University Of Florida

NIH Research Projects · FY 2026 · 2023-02

Project Summary/Abstract We have developed innovative new approaches of administering immunotherapeutic enzymes that robustly directs immune cells away from chronic inflammation toward homeostasis through metabolic programming. Innovative fusion protein enzyme constructs in both tissue-anchored and circulating forms are investigated, quantifying cellular mechanisms of action in order to determine if different administration routes offer advantages. There is considerable need for new therapeutic options for the chronic auto-inflammatory disease, psoriasis, which irreversibly damage epidermal tissues.

NIH Research Projects · FY 2026 · 2023-02

ABSTRACT Smith-Kingsmore Syndrome (SKS) is a newly discovered genetic disorder caused by mutations in the mechanistic target of rapamycin (MTOR) gene. MTOR functions to coordinate intracellular energy levels with cellular homeostasis and growth. MTOR deregulation is implicated in various pathological conditions, including brain dysfunction. A notable example is tuberous sclerosis, in which MTOR hyperactivation due to Tsc1/2 mutation causes autism, epilepsy, and benign tumors in the brain. Clinical features of SKS include macrocephaly, epilepsy, seizure, intellectual disability, autism spectrum disorder, and developmental delay. Our recent studies also revealed new aspects of SKS, including sleep/wake disruption, hyperphagia, hyperactivity, and self-aggression, all indicative of homeostatic imbalance and hypothalamic dysfunction. Our research has a central focus on the circadian and sleep systems. Sleep/wake disturbance is prevalent in SKS patients and represents a top concern of patients and caregivers. Sleep insufficiency reflects homeostatic imbalance in the brain, exacerbating disease states. Our long-term goal is to advance the understanding of the pathophysiology and mechanisms of SKS. Our central hypothesis is that chronic activation of MTOR in SKS disrupts cell physiological homeostatic, leading to disruption of sleep/wake and other functions. To test this hypothesis, we will generate cellular and mouse SKS models and investigate how the pathogenic SKS variants affect MTOR activity, circadian rhythms, sleep/wake homeostasis, and other behavioral and cognitive functions. We hope to provide proof of principle that a better understanding of causal mechanisms, beyond genotype, enables precision medicine treatment strategies. The MTOR inhibitor rapamycin impacts both sleep time/phase and quality. Notably, low-dose rapamycin, optimized for specific alleles to normalize MTOR activity, was able to restore the patient’s sleep/wake pattern, while improving other clinical features. We will explore rapamycin regimens and test the hypothesis that by normalizing MTOR activity, allele-specific low-dose rapamycin can improve sleep/wake and other functions. As rapamycin impacts sleep time and quality and other behavioral and cognitive functions, we hope to expand the concept that sleep/wake function represents a novel neurophysiological biomarker for rapamycin dosing, MTOR activity and CNS homeostasis. This research will lay the groundwork for future mechanistic and therapeutic research. Further, this research has direct and broader implications for other MTORopathies including TSC.

NIH Research Projects · FY 2026 · 2023-02

Project Summary Dementia or major neurocognitive disorder is a condition of significant cognitive decline that impairs independent living. Learning and memory, executive function, perceptual-motor function, social cognition, and language may be affected. Although dementia is typically associated with aging, it is not a natural component of the aging process. Dementia is associated with several diseases, but most commonly associated with AD (60-80%). AD is a progressive neurodegenerative disease with clinical features that include memory loss, cognitive impairment and dementia. More than 5 million Americans currently live with AD and it is expected to increase to as much as 16 million by 2050. Ten percent of individuals over the age of 65 in the U.S. currently have AD. Current treatments for AD have limited efficacy and there is a significant need for improved pharmacological therapies. AD is characterized by the formation of senile plaques and neurofibrillary tangles in the grey matter of affected individuals. The senile plaques are composed of extracellular deposition of insoluble amyloid beta (Aβ) peptides that are typically associated with a wealth of microglia (brain resident macrophages) and astrocytes. ERRs are orphan receptors that play a key role in regulation of oxidative metabolism, and we have discovered that they function as exercise mimetics and enhance cognitive function in normal and aged mice as well as decrease amyloid plaques in animal models of AD. The goal of this project is to develop optimized ERR agonists that may be effective agents in treatment of dementia and Alzheimer's disease.

NIH Research Projects · FY 2026 · 2023-02

Project Summary/Abstract: Type 1 diabetes (T1D) is a complex autoimmune disease resulting from immune-mediated destruction of pancreatic beta-cells within the islets of Langerhans. Unfortunately, gaps in our understanding exist on the exact mechanisms triggering the initial break of immune tolerance in T1D that leads to beta-cell loss. Increasing lines of evidence support posttranslational modifications (PTM) as a key mechanism in production of beta-cell-specific neoantigens and neoepitopes that may play a prominent role in triggering T1D. Beta cell neoepitopes, despite being significant, have not been experimentally confirmed in situ; thereby highlighting the importance of their discovery and characterization in the islets of at-risk individuals as early triggers. The overall objectives of this application are to achieve a broad discovery of in situ islet PTM as potential neoepitope candidates through direct characterization of pancreatic islets from at-risk and recent-onset T1D donors by ultrasensitive proteomics. Novel beta cell neoepitopes will be functionally validated using allele-specific binding predictions and neoepitope- reactive T cell characterization from patient samples. Our hypothesis is that inflammation in the islet microenvironment leads to the production of neoepitopes through PTM of beta cell proteins, which exhibit favored loading into disease-predisposing HLA molecules in at-risk individuals. To discover and validate such in situ PTM neoepitopes, we pursue an innovative strategy consisting of three main aims: 1) in situ PTM discovery by ultrasensitive proteomics; 2) allele-specific HLA binding prediction, affinity analysis, and production of stable HLA complex tetramers; and 3) characterization of neoepitope T-cell reactivity and specificities using essential T1D patient samples and determine if these specificities can serve as biomarkers of T1D. Specifically, in Aim 1 we pursue in situ PTM discovery, which is enabled by our recently developed nanoPOTS (Nanodroplet Processing in One-pot for Trace Samples) technology for single islet proteomics and deep proteome profiling. The achievable deep coverage allows the direct identification of different PTMs (e.g., phosphorylation, deamidation, citrullination, oxidation, etc.). In Aim 2, we focus on PTM-neopeptide/HLA binding prediction and affinity confirmation of promising candidates and generate stable HLA tetramers with synthetic PTM-neopeptides for identifying specific reactive T cells. In Aim 3, we will identify PTM-neoepitope reactive T-cells in patient tissues, confirm the neoepitope T cell reactivity and specificities, reconstruct the human T cell receptor (TCR) alpha/beta sequences in primary T cells, and further validate the T-cell specificities as biomarkers for T1D. Statement of Impact: We anticipate the overall project will not only establish a first-of-its-kind patient islet database resource potential islet neoepitopes, but also confirm novel functional in situ neoepitopes from human patients, identify novel biomarkers, and provide important mechanistic insights into the initiation of T1D and potential prevention strategies for at-risk individuals.

