking a range of MIA insults with changes in offspring brain neurodevelopment through re-coding of CS/DS-GAGs. From these results, we propose to 1) determine how MIA exposure affects spatiotemporal expression of offspring CS/DS-GAGs and link these changes to NDDs later in life, 2) mechanistically investigate how these MIA-induced changes in offspring CS/DS-GAGs influence glycan- protein interactions involved in neurodevelopment, and 3) engineer a state-of-the-art nanopore sequencing technology capable of single-molecule sequencing of biological CS/DS-GAGs to discover glycan-protein binding elements. This multidisciplinary proposal has important translational potential to clarify how MIA exposure leads to neuropsychiatric illness through changes in CS/DS-GAG sulfation patterning during childhood neurodevelopment and provides valuable targets in the prevention and treatment of mental health diseases.

NIH Research Projects · FY 2026 · 2023-08

Abstract The long-term goal of this study is to determine how vagus nerve stimulation (VNS) regulates basal dopamine transmission and methamphetamine regulation of dopamine neurons. Methamphetamine abuse is a major public health issue around the world, yet there are no effective pharmacotherapies for the treatment of methamphetamine addiction. Methamphetamine is a potent psychostimulant that increases extracellular dopamine levels by targeting the dopamine transporter (DAT) in the midbrain and striatum. In the previous cycle of this grant, we shown that methamphetamine competes with the DAT-mediated dopamine uptake, increases dopamine efflux via the DAT, increases the DAT mediated inward depolarizing current leading to increased firing activity of dopamine neurons. Methamphetamine increases Ca2+ levels in the dopamine neurons that enhances both action potential dependent and independent dopamine release (i.e., dopamine efflux). We also found that neuronal depolarization induces DAT internalization leading to decreased dopamine and methamphetamine uptake. Multiple studies have shown that VNS increases dopamine levels in the midbrain region and reduces cocaine seeking behavior, albeit with an unknown mechanism via a multi-synaptic connection between vagus nerve and midbrain region. We found that optogenetic stimulation of vagal sensory neurons innervating the upper gastrointestinal tract depolarizes dopamine neurons and increases basal firing activity of midbrain dopamine neurons lasting for at least 30 minutes. Histological analyses revealed a reduction in somatodendritic DAT in the c-fos positive neurons. These data are consistent with our previous report showing neuronal depolarization induces DAT internalization, that can reduce the efficacy of methamphetamine regulation of dopamine transmission. Our preliminary data also revealed that optogenetic VNS increases extracellular dopamine levels that does not reach ceiling levels, but it reduces the methamphetamine stimulation of dopamine neurons, by depolarizing dopamine neurons, reducing DAT levels. These preliminary data and the literature support the overarching hypothesis that VNS increases basal dopamine levels by depolarizing dopamine neurons, reducing DAT levels, and decreasing methamphetamine-stimulation of dopamine transmission thereby attenuating methamphetamine’s behavioral and cellular responses. Our proposed studies will determine the underlying cellular mechanism for VNS-regulation of dopamine transmission and methamphetamine-induced responses.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY To find therapies for altering the course of age-related diseases, experimental methods that discriminate between normal and pathological aging processes are needed. Nowhere is this need more urgent than in the quest for effective treatments for Alzheimer’s disease (AD). Despite decades of research, AD remains a debilitating, progressive, and ultimately fatal dementia with few and ineffective disease-modifying treatment options. The vast majority of cases (~95%) are sporadic, with no known cause aside from advanced age. As the population over age 65 grows, the burden of AD is destined to grow in lockstep. Avoiding this fate requires rethinking why existing efforts have been ineffective. Although AD is a disease of aged human brains, many AD studies fail to parse aging from disease. We have developed a system that lets us model aspects of human age in vitro: induced neurons (iNs). Once matured, neurons