University Of Connecticut Sch Of Med/Dnt

NIH Research Projects · FY 2025 · 2023-08

Most people living with Alzheimer’s disease and Alzheimer’s disease-related dementias (PLWD) prefer to remain at home in the community, yet research shows that they are less likely than people without Alzheimer’s disease and Alzheimer’s disease-related dementias (AD/ADRD) to successfully return to the community following nursing facility stays. Medicaid’s Money Follows the Person (MFP) program has successfully helped over 100,000 individuals return to community living after nursing facility stays, and Connecticut has one of the highest rates of participation among over 40 participating states. Research finds PLWD have decreased access to the MFP program and lower odds of successfully moving out of nursing facilities, yet it is unknown what mechanisms are leading to these disparities. This study asks why these differences exist and what is causing them, in order to identify policy levers to improve outcomes for PLWD. We will also analyze differences by race and ethnicity, based on evidence that in Connecticut non-Hispanic Black and Hispanic MFP participants are more likely than non-Hispanic White participants to complete moves back to the community. Using a mixed-methods approach we will identify individual, family, organizational, and community factors which influence three distinct outcomes: applications submitted to the MFP program, completed moves from nursing facility to community homes, and sustained community living. The study’s three aims include: Aim 1: in-depth interviews with individuals with and without AD/ADRD and their informal caregivers to identify barriers and facilitators of their participation and success in MFP; Aim 2: asking staff involved in each step of the MFP program about mechanisms contributing to different experiences for PLWD and potential levers for change; and Aim 3: identifying how mechanisms such as individual health, family support, and organizational practices contribute to disparities in MFP applications, completed moves, and sustained community living through quantitative analysis of approximately 55,000 Connecticut Medicaid nursing facility residents. This study builds upon our team’s 14+ years as the evaluator of the state’s MFP program, our long-standing relationship with Connecticut’s Department of Social Services (the state Medicaid administrator) which is enthusiastic about improving outcomes for PLWD in the MFP program. The study also leverages a constellation of rich MFP data collected by and uniquely accessible to our team and merges it with Medicaid and Medicare claims data and Minimum Data Set (MDS) data to create a powerful analytic dataset for analyzing MFP outcomes. The study is innovative in its conceptualization of three outcomes representing different potential points where disparities emerge along the MFP process continuum, taking advantage of rich untapped and unique data on the MFP program and participants, and an academic-government research partnership with a commitment from our state policy partner who is ready to modify Connecticut’s MFP program to improve MFP outcomes for PLWD.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen that can withstand treatment with bactericidal antibiotics even when lacking identifiable resistance genes. It is thought that these recalcitrant infections are attributable to phenotypically antibiotic-tolerant cells called persisters. Despite the acknowledged contribution of P. aeruginosa to chronic and recurrent infections, there is a lack of basic research into cellular mechanisms that underlie P. aeruginosa antibiotic persistence. My central hypotheses in this research proposal are that the cellular responses following treatment will govern P. aeruginosa antibiotic persistence and resistance development (Aim 1), and that increased duration of coexistence with common co-isolate, S. aureus, will increase P. aeruginosa persistence by priming it in a more stress-tolerant state (Aim 2). This fellowship research will provide critical insight into persister physiology as well as the opportunity to learn cutting-edge techniques, analyses, and skills that will prepare me to lead independent research efforts in the future as a dentist-scientist interested in bacteria of the oral cavity. In Aim 1, I will investigate how P. aeruginosa persisters reawaken after drug treatment. I will perform RNA- seq to screen for genes that are differentially expressed between untreated cultures and cells that are viable after antibiotic treatment. Hits from this screen will be functionally validated by conducting persister assays with multiple genetic models for the genes of interest, including transcriptional reporters, knockout mutants, and inducible complementation strains. Significant genes of interest will be tested in biofilm cultures and in host- mimicking media to add clinical relevance. In Aim 2, I will determine how the duration of co-culture with S. aureus affects P. aeruginosa persistence and the physiologies of surviving cells. To efficiently passage and assay P. aeruginosa in co-culture, I am implementing a novel, dual-chambered apparatus that I designed, called the “H- Cell.” The H-Cell allows dynamic crosstalk between species while maintaining segregated populations for efficient sampling. I will determine the transcriptomic changes between P. aeruginosa persisters grown in monoculture or in H-Cell co-culture with S. aureus by RNA-seq. I will validate the hits by testing genetic constructs. Furthermore, I will test P. aeruginosa persistence in strains that are co-isolated with S. aureus from clinical sputum samples and thus have co-existed in a host environment. From my use of multiple bacterial strains, antimicrobials, and culture conditions, I aim to reveal the generalizability or specificity of P. aeruginosa persistence mechanisms and how they contribute to the development of antibiotic resistant progeny. Furthermore, I can apply the investigative approach, technical skills, and tools that I develop throughout this proposal to my future research on bacterial physiology in multispecies communities of the oral cavity. Altogether, completion of this research can inspire anti-persister strategies to reduce the burden of recalcitrant infections and their contributions to the broader antibiotic resistance crisis.

NIH Research Projects · FY 2026 · 2023-07

ABSTRACT An estimated 44% of patients with cancer in the United States are eligible to receive immune checkpoint blockade (ICB). FDA-approved ICB agents include α-PD-1 and α-CTLA-4 antibodies, but the majority of patients do not benefit because their tumors are resistant to these agents. ICB treatment is expensive and may lead to serious toxicity. Prospective identification of patients with ICB-resistant cancers would reduce unnecessary risk and cost and give patients opportunities to seek more appropriate treatment options. To address the unmet need for a peripheral blood biomarker for ICB effectiveness, we performed immune profiling of ICB-treated patients with melanoma, using multiparametric flow cytometry to characterize immune cells in pretreatment peripheral blood. Our analyses revealed a new peripheral blood immune profile—which we called Immunotype-1 (IT-1), defined in part by the presence LAG-3+CD8+ T cells—as a promising biomarker of ICB resistance. This finding was validated in an independent dataset of metastatic urothelial cancer. Patients with IT-1 have inferior overall survival, progression-free survival, and response rates to α-PD-1 blockade. Leveraging our leadership in the clinical development of ICBs, we have assembled one of the largest biobanks of peripheral blood samples from >600 ICB-treated patients across cancer types. In this proposal, we aim to test the hypothesis that IT-1 is a pan- cancer biomarker for ICB, reflecting an exhausted, tumor-specific LAG-3+CD8+ T cell population whose function can be recovered for therapeutic benefit using α-LAG-3 blockade. The Specific Aims are to: 1) Phenotypically and functionally characterize the peripheral blood LAG-3+CD8+ T cell population and determine if this population is represented in the tumor microenvironment in patients with the IT-1 phenotype; 2) Determine the association between IT-1 and clinical outcome in ICB-treated patients across cancer types; and 3) Assess whether IT-1 identifies patients who will respond to relatlimab (α-LAG-3) + nivolumab (α-PD-1). Our project is rooted in strong clinical data and thus likely to identify a biomarker that is mechanism-based, clinically implementable, and most importantly, therapeutically actionable.