NIH Research Projects · FY 2026 · 2023-01

Project Summary/Abstract In P01 AI123036, we were able to generate an algorithm that ranked single agents for Mycobacterium tuberculosis (MTB), identified promising 2-drug combinations and, with a completely novel mathematical approach, identified 3-drug regimens predicted to be significantly better than 2-drug regimens. These predictions were prospectively validated in a BALB/c model (H37Rv) and in a Non-Human Primate model of MTB (Erdman strain). In this proposal, we will extend our previous work. There is a large number of new MTB agents, many with novel mechanisms of action. We have 4 Specific Aims (SA) that, when complete, will allow us to identify multi-drug combinations that will optimize rate of kill for organisms in 3 different metabolic states and will suppress resistance emergence. In the Hollow Fiber Infection Model [HFIM] (SA#1), we will be able to rank new agents on the bases of potency and physicochemical properties. The HFIM provides insight into the drug’s exposure-response for kill and resistance suppression. We identified a near optimal 3-drug regimen (PMD/MFX/BDQ). With new single agents, we can examine substituting a new agent for an older agent AND we can expand the regimens to identify a near- optimal 4-drug regimen. This will be particularly important for patients with high bacterial burdens. In SA #2, we will test regimens from SA#1 in two murine models (BALB/c & C3HeB/FeJ mice). These will give somewhat different information. Both give information regarding kill and resistance suppression. Kramnik mice have pathology more closely resembling that in humans. We will use Matrix-Assisted Laser Desorption Ionization-MS Imaging and Laser Capture Microdissection LCMS. This allows identification of spatial distribution and quantification of drugs. A question regarding cure is how long to wait to sacrifice animals to document eradication. Some agents (BDQ) have long tissue half-lives. We will document rates of ingress/egress of drugs into the infection site, allowing determination when animal cohorts may be sacrificed to document eradication. In SA #3, we will document mechanisms of antimicrobial effect quantitatively. We have generated a first-of-a- kind dynamic model for PBP-binding in MTB, and will link this to rates of cell kill. We have also developed AMP/ADP/ATP intracellular assays. These will be employed for agents like diarylquinolines (e.g. BDQ) and PMD that act as energy poisons (for PMD, this occurs under anaerobic/non-replicative conditions. We will measure intracellular (MTB) drug concentrations, linking them to effect alone and in combination therapy experiments. Proposal success rests on modeling of the data. In SA #4, we have written code to extend earlier analyses, going from 3- to 4-drug regimens. For these high dimensional models, we developed several approaches to speed up analysis making them computationally tractable. At proposal end, we shall develop a 4-drug algorithm allowing rapid identification of near optimal regimens that work for both susceptible and less-susceptible organisms. The algorithm will be general. It will work well for today’s agents but also for agents as discovered.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY A single toxicant exposure during development can produce negative outcomes in adulthood and subsequent generations, presenting a major hurdle in the prevention and treatment of disease. In addition, given the susceptibility to toxicants amid degenerative biological and genetic processes, exposure during old age is a critical sensitive window. Despite their significance, however, the mechanisms that mediate both processes are poorly understood. Lead (Pb) remains one of ten World Health Organization-identified toxicants of major public health concern, even though there have been decades-long efforts to manage the routes of environmental exposure. Numerous studies have demonstrated potent neurotoxic effects of lead exposure on gene expression and the epigenome, resulting in outcomes such as impaired I.Q., behavioral dysregulation, and speech and learning deficits. Our long-term goal is to determine how environmental toxicants interfere with neurobehavior during critical windows so that evidence-based strategies to prevent and treat adult-onset and transgenerational disease can be developed. The overall objective for this NIEHS R01 Award (PA-20-185) application is to determine genome function alterations and epigenetic regulation of environmentally-influenced neurobehavioral phenotypes. The central hypothesis is that environmentally relevant Pb exposure during critical sensitive windows (early development and old age) lead to genomic and epigenetic dysregulation that alters neurogenesis pathway function in the exposed and subsequent generations. The rationale for the proposed research is that investigation of the mechanisms underlying Pb-induced outcomes will advance prevention, risk-assessment, diagnostic, and treatment strategies. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine life stage-specific transcriptomic changes in neurogenesis pathways following developmental and geriatric exposure to environmentally relevant Pb levels; 2) Determine emergent changes in the epigenome related to phenotypic and genetic endpoints; 3) Determine multigenerational and transgenerational transcriptomic and epigenetic changes induced by ancestral exposure. Ultimately, these results will identify critical windows for biomarkers of effect, and inform the interplay among pathways mediating toxic endpoints.