of the brain never again replicate their genome or divide. As such, post-mitotic neurons downregulate cell-cycle mediated metabolic pathways used for the synthesis of DNA building blocks, deoxyribonucleotides (dNTs) and become reliant on the salvage of ribonucleotides (rNTs). I asked if these fundamentally important homeostatic processes are maintained over the human lifespan or in AD. Multiple experimental approaches on iNs derived from 13 sporadic AD (sAD) patients against 13 cognitively normal, age-match individuals (CN), revealed dramatic differences in NT pools, with a significant increase in rNTs in CNs relative to young healthy iNs. This effect was even further exacerbated in AD iNs. I ask here if the dramatic increases in rNTs result in increased ribo-substitution in nuclear and mitochondrial DNA, thus promoting increased DNA damage and bioenergetic dysfunction in age and AD. Further, I observed significant differential expression of key cell cycle and metabolic genes such as CDK1 and RRM1, which I have confirmed at the protein level. Our proposal, for the first time, incorporates long noted pathological observations of chromosomal instability and presence of cell-cycle markers in AD into a testable model system that provides a mechanistic basis for the development of sporadic AD. In addition to the critical scientific advances this proposal aims to achieve, I will also contribute new powerful new tools and datasets to the fields of aging and AD. I have adapted our recently developed repair-seq technology to quantify ribo-substitution in aged and AD iNs. I will further map epigenetic drift over the human lifespan in neurons and quantify the neuronal capacity to generate new methyl groups for non-genetic regulation of transcription.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT As the leading cause of numerous preventable diseases, smoking results in half a million premature deaths each year in the US and another 16 million American adults living with a serious illness. Indeed, about half of smokers will die of smoking-related problems if they do not quit. Most smokers are aware of such deleterious health effects and have the intention to quit. Tobacco cessation, however, is very challenging, partly due to abstinence-associated stress and insomnia. Current tobacco cessation medications are not designed to address these problems, which have contributed to their limited success in enabling tobacco cessation. There are currently about 34 million American adult smokers and the number is not expected to decrease significantly in the near future. Novel interventions are thus urgently needed that would resolve these challenges, which may significantly improve the success rate of tobacco cessation. On the basis of its traditional history and our preliminary data, kava is such a candidate. Kava is a traditional beverage consumed among the South Pacific Islanders for relaxation, stress reduction, and sleep improvement. It is also marketed as a dietary supplement in the US. Incorporating rigorous safety measures, we completed a pilot trial among active smokers with a one-week ingestion of a kava supplement. The results for the first time revealed kava’s potential in enabling tobacco cessation with promising effects on a panel of biological signatures associated with tobacco use, stress, and sleep. The main goal of this R33 application is to replicate the effects of kava on the biological signatures of tobacco use, stress, and sleep in addition to its compliance and safety among smokers. We propose to perform a double-blind randomized placebo controlled two-arm trial among 76 smokers with intention to quit, who will consume AB-free kava at a dietary supplement dose or placebo, 3 times per day for 4 weeks with two follow-ups. Aim 1 will evaluate the compliance and safety of AB-free kava use among smokers and assess changes in smoking behaviors. Aim 2 will quantify a panel of non-invasive translatable biomarkers to objectively evaluate AB-free kava’s holistic effects on biological signatures associated with tobacco use, stress, and sleep. We hypothesize that AB-free kava is a novel and promising intervention to facilitate tobacco cessation via its holistic effects in managing stress and insomnia associated with abstinence. If the results from this study support our hypothesis, kava could emerge as an affordable and accessible dietary supplement candidate for tobacco cessation.