NIH Research Projects · FY 2025 · 2023-07

Abstract/Project Summary: This is a proposal for the seeking support for the Institutional Training Program in Skeletal, Craniofacial and Oral Biology (SCOB) as a T90/R90 training program at the UConn Health, School of Dental Medicine. The SCOB program aims to train the next generation of scientists dedicated to addressing important problems in dental, oral, and craniofacial health through the application of state-of-the­ art approaches to solve these problems. Our program incorporates didactic, research, and career development components to prepare an outstanding cadre of individuals for productive research positions at academic, non-profit, government, biotechnology, or industry institutions. The program has and will continue to train scholars who can initiate and maintain funded research programs, who understand multidisciplinary research and who are prepared for the evolution of their research programs into new directions. Areas of research training include Advanced Materials Science, Behavioral and Social Sciences/Medicine, Cancer Biology, Computational Biology and Bioinformatics, Embryonic Stem Cell and Mesenchymal Stem Cell, Craniofacial and Skeletal Biology, Mathematical Modeling and Imaging Techniques, Mucosal Immunology and Microbiome and Regenerative Medicine. The major tracks of the training program are (i) DMD/PhD (7-8 yrs of training with 3-yrs of support from this training grant), (ii) PhD (4-5 yrs of training with 3 yrs of support from this training grant, and (iii) postdoctoral research (3 yrs training and support). The postdoctoral track supports individuals in different areas of research endeavor including Traditional post-PhD, Post-DMD in PhD training [including non-citizen], and post-DMD involved in post-doctoral training [including non- citizen]. After a rigorous application process that culminates in personal interviews, students are enrolled annually. To provide a solid, common foundation for our scholars/trainees, we have developed Core Activities required of all trainees in each training track. UConn Health has a dynamic group of faculties in various areas of research with highly successful collaborations among faculty throughout the Schools of Dental Medicine and Medicine and The Jackson Laboratory for Genomic Medicine. The Biomedical Science PhD program graduate faculty, active and new institutional research centers and clinical programs provide contemporary laboratory, translational and patient oriented research opportunities that enable a diversified training environment for the program tracks, allow flexibility for the individual needs of trainees, and ensure successful progress through the tracks. The institution has vigorous trainee recruitment programs, with several directed towards recruitment of outstanding cadre of individuals for productive research positions at a variety of settings. We will continue to provide training that is tailored to each candidate through symposia, seminars, courses, clinical research centers and collaborative research experiences.

NIH Research Projects · FY 2025 · 2023-07

The development of pathological cardiac hypertrophy requires the stimulation of gene transcription activated by Ca2+-dependent signaling pathways. However, targeting specifically these Ca2+-dependent pathways is difficult due to the multiple functions of Ca2+ in myocyte physiology. Recent evidence suggests that formation of distinct Ca2+ microdomains provide the molecular mechanism that allows for specificity in Ca2+ signaling. Therefore, understanding the components and regulation of each microdomain is key for the development of novel therapeutics for the prevention and treatment of pathological hypertrophy. We have previously demonstrated that binding of the Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN) to the muscle-specific A Kinase Anchoring Protein mAKAPβ mediates the induction of myocyte hypertrophy. New data show that the mAKAPβ signalosome is also required for a perinuclear Ca2+ transient required for the activation of transcription factors responsible for myocyte hypertrophy. We hypothesize that a pool of perinuclear RyR2 localized to the mAKAPβ signalosome is responsible for this Ca2+ microdomain, and importantly, that these perinuclear RyR2 are segregated from those involved in excitation-contraction coupling (E-C coupling). The central hypothesis of this proposal is that RyR2 localized to mAKAPb signalosomes induces perinuclear Ca2+ transients required for CaN-dependent gene expression that are independent of the canonical function of RyR2 in E-C coupling. Aim 1: Perinuclear RyR2 associated with mAKAPβ signalosomes are within an independent Ca2+ signaling compartment that regulates myocyte hypertrophy. The goal of Aim 1 is to demonstrate that mAKAPb- signalosome associated RyR2 is responsible for bAR-stimulated perinuclear Ca2+ transients that induce myocyte hypertrophy and that this pool of RyR2 is regulated independently from RyR2 involved in E-C coupling. Using novel, targeted activators and inhibitors of the perinuclear RyR2, we will demonstrate the importance of perinuclear RyR2 for the regulation of pathological gene transcription, and show that modulation of perinuclear RyR2 does not impact contractility. Furthermore, the importance of PKA-mediated phosphorylation of RyR2 at several sites will be investigated. Aim 2: The dimensions of the mAKAPβ Ca2+/CaN compartment. Aim 2 will map the perinuclear Ca2+ domain that is specified by the mAKAPb signalosome and demonstrate that this perinuclear compartment does not affect cytosolic CaN activity, but functions to maintain perinuclear CaN activity. Aim 3: Requirement of perinuclear RyR2 signaling for pathological remodeling in vivo. The therapeutic potential of targeting perinuclear RyR2 in mouse models of cardiac hypertrophy will be investigated in Aim 3. Through these Aims, this proposal will define a novel signaling compartment orchestrated by mAKAPb that is required for pathological gene transcription and induction of cardiac disease, but does not affect E-C coupling. Furthermore, completion of this project will reveal how targeting mAKAPb signalosomes can be therapeutically beneficial in the prevention of cardiac remodeling and heart failure.