NIH Research Projects · FY 2026 · 2023-01

Project Summary-Abstract Renal cell carcinomas (RCC) are among the 10 most common cancer types in man (6th) and woman (8th) worldwide. Most RCC are clear cell carcinomas (>80%) that represent an atypical cancer type in many ways including the intrinsic resistance to many chemotherapeutics, sensitivity to anti-neoangiogenesis, and sensitivity to immune checkpoint inhibitors but with very low mutational burden. The treatment landscape of RCC has been changing rapidly in the last couple of years but still with very low complete response rate even with the most updated treatment regimens. Those “atypical” features of RCC prompted us to perform a comprehensive single cell RNA sequencing for human ccRCCs and paired bloods. We found that tumor induced Tregs are highly suppressive to effector T cells and could be the major immune cells conferring the immune suppressive tumor microenvironment (TME). These TI-Tregs have the typical signs of cellular senescence, including the expression of p16, p21, BCL-XL, senescence-associated secretory phenotype (SASP), and the activation of β-galactosidase (β-Gal). The group recently reported the development of the first-in-class BCL-XL degraders, i.e. the Proteolysis Targeting Chimeras (PROTACs, referred to as BCL-XL-Ps). DT2216, a lead BCL-XL-P, targets BCL-XL to the von Hippel-Lindau (VHL) E3 ligase; and PZ15227 targets BCL-XL to the cereblon (CRBN) E3 ligase for ubiquitination and subsequent degradation by the proteasome. We have found these two senolytics are very effective in eliminating TI-Tregs and activate anti-tumor immunity in our recent publication. Based on these novel findings, we hypothesize that aging- and cancer-associated cellular senescence – including senescence from immune cells – is critical in RCC pathogenesis and cancer progression via modulating the tumor immune microenvironment. Understanding these fundamental questions is important for rational design of therapeutic regimens towards to RCC that remains an urgent and unmet clinical need. Specific Aim 1 is to establish the mechanistic connection between cellular senescence – in particular Treg senescence – and RCC with special focus on the modulation of tumor immune microenvironment. Aim 2 is to determine the therapeutic efficacy of senolytic depletion of TI-Tregs and common therapeutics in RCC. The team has expertise in clinical renal cancer, cellular senescence, in particular the senolytic drug development and senescence tracing, cancer models and cancer immunology. We will establish the role of tumor infiltrating Tregs in immune modulation within the tumor microenvironment and determine the efficacy of targeting senescent Tregs in RCC cancer therapy, by combining with anti-neoangiogenesis drugs or immune checkpoint inhibitors.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY. Type 1 diabetes (T1D) is historically described as an endocrine (β-cell) specific autoimmune disease. However, reduced pancreatic size and subclinical exocrine insufficiency are also present at T1D diagnosis. The mechanisms, natural history, and role of these findings in T1D pathogenesis remains unclear. Exocrine atrophy may even precede the onset of multiple islet autoantibodies (Stage 1 T1D) in some subjects, signifying that clinical measures of exocrine mass and function could be helpful early T1D biomarkers. The primary objective of this proposal is to establish the natural history of exocrine loss in pre-T1D and to identify exocrine T1D- predictive biomarkers. We will measure fecal elastase (FE-1), a clinical marker of exocrine function, throughout the course of pre-T1D within TEDDY (The Environmental Determinants of Diabetes in the Young) subject banked samples (Aim 1A). An enrolling R01-funded study of TrialNet (TN) subjects (Campbell-Thompson and Haller, mPIs) will prospectively examine pancreas volume by MRI in single islet autoantibody positive (AAb+), multiple AAb+, and AAb- T1D first degree relatives (FDRs) to evaluate its prognostic utility. Herein we propose to add evaluation of serum and stool exocrine functional markers in this complementary population to evaluate their prognostic utility and determine timing of exocrine loss (Aim 1B). We hypothesize that exocrine markers will be reduced prior to Stage 1 T1D in those destined for progression and that their rate of decline can be used to predict disease onset. Lastly, the mechanisms underlying reduced exocrine pancreatic mass and function in T1D remain unclear; leading hypotheses include a lack of insulin secretion or autoimmunity to exocrine tissue leading to pancreas atrophy. The secondary objective of this study will use samples from the Network for Pancreatic Organ donors with Diabetes (nPOD) cohort to investigate these potential mechanisms and evaluate exocrine pancreatic autoantibodies as biomarkers of risk (Aim 2). We hypothesize that exocrine autoimmunity is present in subjects with multiple islet AAb+ and those with clinical T1D and leads to diminished exocrine mass and function. We hypothesize that insulinopenia is present in those with clinical T1D and leads to further exocrine atrophy. If our hypothesis is proven, this will represent a paradigm shift in our traditional understanding of the pathogenesis of T1D as an endocrine-specific autoimmune disease. This proposal will advance my early career goal to better understanding the timing and role of T1D exocrine pancreas changes and apply this knowledge in prevention and intervention trials. The skills set forth within this research proposal and career development plan will promote independence as a clinical investigator and include collaborations in several large T1D research consortia and training in 1) human subjects research trial design, implementation, and analysis 2) biomarker evaluation and 3) translational science design and technique. The collaborative rapport, excellent mentorship, and mission of training the next generation of investigators across University of Florida institutes and departments provides an ideal environment for career success.

NIH Research Projects · FY 2025 · 2023-01

Project Summary / Abstract It has recently been discovered that G-protein coupled receptors (GPCR) can be regulated by extracellular interactions. The mechanisms behind this phenomenon remain poorly understood. One such interaction is the trans-synaptic connection between presynaptic group III metabotropic glutamate receptors (mGluR) and postsynaptic cell-adhesion molecules of the extracellular leucine-rich repeat and fibronectin type III domain-containing (ELFN) family. Group III mGluRs sense glutamate release and inhibit further release in a negative feedback mechanism. A great body of evidence points to the essential role of synaptic glutamate homeostasis in a range of cognitive and motor functions with dysregulation leading to movement disorders and epilepsy. Binding and stabilization of mGluRs by trans-synaptic interactions with ELFN proteins is critical for regulating glutamate homeostasis. Loss of ELFN proteins in mice tremendously augments glutamate release and results in seizures and hyperactivity. Understanding how these proteins interact in the synaptic environment will provide valuable insight into how neurons maintain control over glutamate levels in the synapse and has implications for physiology and disease. Importantly, both mGluRs and ELFN proteins have been shown to form homo- and hetero-dimers. This proposal aims to test the hypothesis that apart from positioning and stabilizing group III mGluRs at the synapses, ELFN proteins act to allosterically modulate mGluR activity in part by influencing the dimerization dynamics of mGluR subunits. To test this hypothesis, I will build a structural model of the ELFN-mGluR interaction using crosslinking coupled with mass spectrometry, hydrogen/deuterium exchange, and cryo-electron microscopy to understand what binding determinants govern the ELFN-mGluR binding interaction. Additionally, I will investigate the mechanisms of mGluR allosteric modulation by ELFN proteins using a variety of cell-based signaling assays. Together, these experiments will provide a clear foundation for understanding how the brain maintains glutamate homeostasis inside the synapse.

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT: Acute myeloid leukemia (AML) is a rare, devastating, and understudied malignancy with ~20,00 new cases and around 61,000 cases in US. Though intensive chemotherapy primarily consisting of ara C (also known as cytarabine), daunorubicin, and etopside have been used to treat AML for over 4 decades, only 65%, 40%, and 10% of pediatric (age <21), adult (age 21-65), and elderly (age > 65) patients survive 5 years after diagnosis, respectively. Application co-PIs, Drs. Lamba (pharmacology) and Pounds (biostatistician specializing in cancer genomics) have successfully collaborated for over a decade to develop methods and discover molecular prognostic factors for AML. We and other investigators from Children’s Oncology group have recently characterized the genome, methylome, and transcriptome of pediatric AML and associated each of these with prognosis in pediatric AML. Dr. Pounds developed the innovative integrative analysis procedure, canonical correlation with projection onto the most interesting statistical evidence (CC-PROMISE), that dramatically increases statistical power for meaningful biological discovery in a rare-disease small sample size setting; using CC-PROMISE, we discovered that reduced methylation and increased expression of the DNMT3B associates with greater genome-wide methylation burden and worse prognosis; translating the DNMT3B discovery into evaluation of demethylating agents in the ongoing AML16 clinical trial (NCT03164057). These genomic, epigenomic, and transcriptomic features, along with microenvironmental and other factors, must impact the proteome and metabolome of AML in clinically relevant ways which unfortunately are not well understood. There has been essentially no study focused on comprehensive evaluation of the proteome and metabolome of pediatric AML in a reasonable cohort of uniformly treated patients. Noting the marked genomic, transcriptomic, methylomic, and prognostic differences between pediatric and adult AML, it is not plausible to extrapolate finding from adult AML patients to pediatric. Thus an integrated systems-level understanding of the molecular disease biology is needed to develop effective strategies and improve the prognosis of pediatric AML. As pioneers in the collection and integrated analysis of the pediatric AML genome, methylome, and transcriptome, application co-PIs Drs. Lamba and Pounds are uniquely positioned to characterize the proteome and metabolome of pediatric AML and integrate them with our large repository of previously collected molecular, treatment, and outcome data for a series of multi-center clinical trials. Thus, in this application we propose to characterize global metabolome (aim 1) and proteome (aim 2) the leukemic cell obtained at diagnosis for risk stratification and prognosis by evaluating impact on outcome in three St Jude led multi-institute clinical trials (AML02, AML08 and AML16, total patients n=400). In aim 3, we plan to develop a comprehensive integrated view of the genome, methylome, transcriptome, proteome, metabolome, and clinical prognosis of pediatric AML using novel methods. These innovative and exceptionally rigorous studies will be the first comprehensive evaluation of the pediatric AML metabolome and proteome and develop an innovative integrated analysis method to perform the first integrated analysis of five forms of omic data with multiple clinical endpoints to obtain the most complete understanding of pediatric AML systems biology to date.