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY Non-Communicable Diseases (NCDs), including cardiovascular disease, hypertension, central obesity, type 2 diabetes mellitus and respiratory disease, are responsible for 80% of adult deaths annually, and are responsible for having the greatest impact on health adjusted life expectancy and quality of life. Fetal growth restriction (FGR; estimated fetal weight <10th percentile), which occurs in up to 10% of pregnancies, is associated with increased risk of developing NCDs later in life. This is potentially because FGR results in developmental programming of fetal tissues and organs in order to adapt to the adverse conditions resulting in FGR, which persist into adulthood but ultimately predispose physiological and metabolic dysfunction. We have developed the use of a polymer-based, nanoparticle that facilitates non-viral gene delivery specifically to the placenta. Our placenta-specific, nanoparticle gene therapy is capable of increasing expression of human insulin-like growth factor 1 (hIGF1) in multiple animal and human placenta models. Importantly, our nanoparticle gene therapy is proven to be safe to both mother and fetus. We have consistently demonstrated that treatment increases placental glucose and amino acid transporter, and growth factor expression in diverse models of FGR (surgically-induced, genomic manipulation, maternal nutrient restriction (MNR)) because IGF1 is central to most mechanisms responsible for FGR associated with placental dysfunction, and a major regulator of placental and fetal growth and development. This proposal aims to 1) determine the impact of placental nanoparticle gene therapy treatment on developmental programming in fetal liver and kidney in late pregnancy, in the proven guinea pig MNR model of FGR, 2) Identify the mechanisms by which manipulating placenta signaling with nanoparticle gene therapy affect communication with fetal liver and kidney cells in human cell culture models, and 3) investigate the long-term impact of placenta-specific nanoparticle gene therapy on offspring liver and kidney physiology and metabolic health. Preliminary investigations confirm that placenta-specific, nanoparticle gene therapy increased fetal weight in preexisting FGR using the guinea pig MNR model. Furthermore, short-term placenta-specific nanoparticle gene therapy normalizes changes associated with FGR in fetal liver gene expression and kidney collagen deposition, hereby establishing a model in which further investigations into developmental programming of fetal organs can be investigated. This proposal is innovative and significant as it utilizes a nanoparticle technology currently being trialed in the treatment of cancer, but in the setting of reproductive medicine, thus generating knowledge that will inform clinical innovation in order to set the foundation for a healthy pregnancy and lifelong wellness.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Myocardial infarction (MI) is a leading cause of cardiovascular disease and death. After an MI, limited regeneration occurs, and instead, inflammation and scarring cause the affected myocardial tissue to turn fibrotic and thin. This tissue remodeling results in abnormal tissue mechanics and impaired cardiac function, often leading to heart failure and death. Therefore, a promising therapeutic approach is to reduce inflammation and the adverse tissue remodeling of the infarct zone. Unfortunately, many emerging drug candidates to achieve these goals require prolonged dosing over multiple weeks, greatly limiting their clinical translation. To overcome this challenge, I propose the development of a hydrogel that is catheter-injectable and enables the one-time injection of a drug payload into the myocardium for long-term release. This multidisciplinary project merges my expertise in drug delivery, polymeric materials, and cardiovascular bioengineering. Specifically, I propose a nanoparticle-based, therapy-eluting gel that will be retained within the contractile myocardium to locally deliver the chosen therapy at a controlled rate. This hydrogel will address two challenges in cardiovascular therapies 1) retention in the myocardium due to the mechanically active heart and 2) delivery of a sustained therapeutic dose that preserves the bioactivity in the harsh environment of the infarct zone. In this project, I propose to deliver two potential therapeutics investigated in clinical trials to address inflammation and adverse remodeling. Anakinra delivered daily through subcutaneous injection has emerged as a promising candidate to reduce inflammation and prevent cardiomyocyte apoptosis after MI. Fresolimumab has potential to mitigate heart failure after MI by preventing fibroblast activation into myofibroblasts and thereby limiting fibrosis. However, to elicit a therapeutic effect, these drugs must be present for an extended duration via multiple daily injections. Therefore, novel therapeutics like Anakinra and Fresolimumab are limited in efficacy and clinical use and would greatly benefit from materials science approaches that would reduce the need for painful daily injections by enabling them to be delivered locally within the myocardium in a reservoir that can protect their bioactivity, limit their off-target effects, and offer tunable release kinetics that can match the therapeutic window of the chosen drugs. In the K99 mentored phase of this grant, I will develop the catheter-injectable hydrogel and demonstrate retention within the myocardium (Aim 1), tailor the release kinetics of the nanoparticles to achieve both rapid and sustained payload delivery of Anakinra (Aims 2), and demonstrate the therapeutic effect in a preclinical rat model of MI (Aim 3). In the R00 phase, the modular hydrogel technology will be expanded to include a second type of nanoparticle to enable the combinatorial release of Anakinra and Fresolimumab (hydrophobic and hydrophilic drugs, respectively) (Aim 4) and improve heart function quantitatively after an MI by preventing adverse remodeling in a rat preclinical model (Aim 5).