NIH Research Projects · FY 2025 · 2023-07

Abstract/Project Summary: This is a proposal for the seeking support for the Institutional Training Program in Skeletal, Craniofacial and Oral Biology (SCOB) as a T90/R90 training program at the UConn Health, School of Dental Medicine. The SCOB program aims to train the next generation of scientists dedicated to addressing important problems in dental, oral, and craniofacial health through the application of state-of-the­ art approaches to solve these problems. Our program incorporates didactic, research, and career development components to prepare an outstanding cadre of individuals for productive research positions at academic, non-profit, government, biotechnology, or industry institutions. The program has and will continue to train scholars who can initiate and maintain funded research programs, who understand multidisciplinary research and who are prepared for the evolution of their research programs into new directions. Areas of research training include Advanced Materials Science, Behavioral and Social Sciences/Medicine, Cancer Biology, Computational Biology and Bioinformatics, Embryonic Stem Cell and Mesenchymal Stem Cell, Craniofacial and Skeletal Biology, Mathematical Modeling and Imaging Techniques, Mucosal Immunology and Microbiome and Regenerative Medicine. The major tracks of the training program are (i) DMD/PhD (7-8 yrs of training with 3-yrs of support from this training grant), (ii) PhD (4-5 yrs of training with 3 yrs of support from this training grant, and (iii) postdoctoral research (3 yrs training and support). The postdoctoral track supports individuals in different areas of research endeavor including Traditional post-PhD, Post-DMD in PhD training [including non-citizen], and post-DMD involved in post-doctoral training [including non- citizen]. After a rigorous application process that culminates in personal interviews, students are enrolled annually. To provide a solid, common foundation for our scholars/trainees, we have developed Core Activities required of all trainees in each training track. UConn Health has a dynamic group of faculties in various areas of research with highly successful collaborations among faculty throughout the Schools of Dental Medicine and Medicine and The Jackson Laboratory for Genomic Medicine. The Biomedical Science PhD program graduate faculty, active and new institutional research centers and clinical programs provide contemporary laboratory, translational and patient oriented research opportunities that enable a diversified training environment for the program tracks, allow flexibility for the individual needs of trainees, and ensure successful progress through the tracks. The institution has vigorous trainee recruitment programs, with several directed towards recruitment of outstanding cadre of individuals for productive research positions at a variety of settings. We will continue to provide training that is tailored to each candidate through symposia, seminars, courses, clinical research centers and collaborative research experiences.

NIH Research Projects · FY 2025 · 2023-06

Project Summary/Abstract Aging is a key risk factor for chronic disease development which can affect lifespan and quality of life. Cellular senescence has emerged as a potential target to slow down the aging process. Senescent cells are in a state of proliferative arrest and are highly associated with aging and pathological conditions. They accumulate in multiple tissues and secrete pro-inflammatory cytokines and other kinds of molecules that damage surrounding tissues. The markers p16 and p21 (cyclin dependent kinase inhibitors) are commonly used to identify senescent cells, but emerging evidence has shown that these markers are not entirely sensitive or specific markers for senescence. There is currently a lack of understanding of the exact genetic markers that define senescent cells especially on the single cell level. With studies showing that senescent cell clearance can alleviate various diseases associated with aging it is vital to achieve a greater understanding of senescent cell markers so that precision medicine treatments can be designed to more effectively eliminate senescent cells. In this proposal we aim to examine the transcriptome of senescent cells on the single cell level both with aging and senolytic treatment in human adipose tissues. Senescent cells have been demonstrated to accumulate in adipose tissue with aging and chronic disease and with the vast array of cell types present in adipose tissue it makes an excellent model to study specific cell types and markers associated with senescence. Aim 1 will explore the transcriptome of naturally occurring senescent cell populations in adipose tissue with aging. We will use single nucleus RNA sequencing to capture cell types sensitive to typical single cell dissociation methods and compare the transcriptome differences between cells in aged vs. young tissues. We hypothesize that adipose tissue from older donors will contain more senescent cells and that several novel genetic markers will be identified in these cells. Aim 2 will look at the effects of senolytic treatment on human adipose tissue on the single cell level. Senolytics are drugs that are designed to specifically eliminate senescent cells but we do not know the precise cell types targeted by senolytics. We will use single nucleus RNA sequencing to uncover the transcriptome changes that occur with senescent cell elimination and learn what cell types are removed with senolytic treatment. This project will help advance the field achieving a greater understanding of senescent cell populations in adipose tissue and the markers that define them, which is essential to understanding and potentially treating numerous diseases. Through this fellowship my training will include developing my skills in genetics, aging, computational analysis, communication, presentation, networking, scientific writing, clinical knowledge and mentorship. This training will occur in the environment of UCONN Health as well as the Jackson Laboratory and other research institutions. This work will allow me to take advantage of training opportunities and mentorship to advance my career as a future physician-scientist.