NIH Research Projects · FY 2026 · 2022-12

Untreated anxiety and depression are exceedingly common in people living with HIV (PLWH) in the rural South, a population critically affected by the HIV epidemic. Addressing mental health is essential to improving the disparate HIV burden in this population. Yet outside of routine clinical care, few interventions target mental health in PLWH in the rural South. Mobile health technology (mHealth) and community health worker (CHW)-delivered care are two intervention models that address barriers unique to rural populations. Dr. Manavalan is an assistant professor in the Division of Infectious Diseases and Global Medicine at the University of Florida. She seeks a K23 award to gain skills, experience and preliminary data needed to become an independent physician-scientist focused on developing and implementing novel and scalable interventions in priority communities that will contribute to Ending the HIV Epidemic. Through the training and research proposal outlined in this award, Dr. Manavalan will bridge the fields of HIV and mental health by incorporating evidence-based interventions effective in other settings into existing medical systems in Florida. In this 4-year study Dr. Manavalan will adapt an evidence-based CHW-delivered counseling intervention for delivery through an existing mHealth application, as a strategy to reduce anxiety and depression and maximize viral suppression in PLWH in the rural South. In Aim 1, she will conduct robust qualitative research with key stakeholders, guided by the Consolidated Framework for Implementation Research (CFIR), to discern perspectives to inform adaption and implementation of the intervention. Next, in Aim 2, she will assemble a Design Consultation Team composed of 12 members from key stakeholder groups (PLWH from the target population, HIV and mental health providers, and mHealth and behavioral scientists) to assist in adapting CHW-delivered counseling for mHealth delivery, tailored to a rural population. Adaption of the intervention will occur iteratively, be informed from data from Aim 1 and will be guided by the ADAPT-ITT framework. Lastly, in Aim 3, she will pilot the adapted intervention with a type 1 hybrid effectiveness-implementation trial and assess implementation outcomes (reach, adoptability, implementation, maintenance) and preliminary effectiveness outcomes (viral suppression, HIV care retention, ART adherence, and anxiety and depression symptoms) using the RE-AIM model. The proposed program will enable Dr. Manavalan to gain expertise in 1) adaption and evaluation of behavioral interventions, 2) mHealth science, and 3) implementation science and hybrid trial design through mentorship with leading experts, didactic coursework, professional development programs, and application of research. This study will lay the groundwork for a fully powered R01 trial to evaluate implementation and effectiveness of the adapted intervention in communities throughout the state of Florida.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY Adeno-associated virus (AAV)-mediated gene therapy with broadly (b) neutralizing (n) antibodies (Abs) holds great promise for preventing and treating HIV infection. This approach is unique in that host cells, after receiving the relevant genes through AAV transduction, can immediately begin to secrete bnAbs into the circulation. Because AAV is non-pathogenic and its genome persists in host cells, successful AAV transduction of long-lived cells, such as muscle cells, can result in continuous expression of bnAbs for years, possibly decades. Since realizing this vision in humans would dramatically simplify efforts to combat the HIV/AIDS pandemic, this project focuses on overcoming a key obstacle to the clinical use of AAV/bnAb therapy: the high prevalence of anti-AAV nAbs in humans. Anti-AAV nAbs target the AAV capsid and are mainly induced by natural infection with wild-type (WT) AAV, which is endemic in primates. AAV seroprevalence varies geographically, ranging from 30% to 100%, depending on the AAV serotype. Because of structural similarities among different capsids, nAbs induced by infection with WT AAV often cross-react with other AAV serotypes, including those used for gene therapy. This is problematic because, depending on the nAb titer, Ab-mediated neutralization of AAV vectors can reduce or even abrogate transduction. Hence, AAV seropositive (+) individuals are currently excluded from clinical trials of AAV-based gene therapies. These observations, viewed in the context of the 38 million people living with HIV (PLWH) worldwide who could benefit from AAV/bnAb therapy, highlight the need to develop strategies to overcome pre-existing anti-AAV nAbs. Here we propose a series of approaches to bypass humoral immunity to AAV in AAV(+) individuals, with the ultimate goal of expanding people’s access to AAV/bnAb therapy. This project has four specific aims. Our first aim is to identify the least prevalent muscle-tropic AAV capsid in sub- Saharan Africa, home of two thirds of all PLWH. Knowledge gained from this serosurvey would allow AAV(–) PLWH to benefit from AAV/bnAb therapies without any additional intervention. Aim 1 will also establish the range of pre-existing anti-AAV nAb titers that must be overcome to enable AAV transduction in field settings. Our second and third aims are to assess the extent to which transient depletion of serum IgG Abs can decrease anti-AAV nAb titers in AAV(+) rhesus macaques (RMs). Our final aim is to integrate the findings of Aims 1-3 by testing whether depleting pre-existing anti-AAV nAbs in RMs with pharmacologically controlled simian immunodeficiency virus (SIV) infection can enhance AAV-mediated delivery of eCD4-Ig–a bnAb-like molecule that potently neutralizes both HIV and SIV. Aim 4 will also assess the ability of AAV-expressed eCD4-Ig to maintain antiretroviral therapy- free control of SIV virus replication. Thus, this project will not only establish a blueprint for testing AAV-based HIV immunotherapies in the epicenter of the HIV/AIDS pandemic, but it will also expand our toolkit of interventions for evading pre-existing immunity to AAV. If successful, this research could bring us closer to achieving sustained AAV-driven production of anti-HIV biologics in people, regardless of AAV serostatus.