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Muscular dystrophies (MDs) are heritable neuromuscular diseases that cause progressive weakness and loss of muscle as regenerative processes fail to adequately respond to progressive muscle damage. This ultimately results in the replacement of functional musculature with a pathological extracellular matrix (ECM) composed of fibrosis and fat, known as a fibro-matrix, which is a prominent feature of the most common of the MDs, Duchenne MD (DMD). Effective therapeutics to combat fibro-matrix development and facilitate muscle regeneration are a major unmet clinical need for MD patients, however, the mechanisms responsible for these issues are not well understood. This project investigates cellular mechanisms contributing to the failed regeneration and fibrosis development in dystrophic muscle. Specifically, NAD(P)H oxidase 4 (NOX4) has been identified as an anti- fibrotic and pro-regenerative target in muscle. This was demonstrated by marked reductions in muscle fibrosis and beneficial muscle remodeling of severely dystrophic muscle that models DMD, where NOX4 was targeted using genetic and pharmacological approaches. It is hypothesized that NOX4 expression in myofibroblasts, cells that produce ECM following tissue injury, contributes to muscle fibrosis by preventing myofibroblast clearance following muscle regeneration. This phenomenon is known as myofibroblast persistence. The experiments of this project will rigorously investigate the mechanisms leading to and the pathological consequences resulting from the development of myofibroblast persistence using innovative genetic models, in vitro assays, and transcriptomic analyses. Aim 1 will investigate myofibroblast dynamics in dystrophic skeletal muscle using myofibroblast fate-mapping and conditional ablation of NOX4. Myofibroblast persistence will be assessed in a newly-developed assay using cells isolated from dystrophic muscle, and several in vitro assays will be employed to investigate myofibroblast behavior during differentiation and development of persistence. Aim 2 will evaluate the influence that myofibroblasts exert on gene expression and cellular behavior of myogenic, fibroblastic, and immune cells in dystrophic and regenerating muscle. In vitro models will be used to discern physical versus diffusible cues responsible for these myofibroblast-driven phenotypes. The ultimate goal of the current project is to define the pathological consequences of myofibroblasts in chronic muscle disease and provide solid mechanistic insight responsible for the efficacious impact of NOX4-targeting as a beneficial remodeling therapeutic strategy for the treatment of MDs and, potentially, other forms of muscle pathology.

NIH Research Projects · FY 2024 · 2023-07

Breast cancer remains a major killer of women due to the ineffectiveness of current drugs against metastatic and drug-resistant cancers. Additionally, African American (AA) women suffer disproportionately from breast cancer mortality in part because they develop the aggressive Triple- Negative Breast Cancer (TNBC) subtype more frequently than other ethnic groups. Thus, agents effective against drug-resistant and metastatic cancers and TNBCs may improve the survival of breast cancer patients. These aggressive cancers evade cell death through a variety of mechanisms including overactivation of the pro-survival HER-family of Receptor Tyrosine Kinases (RTKs), including EGFR/HER1, HER2, and HER3 (HER1-3), and inactivation of pro-apoptotic signaling. Tumors expressing HER1-3 are difficult to treat due to the partial redundancy among these receptors, their oncogenic signaling as heterodimers, and their ability to aberrantly heterodimerize with non-HER RTKs such as MET and IGF1R. Thus, resistance to current HER-targeted agents is a significant clinical problem. Defective cancer cell apoptosis can result from inactivation of the TNF Receptor Apoptosis Inducing Ligand (TRAIL)/Death Receptor 4/5 (DR4/5) pathway, which selectively kills cancer cells, while not affecting normal cells. Tumor resistance