NIH Research Projects · FY 2025 · 2023-06

ABSTRACT Type 2 diabetes mellitus (T2DM) is an increasingly prevalent chronic disease that affects more than 400 million people worldwide. One of the major complications of T2DM is exacerbated atherosclerotic cardiovascular disease (CVD). Even when modern lipid and glucose control strategies are applied, T2DM is associated with a two- to four-fold increase in CVD risk, suggesting the effect of additional pathologies, such as inflammation. However, current tools to predict CVD outcomes for T2DM patients incorporate only clinical and demographic variables into their models, and they thus attain only a moderate ability to discriminate the highest-risk patients in need of targeted clinical intervention. Our lab recently discovered that monocyte-derived foam cells, which are well-known to play a central role in atherosclerotic CVD, can undergo both homeostatic (non-inflammatory) and pathogenic (inflammatory) foaming. Using a transcriptomic signature from pathogenic foam cells, our lab developed a CVD prediction model called CR30 which outperformed existing tools. To address the critical knowledge gap of identifying CVD risk specifically in T2DM patients, I analyzed monocyte transcriptomic data from the Multi-Ethnic Study on Atherosclerosis (MESA). From this preliminary analysis, I identified a transcriptomic signature unique to T2DM patients with CVD, containing a super-network downstream of the co- regulator proteins SNW1, NCOR2, and CITED2. We hypothesize that this transcriptomic super-network represents a unique molecular signature which can be used to improve prediction of atherosclerotic cardiovascular events in individuals with T2DM. In this proposal, I will test this hypothesis by applying two different strategies to develop predictive models. In Aim 1, I will apply supervised machine learning approaches to select a set of genes from my preliminary analysis which are predictive of T2DM-CVD outcomes. I will then test several modeling strategies in training and building a T2DM-CVD prediction model incorporating this gene set combined with clinical data. In Aim 2, I will use another approach to incorporate T2DM-CVD molecular signature into modeling by focusing on the transcriptomic super-network. I will generate enrichment scores for the super-network, then incorporate the scores as variables into model development. The long-term goal of this project is to identify biological risk factors for CVD in patients with T2DM. The anticipated impacts are the identification of novel targets for mechanistic studies and the advancement of biology-informed approaches to clinical outcomes prediction. The training goals of this proposal will provide me with biologically-informed quantitative skills. This interdisciplinary, highly translational project will leverage the innovative environment and unique opportunities in the sponsor’s lab and the University of Connecticut School of Medicine. The expected outcomes from this project will promote my career goals of becoming a next-generation physician-scientist capable of integrating biological knowledge and quantitative skills to solve clinical problems for patients with chronic disease.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT Anti-programmed death-1 antibody (αPD-1 Ab) as a single agent for treating human hepatocellular cancer (HCC) was withdrawn from the US market on July 26, 2021, because a multi-center phase III study did not demonstrate its efficacy in improving patient survival over controls. Thus, there is an urgent need to identify and target critical cellular and molecular regulators to design new immune checkpoint therapy (ICT) strategies against HCC. A unique, orthotopic, and clinically relevant murine HCC model was established that mimics HCC initiation and progression in humans and reflects the tumor biology, immunology, and histology typical of human disease. In this model, SV40 T antigen (TAg) is expressed solely in tumors as a trackable tumor-specific antigen (TSA), enabling TSA immunity study during tumor initiation, progression, and response to treatments. Using this model, several immune-based therapeutic strategies for HCC were developed and a novel microbe-based strategy was recently established that significantly improves the therapeutic efficacy of αPD-1 Ab for HCC. Specifically, Bacteroides thetaiotaomicron (B.th), one member of genus Bacteroides, with CpG-rich nucleic acid which functions as TLR9 agonist, was identified as a microbial regulator to significantly boost αPD-1 Ab therapeutic efficacy for HCC. This exciting finding led to studies of the underlying cellular and molecular mechanisms. Single- cell RNA sequencing (scRNA-seq) revealed that HCC growth upregulated Kruppel like factor-2 (KLF2) in tumor- associated macrophages (TAMs), and B.th addition activated effector CD8+ T cells and improved αPD-1 therapeutic effect against HCC, which was associated with reduced KLF2 expression in TAMs. Moreover, adoptive cell transfer (ACT) of macrophages (MΦs) with KLF2 knockdown (KD) enabled TSA immunization to significantly suppress HCC growth. Conversely, KLF2 overexpression (OE) in MΦs compromised B.th/αPD-1- induced therapeutic suppression of HCC. These compelling results highlight KLF2 as a key regulator mediating microbes’ impact on hepatocarcinogenesis and B.th/αPD-1 immunotherapy by modulating MΦs. Further studies indicate that KLF2 controls MΦ expression of TLR9 and signal-regulatory protein α (SIRPα) to regulate MΦ tumor phagocytosis and immune regulatory function. These findings generate the following hypothesis: B.th, with CpG-rich nucleic acid, reinvigorates αPD-1 Ab ICT in HCC by phenotypically and functionally programming MΦ via KLF2-controlled expression of TLR9 and SIRPα. In Aim 1, KLF2-directed MΦs as the cellular basis mediating HCC pathogenesis and immune tolerance will be studied toward the development of a new therapeutic approach by integrating KLF2-KO MΦs with αPD-1 Ab. In Aim 2, the molecular mechanism and regulators by which B.th/αPD-1 suppress KLF2 to reprogram MΦ by controlling TLR9 and SIRPα expression will be studied. The knowledge generated from this study will not only identify unrecognized endogenous regulators with a role in programming MΦ in HCC, but also make a case that these factors are effective targets to trigger MΦs and lead to improved ICT for HCC.

NIH Research Projects · FY 2026 · 2023-05

ABSTRACT An estimated 70% of all eukaryotic cellular proteins are regulated by phosphorylation. Strict temporal and spatial control are essential for the fidelity of this process, as derailed signaling cascades lead to disease. While the importance of phosphorylation is clear, knowledge gaps remain in the mechanisms that regulate key proteins involved in this process, especially phosphoprotein phosphatases (PPP). Our long-term goal is to understand the structural and functional mechanisms that control PPP activity in health and disease. Here, we focus on the function of protein phosphatase 1 (PP1) and PP2A, both of which have major roles in cell division and cancer. Our aims are designed to define the mechanisms of PP1- and PP2A:B55-based substrate recruitment to obtain a systems biology understanding of the proteomes and phosphatomes directed by these enzymes. For the PP2A family of enzymes, it is established that substrates are recruited by their variable B- subunits. We recently showed that the PP2A B56 subunit binds specifically to its substrates via a newly identified short linear motif (SLiM), LpSPIxE. This has led to the discovery of scores of novel B56-specific substrates and the development of the first PP2A:B56-specific regulator. Here, we investigate PP2A:B55, the most abundant PP2A holoenzyme in cells and the primary enzyme responsible for dephosphorylating CDK1 targets to initiate mitotic exit. Consistent with this, at mitotic entry, PP2A:B55 activity is inhibited. This is achieved by two B55-specific inhibitors: FAM122A and ARPP19. To molecularly define how these inhibitors block PP2A:B55 activity and to elucidate the molecular basis of B55 substrate recruitment via a B55-specific SLiM, we will determine both holoenzyme (quadruple complexes) structures. This is technically challenging, as these PPPs cannot be functionally expressed in E. coli or insect cells, a problem we have successfully overcome. Furthermore, we have developed a unique PP1 regulator (PhosTAP), which we show can be successfully leveraged to fully define the PP1 interactome and phosphatome. Due to its 100% specificity and exceptional affinity for only PP1, this novel PP1 PhosTAP can also be leveraged to specifically recruit PP1 to its point of action within the cell, in a manner similar to that used by PROTACs for targeted degradation. Together, the proposed aims will provide the much-needed molecular data that demonstrate how key PPP holoenzymes, especially PP1 and PP2A holoenzymes, bind their substrates and how these interactions are regulated during the cell cycle. Because these holoenzymes have critical roles in multiple human diseases, especially cancer, the proposed work will establish these holoenzymes specifically, and PPPs generally, as potent and specific drug targets.