NIH Research Projects · FY 2026 · 2022-12

Summary During development and throughout adulthood, humans display strong neuroplasticity, the capacity of adaptive changes of neurons and neural circuits in response to social environments. Plasticity in chemosensory system is essential for learning and memory, social communication and quality of life. Loss of olfactory sensing (anosmia) leads to social isolation, neurological diseases, such as schizophrenia, Alzheimer’s disease (AD) and Parkinson’s disease (PD), and social disorders such as autism. However, the role of neuroplasticity in olfactory- mediated social behaviors is unclear and understudied. We and other labs have developed genetic tools in ants, thereby providing a novel model to study olfactory neuroplasticity in social organisms, as (a) ants are highly social and display complex social behavior; (b) ants display striking neuroplasticity during development and throughout adulthood; (c) neuronal activity is likely required for proper development of olfactory neurons in ants, which is reminiscent of activity-dependent neuronal survival in mammals. The Drosophila genome contains 60 odorant receptor (Or) genes, while 300-500 Or genes have been identified in several ant genomes. In the ant Harpegnathos saltator, mutation in the gene odorant receptor co- receptor (orco), which disrupts the function of all ORs, significantly impacts ant olfaction, and mutant animals display a wide range of abnormal social behaviors. Surprisingly, and unlike other insects, such as Drosophila, this loss of OR functionality during development dramatically reduces the number of odorant receptor neurons (ORNs) and antennal lobe (AL) glomeruli where ORNs project. Further transcriptome analysis suggests that there are two types of ORNs: activity-dependent and activity-independent ORNs. We will use these features in the orco-/- ants and perform a series of molecular and cellular experiments to determine (a) the role of neuronal activity and receptor trafficking in olfactory neuronal survival, and (b) the role of gustatory and ionotropic receptors in neural development.

NIH Research Projects · FY 2026 · 2022-12

Alzheimer’s disease (AD) is an incurable neurodegenerative brain disorder that causes progressive memory loss and cognitive decline, and is the No.1 cause of dementia. It is characterized by the coexistence of extracellular amyloid plaques, mainly formed by the amyloid beta-42 (Abeta) peptide, and intracellular neurofibrillary tangles containing aggregates of abnormal tau. Abeta and tau were considered as disconnected culprits for many years, but in view of recent studies, it is clear that they are intimately related and possess synergistic activities. Sadly, very little is known about how Abeta and tau interactions trigger AD pathogenesis, which significantly hinders the development of effective treatments. To address this, we generated a new fly model of AD that genetically produces both human Abeta and tau resulting in synergistic pathology. These flies display extracellular deposits of thioflavin-S-positive Abeta, intracellular aggregation and phosphorylation of wild-type tau, and progressive loss of neuronal cells. The robust and consistent pathology of these flies provides a unique opportunity for gene discovery efforts and thus we performed a massive loss-of-function RNAi screen in the fly eye, which provides a useful and easy-to-score phenotype. Out of 6,600 RNAi stocks tested, we identified 31 suppressors and 119 enhancers, including multiple genes not previously known to be associated with AD. Most suppressors are linked to protein modification or cleavage, ribosomal function, cell metabolism, transcription, chromatin modulation, and transport to name a few. Here, we will employ a strategically designed pipeline that integrates genetics with high-throughput behavioral platforms and target prioritization to identify robust late-stage modifiers of the disease (Aim 1). On the other hand, we will fast-track a mechanistic and therapeutic analysis of one of the strongest suppressors along with its human homologue (Aim 2). This suppressor encodes a highly disordered protein of uncharacterized function and was also found in two other genetic screens performed by us. Thus, we have labelled it as a high-priority target. We strongly believe that manipulation of the 150 modifiers of Abeta+tau toxicity presented here will provide the foundation for new types of targets or therapeutics. Therefore, this work may contribute significantly to the goals of the National Plan to Address Alzheimer’s Disease.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT The Southeastern Coastal Center for Agricultural Safety and Health (SCCAHS) builds on strong partnerships among health disparate farmworker, fisher and forestry communities, frontline public health practitioners, and scientists engaged in transdisciplinary occupational health research across the Southeast and U.S. Caribbean regions. The multidisciplinary, inter-institutional network of projects, programs and Cores SCCAHS will implement innovative research approaches and develop tailored, system-driven translation and dissemination strategies drawing from environmental and human toxicology, occupational medicine, public health and biomedical informatics research. The proposed Center includes an Evaluation and Planning Core (with Administration; Evaluation; Diversity Equity and Inclusion; and Emerging Issues Programs), an Outreach Core, and a Research Core. Proposed research projects and the focus of the Pilot/Feasibility Program include activities within all four categories detailed in the PAR (basic/etiologic, intervention, translation, and surveillance). Targets areas of concern include respiratory health, chronic kidney disease and musculoskeletal disorders; cross-cutting themes include surveillance and mental health/substance abuse. Research projects include the following: Category I: Basic/Etiological • Assess Personal Air Particulate and Pesticide Exposure and Respiratory Health Outcomes among Farmworkers in the Southeast (Tara Sabo-Attwood, Environmental and Global Health, UF College of Public Health and Health Professions). • Development of urinary biomarkers of occupational stress in agricultural workers (Christopher Vulpe, Center for Environmental and Human Toxicology, UF College of Veterinary Medicine) Category II: Intervention • Effectiveness and implementation of self-management strategies for low back pain among aquaculture and horticulture workers (Kimberly Dunleavy, Physical Therapy, UF College of Public Health and Health Professions) Category III: Surveillance • Detection of Chronic Kidney Disease of Unknown Etiology in Florida by Repurposing a Statewide Data Infrastructure for Surveillance (William Hogan, Health Outcomes and Biomedical Informatics, UF College of Medicine) Category IV: Public Health Translation The Pilot/Feasibility Program will seek to fund new- and early- stage investigators along the translational science spectrum to study health outcomes at the population level (annual competitive seed funding)

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Rod and cone photoreceptors are indispensable for our vision. Their death or dysfunction is an underlying cause for a vast majority of blinding retina conditions. Key to photoreceptor function is the ability to transmit the signal that they generate in response to light to other neurons in the retina for processing of visual signals and their communication to the brain. For this to occur, photoreceptors form elaborate synapses with the downstream neurons, the bipolar cells (BC). Deficits in synaptic communication between photoreceptors and bipolar cells are known to cause congenital stationary blindness in humans, various forms of rod/cone dystrophies and frequent co-morbidity with many other ocular conditions. The long term goal of our collaborative program is to obtain atomic level view of molecular organization of machinery that enable synaptic communication of the photoreceptors with the hope to better understand blinding conditions and devising strategies for their treatment. Recent research from our laboratories and others have identified several molecules critical for the synaptic communication of photoreceptors. We have further discovered that many of these components are scaffolded into macromolecular assemblies that span the synaptic cleft and physically integrate pre-synaptic elements of photoreceptors with post-synaptic receptors in BC. Specifically, we found that the postsynaptic receptor on BC: mGluR6 interacts with two cell-adhesion molecules in photoreceptors: ELFN1 and ELFN2. Furthermore, the machinery that drives excitation of BC in response to synaptic photoreceptor inputs is associated with an orphan receptor GPR179 which in turn is integrated with pre-synaptic cell adhesion-like molecule pikachurin (Pika) in photoreceptors. We also documented that loss of this organization abolishes synaptic transmission leading to night blindness. However, at the moment we know absolutely nothing about structural basis of these trans-synaptic complexes. Proposed studies aim to fill this gap by determining the atomic structures of the key trans-synaptic scaffolds: ELFN1-mGluR6 and Pika-GPR179 complexes and probing their biochemical mechanisms. This will be achieved by highly synergistic international collaboration leveraging expertise in biochemistry and cell biology of photoreceptor synaptic proteins and recent advances in high resolution cryogenic electron microscopy (CryoEM) to obtain high resolution molecular structures of the complexes probing their mechanisms at exceedingly precise level. The premise of this proposal is that understanding synaptic organization of photoreceptors would lead to novel therapeutic strategies for ameliorating blindness.