to TRAIL and other DR4/5 agonists results primarily from poor pharmacological properties of the agonists and the ability of cancer cells to downregulate DR4/5. Consequently, agents that could inactivate the EGFR/HER2/HER3 signaling axis and upregulate and activate DR4/5 independently of the TRAIL ligand may be efficacious against breast cancers unresponsive to current medicines. Disulfide bond Disrupting Agents (DDAs) are a new class of anti-cancer agents that induce regression of primary tumors and metastatic lesions of drug-resistant patient-derived tumors in animal models. In addition to the structural uniqueness of DDAs, recent studies indicate that DDAs are the first identified active site inhibitors of the Protein Disulfide Isomerases (PDIs) ERp44 and AGR2. Further, DDA inhibition of the PDIs ERp44, AGR2, and PDIA1 alters the disulfide bonding of HER1-3 and DR4/5, resulting in HER1-3 downregulation, DR5 upregulation, and disulfide bond-mediated oligomerization and activation of DR4/5. The objective of the current project is to move DDAs toward clinical trials. The two Specific Aims proposed to achieve this objective are to 1) optimize DDA pharmacological properties and dosing for future IND-enabling studies, and 2) validate biomarkers to predict tumor sensitivity to DDAs and to monitor target engagement, and thoroughly evaluate any adverse effects of DDAs on normal tissues or animal health. Based on their unique mechanisms of action and preclinical efficacy, we expect DDAs to benefit breast cancer patients with treatment-refractory breast cancers.

NIH Research Projects · FY 2025 · 2023-07

Due in part to recent advances in screening and treatment, the 5-year relative survival rate for patients with early-stage non-small cell lung cancer (NSCLC), the leading cause of cancer death worldwide, continues to increase each year. The uptake of guideline-recommended computed tomography (CT) imaging surveillance semiannually for 2 years and annually for up to 5 years following curative-intent therapy is increasing rapidly in the U.S., despite unclear evidence regarding its benefit in reducing mortality. Furthermore, clinical trials of CT imaging surveillance have not been reported among U.S. populations with NSCLC. This gap in research is alarming and portends a low quality of evidence in clinical guidelines. Currently, no comprehensive lung cancer surveillance data source exists that catalogs real-world lung cancer surveillance utilization patterns and downstream outcomes, both of which are necessary to develop evidence-based recommendations for surveillance following curative-intent therapy. This project, Advancing Precision Lung Cancer Surveillance and Outcomes in Diverse Populations (PLUS2), will create this unique data source to study, understand, and optimize lung cancer surveillance and downstream outcomes. Building on the extant infrastructure and preliminary data from the lung cancer screening registry of the PCORI- and NCI-funded OneFlorida+ Clinical Research Consortium, a network of community practices that serve Florida, Georgia, and Alabama, PLUS2 will leverage multilevel data from electronic health records, claims, and system-level factors for patients with early-stage NSCLC who have completed curative-intent therapy (n~27,217; median age 70) from 2012-2022 (retrospective cohort) and 2022-2025 (prospective cohort). The overarching goal of the project is to evaluate the comparative effectiveness of lung cancer surveillance strategies, principally semi-annual versus annual CT surveillance, in relation to long-term outcomes among diverse patients with early-stage NSCLC within the U.S. population. By generating previously unavailable real-world data from NCI’s Lung Cancer Intervention and Surveillance Modeling Network (CISNET) for use in validated simulation models, this proposal responds directly to calls to improve patient-centered decision-making in lung cancer surveillance candidates for whom the net benefits of surveillance are currently uncertain. This study is foundational for lung cancer surveillance practice change.