NIH Research Projects · FY 2026 · 2023-04

Periosteum, a primary site of the fracture healing response, is highly vascularized and densely innervated. Studies on bone fracture have primarily focused on the role of growth factors and vascularization in the healing process, but we postulate that sensory nerve signals are a critical part of the regulatory mechanism that initiate the stem/progenitor cell responses required for fracture callus formation. A number of lines of evidence point to sensory innervation, or signals associated with sensory nerves as promoters of bone accrual and healing. For example, fracture healing is impaired following chemical sensory denervation, but the sensory nerve-derived signals that promote healing as not yet defined. We hypothesize that damage to sensory nerves in the periosteum orchestrates the bone-healing cascade through calcitonin gene-related peptide (CGRP) – calcitonin like receptor (CLR) signaling. This is important given that multiple CGRP inhibitors were recently approved by the FDA for prevention and treatment of migraines. In Aim 1 we will evaluate the effects of CGRP inhibitors on bone healing. Given that CGRP plays a role in bone turnover and potentially healing, it is important to understand the impact of CGRP inhibition on fracture healing. In the Aim 2, we will determine which cell lineage or lineages responsive to CGRP signals during healing using targeted deletion of the CLR receptor. CLR deletion in early fracture healing will be targeted to the following lineages using inducible Cre’s: MPCs (αSMA-CreER), chondrocyte (Acan)-CreER, osteoblasts using Col2.3CreER and in endothelial (Cdh5-CreER) during fracture healing. The effects on callus formation and strength, as well as differentiation in the callus will be determined. We will examine cellular mechanisms of CGRP/CLR action utilizing in vivo approaches to study MPC expansion, differentiation and vascularization. We propose to dissect CLR signaling by distinguishing effects of the ligands. The main ligands are CGRP and Adrenomedullin that act via CLR and RAMP, of which CLR-RAMP1 is main complex for CGRP signaling while CLR-RAMP2 and RAMP3 is responsible for ADM signal activation. In Aim 3 we will evaluate effects of ADM deletion in mesenchymal population using ADMfl/fl mice. We will also define downstream signaling mechanism of CLR deletion in MSCs and endothelial cells using 10x genomics. Our study will also provide critical information on the effects that newly approved inhibitors of CGRP signaling exhibit on bone healing and what cellular mechanisms affect healing via CLR receptor Finally, testing approaches to modulate CGRP/CLR sensory singling may lead to a therapeutic strategy to enhance bone healing.

NIH Research Projects · FY 2026 · 2023-03

Abstract Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system affecting 1 in 100,000 live births with no cure or effective long-term treatment. GLD is caused by loss-of-function mutations in the galactosylceramidase (galc) gene, where loss of GALC enzymatic function results in toxic accumulation of its substrate, a lipid called galactosylsphingosine or `psychosine'. Psychosine cytotoxicity is considered the basis of several key pathologies in GLD. Neuropathology in GLD is marked by profound demyelination and inflammation. However, molecular details of these processes are limited, leaving few therapeutic options. Early histological evidence for CD8+ T cells within demyelinated lesions in both the twi mouse brain and human GLD brain had suggested a role for adaptive immunity in this disease. However, the function of CD8+ T cells in GLD has not been previously determined. To address this gap in our knowledge, we analyzed the timing of T cell population changes by flow cytometry in CNS tissues from the twitcher mouse model of GLD and compared with wt littermates. We identified a rapid and protracted elevation of T cells in the twi CNS that was coincident with the onset of clinical disease at postnatal day 21 (P21). These data were confirmed using single-cell RNA sequencing on twi and wt littermate brain tissues from which identified a 9-fold increase in CD8+ T cells in twi mouse brains at P21. Our transcriptomic data also defined the CD8+ population as cytotoxic T lymphocytes (CTLs). To test the function of CD8+ CTLs in GLD-like disease in twi mice, we depleted CD8+ T cells by administering anti-CD8 antibody to twi mice and found that this treatment effectively prevented disease onset, preserved wellness and completely prevented CNS demyelination while also attenuating pro-inflammatory cytokine levels in brain and blood. These novel and highly translational data portend a pathogenic role for CD8+ T cells in GLD and underlie the basis for our overall hypothesis that CD8+ T cells are pathogenic in GLD and directly contribute to disease. Accordingly, Aim 1 will characterize the spatial and temporal development of CD8+ T cells in twi mice and challenge the contribution of CD8+ T cells using knockout mouse lines to then assess clinical, biochemical, and pathological outcomes. Aim 2 will define the overall nature of adaptive immunity by evaluating contribution of CD4+ T cells to CD8+ T cell-mediated immunity in twi mice using targeted depletion and genetic strategies. Aim 3 will identify and profile the timing, and location T cells responding to authentic antigen, the clonality of the T cells using T cell β chain VDJ phenotyping, and then identify the nature of the antigen presenting cell types using transgenic reporter mice. These multi-disciplinary studies will interrogate a previously unrecognized CD8+ T cell neuroinflammatory response in GLD. Outcomes of these studies are expected to fill an important gap in our understanding on the fundamental cellular pathological mechanisms underlying the development CD8+ T cell mediated neuropathology and disease in GLD.