NIH Research Projects · FY 2024 · 2022-09

Title: Networking and Expanding Undergraduate Research On the Neurobiology of Aging to Advance Diversity (NEURON-Aging) ABSTRACT: The goal of this NIA R25 proposal “Networking and Expanding Undergraduate Research On the Neurobiology of Aging to Advance Diversity (NEURON-Aging)” is to expand the pool of researchers interested in biomedical, behavioral, and clinical aspects of brain aging and Alzheimer’s disease and related dementias (ADRDs) through undergraduate research activities that enhance diversity. Minority health disparities for cognitive outcomes in advanced age are well documented, with older adults from minority groups being more likely to self-report impairments and to develop dementia. Healthcare and research professions that are critical for addressing clinical needs in diverse elderly populations, however, have inadequate representation from diverse and disadvantaged groups. This is a significant barrier for mitigating these health inequities. Thus, there is an urgent and unmet need to support educational pipeline programs that increase the diversity of the research and clinical workforce focused on understanding and treating brain aging and ADRD to reduce minority health disparities. The current proposal will expand the pool of undergraduate trainees interested in pursuing research and clinical careers related to understanding and treating brain aging and ADRD with 3 Specific Aims. Aim 1 will increase the participation of University of Florida (UF) underrepresented minority (URM) undergraduate students conducting mentored research on the neurobiology of aging and ADRDs in NIH-funded laboratories and expand their research network to facilitate an interest in brain aging. Aim 2 will increase the representation of URM undergraduates from outside UF and non-Research I Institutions conducting mentored research in NIH-funded laboratories examining brain aging and ADRDs. Finally, using institutional matching funds provided by Centers and Institutes at the University of Florida, Aim 3 will increase the numbers of diverse students proceeding on to top graduate programs focused on brain aging by proving 12-24 months of postbaccalaureate training as a bridge to PhD training. The proposed education and research training program will have the following measurable objectives: (1) NEURON-Aging students will develop an appreciation for brain aging and related diseases through year-round mentorship and networking; (2) NEURON-Aging students will have an increased sense of scientific efficacy through mentored, hands-on research training; 3) NEURON-Aging students will have an enhanced understanding of experimental design and data interpretation in brain aging-research areas, (4) NEURON-Aging students will develop professional communication skills by presenting their research locally and nationally; (5) NEURON-Aging students will develop awareness of graduate and professional school program application processes; and (6) The majority (>50%) of NEURON-Aging students will matriculate into graduate or clinical training programs related to aging within two years of receiving their Bachelor's degree.

NIH Research Projects · FY 2026 · 2022-09

Abstract The clinical symptoms of Alzheimer disease (AD) dementia occur downstream of pathological deposition of Aβ peptides in extracellular cored-neuritic plaques and aggregated tau protein in intracellular neurofibrillary tangles (NFT) in the brain. Since deposition of Aβ precedes tauopathy in early-onset familial AD (fAD), it is accepted that Aβ can trigger tau misfolding into NFT, initiating a cascade of cumulative pathology that progressively leads to dementia. In sporadic AD, the coincident deposition of Aβ appears to correlate with tau misfolding and the severity of NFT pathology. Collectively, these findings suggest that Aβ deposition can exacerbate tau misfolding and NFT formation leading to cognitive deficits and dementia. However, the underlying mechanisms and characteristics of Aβ and tau that synergize resulting in NFT pathology and pathological sequelae is still unclear. Our proposal is designed to provide experimental insights into the individual contribution of tau (Aim 1) and Aβ (Aim 2) in driving Aβ-tau synergy in mouse models of AD. Evidence suggests that a major mechanism by which Aβ synergizes with tau misfolding involves the hyper-phosphorylation of tau. A recent study of AD patients that quantitatively mapped the progressive emergence of phosphorylated epitopes in tau identified 19 Ser/Thr residues that are most frequently phosphorylated in individuals that exhibit concurrent Aβ pathology. The main objective in Aim 1 is to dissect the contribution of these phosphorylation events in the misfolding and aggregation of tau that occurs in the presence of concurrent Aβ pathology. Using AAV technology, we have the capability to generate and express a large number of tau phospho-mimetic variants in APP TgCRND8 mice. Using this mouse model, in Aim 1 we propose a broad study to systematically dissect the phosphorylation events that drive tau misfolding and NFT formation in the presence of Aβ. Over many years of research, our laboratories have created mouse models that exhibit a spectrum of Aβ pathologies, including mice that develop primarily diffuse Aβ pathology and mice that primarily develop cored- neuritic pathology. Given that there are questions regarding the type of Aβ pathology that underlie Aβ-tau synergy, in Aim 2, we propose to use our AAV approach to examine Aβ-tau interactions in this diverse collection of APP transgenic models that exhibit different types of Aβ pathology. Additionally, in Aim 2, we will use pharmacologics and inducible APP models to examine the role of newly-made soluble Aβ vs long-lived insoluble Aβ in tau phosphorylation/aggregation process. Phospho-proteomic analysis will help us determine the relationship of different types of Aβ to the resulting tau phosphorylation profile. Collectively, this work will improve our understanding of the Aβ-driven phosphorylation cascade that appears to promote tau misfolding and aggregation into NFT.