NIH Research Projects · FY 2025 · 2023-07

ABSTRACT The human breast is a highly organized, complex organ that consists of an epithelial tissue surrounded by stroma that regulates its proliferation, differentiation, and survival. Stroma is responsible for sustaining normal breast tissue structure and function via a variety of signaling mechanisms that control and regulate normal processes and suppress malignant transformation. The role of stroma in breast tumor initiation, progression, and responsiveness to treatment as well as potential utility for targeted therapies has been widely discussed in recent reviews. While cancer tissue stroma has been widely explored, the evidence of stromal contributions to early stages of carcinogenesis is extremely limited. To fill these gaps, we propose a conceptually and methodologically novel investigation that will focus on benign breast disease (BBD) and high mammographic breast density (MBD), strong risk factors both independently associated with increased breast cancer (BCa) risk, which presents a unique opportunity for studying early changes in the breast and elucidating underlying molecular mechanisms. The study will be conducted by an interdisciplinary team of experts in BCa epidemiology, breast pathology/image analysis, BCa biology, and biostatistics, with a history of long and productive collaboration. We will use data and breast biopsy samples from three established prospective cohorts (Nurses’ Health Study, Nurses’ Health Study II, and Women’s Health Repository) to address the following aims: (1) prospectively examine associations of reproductive risk factors (e.g., parity, age at first birth) with expression of stromal markers (αSMA, FAP, MMP14, TNC, and S100A6) in benign biopsy samples from cancer-free women (n~1,350); (2) examine associations of stromal markers with MBD (n~1,350); and (3) examine associations of stromal markers in women with a previous benign biopsy and the risk of subsequent BCa in a nested case-control design (~400 cases/~975 controls). This proposal leverages established tissue resources, use of validated multiplex immunoflourence for stromal markers, and automated image analysis for MBD assessment. Understanding the associations of BCa risk factors with stromal markers will advance our knowledge on its role in breast carcinogenesis in epidemiologic studies. We will generate the first comprehensive data on the stromal markers’ expression in non-cancer breast and will identify markers that could significantly advance future risk prediction. Stromal activity is potentially modifiable via a variety of targeted therapies; if our project successfully demonstrates an association between stromal markers in benign breast tissue and increased BCa risk and/or high MBD, these findings could eventually translate into pharmaceutical interventions aimed at primary BCa prevention in high-risk women with high MBD and/or BBD. Importantly, these findings would apply to a large segment of women undergoing routine biopsies and those with high MBD in whom novel prevention strategies, improved risk prediction, and tailored clinical management are urgently needed.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Gene expression, the flow of genetic information from DNA to messenger RNA (mRNA) to protein, involves delicate regulation by a group of small RNAs named microRNAs. MicroRNAs can inhibit gene expression by binding to mRNAs and prevent them from being translated into proteins. MicroRNA levels in cancer cells are usually different from the microRNA levels in healthy cells, leading to differential expression of certain cancer-related genes. Controlling microRNA levels therefore offers a promising target for cancer treatment. Recently, we found that when T-cell acute lymphoblastic leukemia (T-ALL) cells are treated with dexamethasone, a glucocorticoid steroid commonly used in leukemia chemotherapy, two highly related and pro-cancer microRNAs (miR-221/222) are degraded by their target mRNA (BIM). This is surprising because microRNAs usually control the levels of their targets, but not the other way around. This is also exciting because it represents an emerging gene regulation mode carried out by the mRNAs and opens up strategies for cancer intervention. In this proposal, we first aim to understand how BIM mRNA triggers miR-221/222 degradation. Our second aim is to explore how miR-221/222 degradation enhances killing of T-ALL cells during chemotherapy. In the final aim, we will develop an innovative biochemical and computational protocol to globally identify sequences in different target mRNAs that can induce miRNA degradation in T-ALL patient samples. Collectively, our efforts will examine a new mechanism of gene regulation in T-ALL, in which mRNAs counteract microRNAs. Because resistance to glucocorticoid is a serious limitation for T-ALL chemotherapy, elucidating the underlying mechanism of resistance may provide the basis for improving current therapeutic interventions. Given that we have discovered a potentially widespread occurrence of the mRNA-induced microRNA degradation pathway in cancer, identifying the mRNAs that can degrade miRNAs and the proteins involved in this process would help develop new therapies to combat T-ALL.