NIH Research Projects · FY 2025 · 2023-02

PROJECT SUMMARY Alzheimer’s Disease (AD), the most common cause of dementia, is a debilitating disease that leads to progressive memory loss, cognitive impairment, and ultimately death. Pathological hallmarks of AD include extracellular amyloid beta (Aβ) plaques. β-secretase-1 (β-site APP cleaving enzyme 1, BACE1) is the rate- limiting enzyme of toxic Aβ generation. Transgenic BACE1 KO mouse models of AD led to suppression of AD pathology, which suggests that inhibiting BACE1 may be a rational strategy for AD treatment. However, human clinical trials have shown that BACE1 inhibitors are inefficacious, even worsening cognitive function, among AD patients. This benchtop-to-bedside translational failure is due to our incomplete understanding of BACE1’s physiological function. In particular, the mechanisms underlying neuronal and synaptic impairments in BACE1 deficiency or inhibition is poorly understood. In this proposal, we will address this knowledge gap by testing the hypothesis that BACE1 modulates intrinsic and synaptic neurophysiological properties in a cell-type- and circuit- specific manner in the hippocampus, a major substrate of memory storage derailed by AD. My preliminary data of whole-cell patch clamp of hippocampal pyramidal neurons (PNs) show that selective BACE1 deletion in excitatory neurons leads to neuronal hyperexcitability, suggesting that BACE1 deletion disrupts intrinsic neuronal function in a cell-autonomous manner. Given my preliminary findings, I hypothesize that BACE1 modulates excitability and synaptic transmission in hippocampal PNs by the regulation of ion channels – the identities of which have yet to be fully elucidated. In Aim 1, I will comprehensively determine the ionic basis underlying the hyperexcitability phenotype in my Excitatory-BACE1-KO mice (mice in which BACE1 is selectively deleted in excitatory PNs), and characterize the synaptic transmission and plasticity deficits in Excitatory-BACE1-KO, through patch clamp electrophysiology methods. In Aim 2, I will delineate behavioral deficits Excitatory-BACE1- KO neurons, and rescue hypothesized cognitive deficits in mutant mice by normalizing PN hyperexcitability through a chemogenetic approach. The findings from this study will provide insight into neuronal and synaptic physiology, mechanisms of learning/memory and behavior, and future AD therapeutic strategies. Importantly, completion of this project will help me master current concepts and state-of-the-art techniques in patch clamp electrophysiology, behavior studies, and in vivo genetic perturbation and increase my scientific communication skills through extensive opportunities to present and publish my studies. As an MD/PhD student at UConn Health, I will have access to mentors and experts that will not only directly facilitate my mastery of the necessary technical skills, but I will also have opportunities to continue honing my clinical skills and gain specialized experience during and after my research phase. Fulfilling my training and development plan will be a crucial step toward my future career as a physician-scientist studying the mechanisms underlying neurodegenerative disease in patients.