NIH Research Projects · FY 2025 · 2022-09

This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-RM- 24-013. Dystonia is a movement disorder characterized by sustained or intermittent muscle contractions causing abnormal, often repetitive, movements or postures. DYT1 early-onset generalized dystonia is the most common type among genetic dystonias. Most of the individuals affected by DYT1 dystonia share a trinucleotide deletion (ΔGAG) located in exon 5 of DYT1 or TOR1A gene, leading to a loss of a glutamate amino acid residue for torsinA (torsinA∆E). The symptoms start in the limbs and then become generalized. Affected individuals could be seriously disabled and need to use a wheelchair. Conditional knockout of torsinA and knockin of mutant torsinA in mice points to the involvement of multiple brain regions and cell types in the pathogenesis of DYT1 dystonia. So far, these and other pathophysiological studies of DYT1 and other dystonias support a circuit or network model of dystonia pathogenesis. However, which brain region and neuronal types play a critical role in pathogenesis is unclear. Preliminary studies of conditional knockin (KI) mouse models of DYT1 dystonia revealed striatal medium spiny neurons (MSNs) play a vital role in the pathogenesis of DYT1 dystonia. However, how torsinA∆E in MSNs leads to dystonia is unknown. These unknowns impede the development of effective treatment for DYT1 patients. The broad, long-term objective is 1) to determine the functional role of torsinA in vivo and the mechanism by which torsinA∆E leads to DYT1 dystonia, 2) to develop a novel and effective treatment. The specific goal of this supplement application is to replicate the behavioral analysis of a newly developed, targeted DYT1 mouse model with an overt dystonia phenotype without growth retardation and use this model to test its suitability for preclinical drug development (part of the original Aim 1). We hypothesize that additional ΔGAG expression in the striatal MSNs in Dyt1 KI mice will accelerate the development of the dystonia phenotype in mice and better recapitulate the phenotype of human dystonia for pathophysiological studies and preclinical drug testing. We plan to collaborate with NIH-assigned CRO to replicate the newly developed novel overt dystonia model. We will generate and characterize this conditional KI line in the heterozygous Dyt1 ΔGAG KI background. We will perform the tail suspension test to determine the overt dystonia phenotype and the treatment effect of trihexyphenidyl, an FDA-approved drug for dystonia patients. We are particularly well prepared to undertake the proposed research because we have developed and characterized the novel Dyt1 conditional KI mice. The successful completion of the proposed study will characterize and validate a novel DYT1 dystonia mouse model that will contribute significantly to the study of the pathophysiology of DYT1 dystonia and the development of anti-dystonia drug treatment. It will also have an impact on other dystonias and related neurological disorders.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Although proteases are widely known to be involved in disease pathophysiology, a consequential challenge in protease drug discovery is to design or isolate a specific ligand that selectively inhibits or activates a target protease to remediate disease states and facilitate mechanistic investigations. However, besides well- studied enzymes such as angiotensin-converting enzyme and HIV protease, developing drugs for new protease targets has proven an iterative, arduous, and often unsuccessful process. Recognizing that the property of a ligand ultimately dictates its modulatory function and binding mechanism, the proposed research postulates two hypotheses. First, if molecules are selected directly based on their modulatory function from large libraries, their properties will directly relate to their function, rather than their binding capabilities. Second, if the binding mechanism a modulator is determined, functional relationships between ligand properties and mechanism can be developed and possibly extended these findings to related proteases. The proposed research pursues three directions, with an overall objective to transform protease ligand discovery and protease biochemistry from iterative endeavors to data-driven, and ultimately predictive processes. The first research direction will establish a machine learning (ML)-guided high-throughput screening platform that isolates protein-based protease modulators directly based on how they alter protease function. Here, property- function relationships will train machine learning algorithms for function prediction and ML-guided library design will significantly reduce the search space for protease modulators while exploring distal regulation diversity more comprehensively. In a second research direction, this platform will be extended to isolate nanobody- based substrate selective modulators of β-secretase and insulin-degrading enzyme, two proteases that are key therapeutic targets in Alzheimer’s disease and Type-2-Diabetes, respectively. The ability to finely reprogram the substrate selectivity of proteases can revolutionize how to study and drug polyspecific enzymes and lead to successfully targeting previously undruggable proteases. The third research direction will implement deep mutational scanning protocols to map the modulatory landscape of proteases and determine how modulators alter protease substrate preference at the molecular and physiological scale. This approach will identify conformational epitopes of modulators, characterize novel distal sites, and uncover long-range distal communication. Taken together, the long-term payoff of these studies is to establish generalizable ligand design guidelines based on ternary relationships between ligand property, binding mechanism/protease structure and modulatory function, enabling one to better understand how proteases work and how to control them. The vast experience of the Denard research lab in high-throughput protease engineering and support from a machine learning expert and neuroscience expert, respectively, strongly supports the feasibility, success, and sustainability of this multidisciplinary, and potentially broadly impactful independent program.

NIH Research Projects · FY 2025 · 2022-09

The over-arching goal for the RE-JOIN Consortium is to define how the neurons that mediate chronic joint pain innervate different articular and peri-articular tissues, with a focus on the knee and temporomandibular joint (TMJ). With an improved understanding of how different neural subtypes distribute through the joint and how these subtypes change with age and disease, new therapies can be developed to reduce the heavy burden of chronic joint pain. To achieve this goal, our team will focus on advancing our understanding of pathology-pain relationships in the knee and TMJ by combining expertise in neural tracing, 3-dimensional imaging, and evaluations of chronic joint pain and disability. Our proposal brings together a highly collaborative team that spans basic science and clinical research with extensive experience in both the knee and TMJ, allowing us to evaluate shared vs. joint-specific shifts in innervation networks and the development of chronic joint pain. Specifically, our team will first use neural tracing dyes to identify the cell bodies in the dorsal root ganglia and trigeminal ganglia that project to the muscle, bone, or intra-articular joint tissues. These neurons will then be evaluated for their function using electrophysiologic tests and their transcriptome using single cell RNA-Seq. By overlapping neural function with gene expression, we will identify promoter targets and design adeno-associated virus (AAV) vectors to produce fluorescent labels alongside the expression of these targets. Importantly, this approach will allow us to develop AAV-based tracers for specific functional neural subtypes, as well as combine traditional markers of functional subtypes with any newly identified markers that describe how the neuron changes with age, sex, and osteoarthritis (OA) severity. Using these tracers, we will then evaluate the distribution of functional neural subtypes throughout the joint (including bone, cartilage, synovium, joint capsule, ligament, tendon, fascia, and muscle) and how these innervation networks change with age, sex, and OA severity. Moreover, these tracers will be used to evaluate how joint innervation adapts following the application of two neural ablation techniques for pain relief in the knee and TMJ. To evaluate the clinical significance of our preclinical studies, innervation changes will be assessed in tissues collected from patients undergoing total joint replacement of the knee or TMJ. In all of our studies, joint innervation will be paired with detailed analyses of joint pain and disability. In rodents, these analyses will include detailed behavioral characterizations; in patients, these analyses will include quantitative sensory tests and other assessments of joint function. Combined, this approach will allow us to evaluate pathology-pain relationships related to joint innervation from the preclinical model to the clinic.