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Mechanotransduction is the process by which cells sense and transduce extracellular mechanical stimuli into intracellular signaling and gene expression. Mechanotransduction is ubiquitous across diverse organisms and has significant influences on cell function and behavior, in parallel with chemical and genetic signal transductions. The mechanics of microenvironments mediate mechanotransduction through cell-microenvironment interactions and its mis-regulation is at the heart of various pathologies. A major knowledge gap in the field is how mechanical stimuli from microenvironments are transduced into cellular signaling and what the relationships are between microenvironmental mechanics, cell signaling, gene expression, and cell functions. We recently discovered that multiple electrically non-excitable epithelial cell lines can initiate and propagate spontaneous long-distance intercellular calcium waves (ICWs), when cells are cultured in 2D/3D soft microenvironments, but not in stiff ones. Because calcium regulates a broad range of essential cell functions, our findings uncover an unprecedented mechanotransduction nexus between microenvironmental mechanics and diverse cellular signals. We hypothesize that the cellular actomyosin contractility that is regulated by soft microenvironments (10s kPa) promotes the initiation and propagation of the long-distance ICWs. In this R35 grant, we propose a cross-disciplinary research program that systematically elucidates this novel mechanotransduction process and establishes a framework to bridge the knowledge gap. This will be accomplished by leveraging innovative approaches such as genetically encoded fluorescent calcium sensors, CRISPR imaging, high-throughput cell selection, and algorithms to pursue two interrelated research themes. The first theme is the identification of regulatory mechanisms that initiate the calcium waves by active modulations and live-cell imaging of contractility- associated proteins. The expected results will provide important molecular insights of the link between mechanotransduction and the ICWs. The second theme is the dissection of mechanisms through which calcium waves enhance cell migration and achieve biological functions by manipulating ICWs and investigating the full transcriptome profiles. The results will advance our understanding of the relationships between microenvironmental mechanics, cell signaling, gene expression, and cell functions. We envision that the fundamental principles uncovered in this project will apply broadly to various cell systems and physiological functions. The long-term objective of our research program is to establish a mechanistic foundation of mechanobiology and promote the creation of therapeutic strategies which leverage these principles. The success of this proposal will enable my group to embark in a long-term research direction to tackle a variety of critical and challenging questions regarding the interplay between mechanotransduction and cell signaling.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder defined by social deficits and restricted and repetitive behaviors. These behaviors have been associated with impaired sensory processing and sensorimotor integration. In addition, ASD is often comorbid with intellectual disability (ID) and cognitive impairment. This is especially true of ASD diagnoses associated with one of several major ASD risk genes. While it is well established that sensory processing is critical for cognitive function in general and learning in particular, it is not clear how deficits in sensory processing in ASD may contribute to cognitive and learning impairments in patients. I hypothesize that learning impairments in ASD patients stem from sensory processing deficits leading to a disruption in cortical functional connectivity critical for learned behaviors. I will test this idea by using a mouse model of major ASD genetic risk that shows a tactile instrumental learning impairment. Importantly, learning deficits require the function of the ASD risk gene in cortical excitatory projection neurons (see preliminary data) highlighting the importance for cortical processing in this phenotype. In addition, these animals have a reduced cortical response to touch. In Aim 1, I will assess the impact of the ASD risk gene on sensory processing in dorsal cortex by monitoring calcium dynamics as mice receive passive sensory stimulation. In addition, I will manipulate the ASD risk gene in sensory cortex to test its autonomous control of calcium dynamics and connectivity. I expect that the ASD risk gene model mice will have reduced responses in sensory cortex and disrupted functional connectivity to downstream regions in dorsal cortex. In Aim 2, I will monitor dorsal cortex calcium dynamics as mice perform an instrumental learning task to test the idea that the major ASD risk gene causes altered dynamics of cortical functional connectivity. Furthermore, I will determine to what extent putative mesoscale connectivity deficits in the mouse model predict learning impairments in the task. Lastly, I will specifically manipulate the ASD risk gene in sensory cortex to tests its autonomous role in regulating functional connectivity during learning. The impact of this proposal will be to understand the neurobiological underpinnings of impaired learning related to a major ASD risk gene, which may serve to inform the development of treatments for cognitive deficits in ASD patients. In conclusion, I am an excellent candidate for a National Research Service Award Fellowship because of my background in ASD risk gene research and the training in cutting-edge systems neuroscience tools provided by the Rumbaugh lab. Altogether, the training and experiments proposed here will enable me to further our understanding of ASD and lay a strong foundation for my career goal of running an independent research laboratory.