NIH Research Projects · FY 2025 · 2023-02

Anxiety disorders in youth are: 1) the most prevalent psychiatric illnesses, 2) associated with severe disability, and 3) considered gateway disorders--as they predict a broad range of adult psychiatric and functional problems. Despite the high prevalence and impairment, less than half of anxious youth receive mental health services and access to evidenced-based interventions lags far behind that of less common psychiatric illnesses, such as attention deficit hyperactivity disorder. This application addresses this mental health service gap and responds to NIH’s priorities in PAR-MH-21-131: Pilot Effectiveness Trials for Treatment, Preventive and Services Interventions (R34) aimed at testing interventions with previous efficacy in community settings using novel service delivery methods. Specifically, we propose to refine and assess the feasibility of the Anxiety Action Plan (AxAP), a brief intervention to reduce pediatric anxiety, delivered by primary care providers (PCPs; defined here as nurse practitioners, physician assistants, and/or pediatricians) in community pediatric primary care clinics. Primary care settings are ideal for addressing pediatric anxiety specifically because: 1) prevalence rates of excessive anxiety are high in primary care (approximately 10-20%), 2) over 90% of anxious youth report physical complaints (e.g., stomach aches) and are “frequent flyers” in primary care settings, 3) children with, compared to without, medical conditions treated by PCPs are more likely to have elevated anxiety, and 4) PCPs are often the first and only health professional children visit. This proposal builds on the PI’s development and feasibility pilot work with PCPs conducted as part of the NIMH-funded Center for Mental Health in Pediatric Primary Care and with school nurses as part of a Department of Education grant. The AxAP, modeled after the Asthma Action Plan familiar to PCPs, is based on the core element of cognitive behavioral therapy for anxiety (i.e., behavioral exposure), was designed to fit within the short primary care visit (20-30 minutes), can be delivered virtually, is brief (1-4 sessions), and can be billed for as an office visit. Uniquely, and in stark contrast to co-location or integrated models, the goal of the AxAP is to enhance the capacity of PCPs to identify and intervene with anxious youth, which will enhance access to care in general and especially in locations with few mental health specialists. The proposal also incorporates several innovative features including pilot tests of measures to conduct: 1) a cost-benefit analysis of the AxAP and 2) an examination of theory-based target mechanisms at the child, parent and PCP levels. If results of this study are positive, findings would support a large effectiveness trial using an intervention that is ready for dissemination and that could significantly improve clinical care for anxious youth, enhance the capacity of PCPs to identify and reduce anxiety, and lower personal and economic costs associated with pediatric anxiety.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY Chronic kidney disease (CKD) is a public health problem that afflicts more than 37 million Americans. The current therapeutic options for this progressive disorder are limited; therefore, novel therapeutic strategies are urgently needed. Common features of CKD are kidney inflammation and fibrosis. Inflammation often triggers fibrosis, and fibrosis is the end result of chronic inflammatory reactions. Renal inflammation is characterized by macrophage activation and proinflammatory molecule production. However, the molecular mechanisms underlying macrophage activation are not fully understood. Therefore, the long-term objective of this proposal is to understand the molecular mechanisms of macrophage activation so that effective strategies can be developed for the treatment of CKD. We have identified PU.1 as a critical factor in the regulation of kidney inflammation during the development of CKD. Specifically, we have demonstrated that PU.1 is induced in the kidney in experimental models of CKD and in human kidney with CKD and PU.1 is obligatory for macrophage activation and inflammatory molecule production and development of CKD. Genetic deletion or pharmacological inhibition of PU.1 prevents macrophage activation and inflammatory molecule production in macrophages in vitro, and pharmacological inhibition of PU.1 suppresses macrophage activation, inflammatory molecule production, and fibrosis development in the kidney with obstructive injury. Furthermore, the proinflammatory effect of PU.1 appears to be mediated by the NOD-like receptor family pyrin domain–containing 3 (NLRP3) inflammasome pathway. In this application, we propose to examine and characterize the role of PU.1-NLRP3 pathway in macrophage activation and polarization and proinflammatory and profibrotic molecule production to further understand the molecular mechanisms of inflammation and development of kidney fibrosis. Our central hypothesis is that PU.1 promotes NLRP3 expression and inflammasome activation in macrophages leading to proinflammatory and profibrotic molecule expression and development of kidney fibrosis. To test this hypothesis, we will pursue the following Specific Aims. Specific Aim 1 is to determine the role of PU.1 in macrophage activation and polarization in vitro and in vivo. Specific Aim 2 is to dissect the molecular mechanisms by which PU.1 promotes macrophage activation and polarization. Specific Aim 3 is to explore the therapeutic potential of a selective PU.1 inhibitor for the treatment of CKD. We plan to utilize molecular, genetic, and pharmacological approaches to examine the role of PU.1- NLRP3 pathway in macrophage activation and polarization and development of kidney fibrosis. Results from these studies will provide novel insights into the molecular mechanisms of kidney inflammation and could lead to the development of novel therapeutic strategies for the treatment of CKD.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT Acute kidney injury (AKI) is characterized by abrupt deterioration in kidney function, manifested by an increase in serum creatinine level, with or without a reduction in the amount of urine output. Tragically, between 2009- 2019, hospitalizations in the US complicated by AKI increased by 42%. The long-term objectives of this application are to better understand the kidney local microenvironment and its impact on AKI with an eye towards development of new treatment strategies. The AKI research field believes that renal tubules are the epicenter of damage, yet little attention has been paid to changes in the renal local microenvironment and associated repair processes, which are certain to impact AKI. The concept of a ‘microenvironment’ has shaped the understanding of the pathogenesis of various diseases. However, the AKI microenvironment is poorly characterized. The kidney local microenvironment in AKI - consisting of injured tubular cells, activated fibroblasts, inflammatory cells (e.g., macrophages), other cellular components, extracellular matrix (ECM), and a variety of secreted factors - is complex, heterotypic, and dynamic. After AKI, in general, renal tubules undergo a repair process of dedifferentiation. During this process, ECM is the major organizing component for microenvironment construction and tubule repair, serving as a scaffold for remodeling. The major cellular source of ECM synthesis in the kidney is interstitial fibroblasts. Several subpopulations of fibroblasts are activated exceptionally early after AKI (1h), far earlier than tubular cell proliferation (3d). This suggests that fibroblast-derived proteins may act early in AKI. To explore this idea in depth, matrix proteins were compared between AKI and control kidneys using ischemic kidney models and proteomics. This identified extracellular matrix protein 1 (ECM1; a secreted glycoprotein) as the earliest and highest activated matrix protein after ischemic AKI. ECM1 was induced rapidly (4-8h) after AKI and localized predominantly to fibroblast-rich foci in the kidney interstitium. It was also found that after AKI, Sonic Hedgehog (Shh) growth factor secreted by renal tubules specifically targets fibroblasts to mediate cell-matrix interactions. Further study revealed that ECM1 binds to Shh in vitro, knockdown of ECM1 aggravates AKI in vivo, and ECM1 peptide prevents tubular cell death in vitro. Based on these observations and the role of macrophages in microenvironment formation, it was hypothesized that after AKI, ECM1 directly recruits Shh, which activates fibroblasts and macrophages to form a favorable microenvironment to promote kidney remodeling. This hypothesis will be tested by determining the mechanistic role of ECM1 in kidney microenvironment formation ex vivo (Aim 1); determining the roles of injured tubules, activated fibroblasts, and macrophages in constructing the kidney microenvironment after AKI (Aim 2); and determining the role of the ECM1-organized cell-matrix interactions in promoting AKI repair in vivo (Aim 3). Our investigations have broad implications for elucidating mechanisms for kidney repair and designing novel therapeutic regimens to prevent or mitigate AKI.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT. Phosphorylation is one of the most ubiquitous, reversible posttranslational modifications in cells, and is a critical component of most signaling cascades. Strict temporal and spatial control are essential for the fidelity of this process, as derailed signaling cascades lead to disease. Here, we continue our long-standing effort to investigate signaling in neurons. If neuronal signaling goes awry, the most prominent results are well known diseases, such as Alzheimer's disease and stroke. Recently, we successfully determined how the most ubiquitous neuronal ser/thr protein phosphatase (PPP) calcineurin (CN) recruits its substrates. Namely, CN binds regulators and substrates via CN-specific recruitment motifs (PxIxIT and LxVP). Further, we also discovered that CN uses an active site recognition sequence (TxxP) to target substrate phosphosites, which, in turn, drives vital biological functions. This is the first defined active site recognition sequence for any PPP, transforming our ability to identify novel CN-specific phosphosites. Here, we will leverage our newly established tools and discoveries to achieve three aims. First, we will establish the CN interactome and substratome in distinct neuronal populations. This will enable us to demonstrate the diversity of CN functions in neurons, define if they differ in response to stimuli as well as identify the molecular substrates that are necessary for these changes to occur. Building further on the success of the previous funding period, we will also advance our molecular understanding of CN substrate recruitment by studying two critical CN substrate signaling platforms: CN-AKAP5 and CN-TAK1. Specifically, we will show that the these signaling platforms utilize multiple, competing PxIxIT/LxVP motifs to recruit CN via different proteins and show how these distinct mechanisms define CN substrate dephosphorylation efficacy. Critically, our preliminary data suggest that posttranslational modification of AKAP5 modulates CN control of PKA activity and ultimately receptor regulation. Finally, we have also recently confirmed our prediction that the transforming growth factor-β activated kinase 1 (TAK1) binds directly to CN. However, unexpectedly the TAK1 regulator TAB2 also binds directly to CN via different LxVP and PxIxIT motifs. We will investigate the mechanisms and consequences of this interaction on CN recruitment and TAK1 function. The modes of action and regulation of CN in the AKAP5 and TAK1 signaling platforms are unexpected and highlight the broad variety of mechanisms used to regulate CN activity. Taken together, the proposed studies leverage a powerful integrated approach that combines atomic resolution techniques with biochemical and cell biology experiments to obtain novel insights into the molecular mechanisms used to direct CN activity. Because CN has critical roles in human diseases generally, and in the brain specifically, and because CN is the only successfully therapeutically targeted PPP (CN is the target of the blockbuster immunosuppressants cyclosporin A and FK-506), our proposed work will provide a critically needed molecular and cellular understanding of CN activity and regulation in neuronal function.