NIH Research Projects · FY 2025 · 2022-09

A substantial missed opportunity to prevent cancers in rural areas of the United States is the low rates of human papillomavirus (HPV) immunization. Among the 50 states, Florida is a particular concern with the second highest number of HPV-related cancers and a rank of 44th in HPV immunization initiation (≥ 1 dose). Even higher cancer risk is present within the rural northern Florida counties that are the focus of this proposal. Due to multiple factors presenting challenges for parents living in rural areas to access timely information and resources about their cancer prevention options a multilevel approach is needed. Guided by the Integrated Behavior Model, the overall objective of this proposal is to ensure all families have access to cancer prevention information to make the best choices for their children’s health. We will use a three-arm cluster randomized trial to assess the added clinical- and associated cost-effectiveness on increasing uptake of proven cancer prevention protocols with three nested evidence-based implementation strategies: (1) clinician-targeted communication training, (2) parent-targeted educational resources designed to support parents - messages and structured phone conversations, (3) community-targeted healthcare access to transportation assistance, mobile clinics, and navigation to healthcare insurance. Community-focused organizations with longstanding relationships within rural communities will support implementation: University of Florida (UF) Institute of Food and Agricultural Sciences Extension, UF Clinical and Translational Science Institute Community Engagement Program, UF Cancer Center Community Advisory Board, UF OneFlorida Clinical Research Consortium, Suwannee River Area Health Education Center, and a federally designated Rural Health Network. The specific aims are: (1) Estimate the added clinical- and associated cost-effectiveness of parent-targeted educational resources alone and when combined with community-targeted healthcare access assistance beyond the effects of clinician-targeted training; (2) Estimate the differential effectiveness of the implementation strategies by patient-level factors; and (3) Measure moderation of implementation strategy effectiveness by clinic-level factors. The proposed research is significant for its potential to increase parents’ informed decision-making about cancer prevention. The project is innovative through its evaluation of the clinical- and cost-effectiveness of layered evidence-based strategies. The proposed project will build evidence on how to support parents and prevent cancers in rural areas.

NIH Research Projects · FY 2025 · 2022-09

Resolving single-cell analysis challenges via data-driven decision frameworks and novel statistical methods Abstract: Despite the power of single-cell RNA-seq, the data presents a multitude of analytical challenges and researchers continue to struggle with data analysis. The long-term goals of this research program are to develop robust, efficient, and scalable statistical methods and tools that enable all scientists to obtain accurate biological inferences. Specifically, we propose to develop interactive data-driven decision-frameworks to guide researchers through analyses and make informed analytical decisions. We also propose developing methods that retain interpretability while accommodating complex experimental designs. All of our approaches will be developed as highly accessible statistical software with interactive visualization and analysis modules available via webservers. Our proposed methods will result in richer analyses and biological insights, as well as, improved reproducibility and reliability of scientific results.

NIH Research Projects · FY 2025 · 2022-09

SUMMARY / ABSTRACT Recent evidence put forth by our group and others suggests that the pancreas of patients with recent-onset type 1 diabetes (T1D) contains a significant number of “empty” beta cells that have lost the ability to secrete mature insulin but retained other hallmark features such as proinsulin expression. The mechanisms that underlie formation of such cells and their fate remain poorly understood. We hypothesize that empty beta cells, which are invisible to standard insulin immunostaining and potentially also to the immune system, play important roles in the pathophysiology of T1D while holding therapeutic potential for reversion of diabetes. We propose in depth experiments to characterize empty beta cells, using MACSima ultra high-content immunofluorescence imaging to assess in situ expression of 78 immune and pancreas cell markers, including vascular and lymphatic annotation with signatures of inflammation, extravasation/trafficking, and immune cell residency in autoantibody positive (AAb+) and T1D donors as compared to donors with type 2 diabetes (T2D) and non-diabetic controls available through the Network for Pancreatic Organ donors with Diabetes (nPOD) repository. We will compare insulin containing versus insulin negative islets, within and across donors, to identify empty beta cells and determine how they correlate with islet, acinar, and immune cell phenotypes. These data will serve as a template for a serial section to undergo laser capture microdissection (LCM) of insulin containing islets, insulin negative islets, and acinar tissue regions, which will be subjected to bulk RNAseq as well as our novel method for quantifying beta cells based on DNA methylation patterns. In addition, we will use a novel mouse model (beta cell-specific, tamoxifen-inducible Adar1-mutant) and cultured human pancreas slices to functionally interrogate molecular pathways underlying the formation of empty beta cells. Specifically, we propose to leverage these two model systems to test therapeutic candidates— including an incretin mimetic (GLP1), an endoplasmic reticulum (ER) stress inhibitor (ISRIB), and multiple biologics targeting specific immune subsets— for their ability to modulate empty beta cells, their insulin content and insulin secretion. We will then correlate these functional data with molecular and cellular features of hormone negative islet cells via MACSima, RNAseq, and DNA methylation. These studies are expected to yield insights into a fundamental yet little understood process taking place in human T1D. We anticipate that empty beta cells can be re-functionalized, paving the way for therapeutic development to restore endogenous beta cell function in T1D. Ultimately, when combined with effective interventions to constrain autoimmunity, it is our hope that the metabolic modalities explored here could provide a means to reduce insulin requirements or even achieve insulin independence after T1D onset, dramatically improving longevity and quality of life for these patients.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract The overall goal of this 7-year UG3/UH3 collaborative project is to develop an interventional training for oral healthcare professionals based on behavioral skills determined to positively impact adult-child interactions in pediatric dental care. The goal of this line of research is to measure the mechanisms involved in the effectiveness of this training on dental provider utilization of these skills, and how skill utilization affects satisfaction, adherence, and behaviors of very young children (under the age of 6) and their parents/caregivers. Two years of funding will be required to align community partners, develop a training protocol, test procedures, and prepare staff. The following five years will be needed to recruit, deliver, and measure outcomes of this unique training. This project will align community- and university-based dental providers (dentists, hygienists, assistants) with a research team to develop and test the effects of a training workshop on outcomes related to provider implementation (acceptability, understanding, feasibility, system climate, and system support), training (provider knowledge, fidelity, skill use, acceptability, satisfaction), and caregiver-child experiences within a dental appointment (child cooperation, distress, and pain, as well as family acceptability and satisfaction). With an innovative training based on a well-established behavior management program for preschoolers, Parent-Child Interaction Therapy (PCIT), findings from this project will reveal if oral health care providers’ responsivity to children’s developmental needs can be changed and effectively impact the experience of dental care in very young children. The valuable connections with community partners in the states of West Virginia, Arkansas, and North Carolina allow this study to be both feasible and representative of a large variety of dental settings. The project is designed to train dental providers on a few key skills that could greatly impact child comfort in the dental office and create a foundation for future curriculum development for all dental staff. The long-term goal is to disseminate an effective training package for students of dentistry and current dental providers on a large scale that helps dental providers reduce child distress. Specifically, the ultimate intent is to disseminate this concise, effective skills training framework to enhance the use of positive and developmentally-sensitive strategies within the oral health treatment of the youngest patients.