NIH Research Projects · FY 2026 · 2022-12

Abstract Alzheimer’s disease (AD) is the most common age-dependent neurodegenerative disease, which is largely recognized by the presence of amyloid plaques, neurofibrillary tangles, and progressive development of neuronal loss. Neuronal loss is an age-associated event, which exacerbates the loss of synapses and causes severe cognitive dysfunction. Therapeutic intervention for AD treatment should not only reduce AD pathological hallmarks such as amyloid deposition and tau aggregation, but also mitigate synaptic impairment and neurodegeneration. This proposal focuses on pre-clinical therapeutic exploration of C-terminal domain of CX3CL1 (CX3CL1-ICD), which has an activity for inducing neurogenesis and neuroprotection. We have recently discovered that a CX3CL1-ICD-derived peptide (Tet34) induces activation of insulin receptor substrate-1 (IRS-1) and IRS-2, and its downstream molecules, Akt and Foxos. Strikingly, neuronal cells treated with this peptide exhibited significantly reduced cell stress, cytochrome C release and cleavage of caspase 3, induced by the oligomeric Aβ treatment. Hence, Tet34 attenuates apoptosis and Aβ-induced cellular toxicity. In this renewal proposal, we will test the hypothesis that peptides derived from C-terminal CX3CL1 have the translational potential for improving cognitive functions by decreasing cellular stress, enhancing neural differentiation and reducing tau-mediated neurodegeneration. Specifically, we will answer the question of whether N- and C-terminal fragments of CX3CL1 have differential cellular functions, which potentially antagonize the beneficial effect of CX3CL1 in neurons. We will also explore the biochemical mechanisms underlying CX3CL1-ICD-dependent neurogenesis in adult and synaptic regulation. The knowledge gained from this study will allow us to explore our long-term and ultimate goal, which is to discover more specific molecules that can be used to treat AD patients. To test our hypothesis, we will employ multiple approaches to address questions in the following three specific aims: Aim 1: To identify potent short peptides derived from CX3CL1 C- terminal domain (CX3CL1-ICD) for reducing AD pathology; Aim 2: To determine whether N- terminal and C-terminal CX3CL1 have differential effects on tau pathology in AD mouse models; Aim 3: To determine the molecular mechanism underlying CX3CL1-ICD in the control of gene expression. By accomplishing experiments as proposed, we will gain knowledge that will reveal the role of CX3CL1-ICD in the control of AD pathogenesis.

NIH Research Projects · FY 2026 · 2022-11

PROJECT SUMMARY Despite widespread vaccination, influenza (flu) remains a leading cause of death among older adults. Vaccination is the most effective way to prevent infectious disease. However, older adults have dysregulated immune responses that reduce vaccine efficacy and leave them at risk for severe infection and death. Older adults have reduced T cell proliferation, impaired B cell responses, and decreased antibody titers following flu vaccination. Current methods to improve vaccine efficacy in older adults target singular deficits in immune responses and fail to completely rescue responses. Vaccination requires a complex coordination of multiple cell types and tissues; thus an approach that targets the overall biology of aging, in line with the geroscience hypothesis, may be more appropriate for improving vaccine protection and immune responses in older adults. Senescent cell accumulation is a hallmark of aging and evident in various tissues with age. Although these cells are characterized by a mostly irreversible state of cell cycle arrest, they remain metabolically active and importantly, secrete a heterogeneous cocktail of inflammatory cytokines and chemokines that contribute to tissue dysfunction and damage that is coined senescence associate secretory phenotype (SASP). Accumulation of senescent cells and SASP create pro-inflammatory environments and have a causal role in many age-related disorders. CD4 T cells and B cells, the main cells responsible for robust vaccination responses, are extremely sensitive to their microenvironments. Thus, we propose that accumulation of senescent cells and their SASP drive diminished vaccination responses with aging. Importantly, drugs that specifically kill senescent cells, termed senolytics, have been developed and require only intermittent administration to eliminate senescence cells and mitigate the SASP. The safety and efficacy of senolytics have been shown in mouse studies and can alleviate a range of age-related diseases. Human pilot studies have supported their safety and clinical utility in certain pathologies. However, the impact of senolytics on vaccination responses in aged populations has not yet been examined. The overall hypothesis in this proposal is that senescent cells and the SASP play a causal role in impaired flu vaccination responses with aging and that pharmacological clearance of senescent cells will improve vaccination responses. We will test this hypothesis by treating young and aged mice with senolytic drugs prior to vaccination. We will utilize two different vaccination methods, recombinant flu nucleoprotein to induce protective immunity and adjuvanted inactivated flu to induce neutralizing immunity, and then infect mice with flu to interrogate both cell-mediated and humoral vaccination responses. Additionally, we will test our hypothesis in human cells by determining how senescent cell conditioned media impact human T and B cells responses in culture. These approaches will allow us to examine the role of cellular senescence in impaired vaccination responses with aging and investigate the translational utility of senolytic drugs as a pre-vaccination adjuvant.

NIH Research Projects · FY 2025 · 2022-09

Emerging adults (ages 16-25) have been particularly impacted by the opioid crisis. Although medications are crucial for reducing mortality, return to opioid use, and other harms associated with opioid use disorder (OUD), persons in recovery who take or who have taken medications for OUDs (MOUDs) have many unmet needs and ongoing risk factors that prevent their return to full functioning and to flourishing. The recovery needs of EAs following stabilization on MOUDs are not well documented and are likely unique to this age group. EAs face developmental milestones characterized by frequent transitions and instability in education, housing, and relationships. The recovery needs of EAs taking MOUDs are further complicated by high rates of co-occurring mental health disorders, polysubstance use, and premature discontinuation of MOUDs. Recovery support services, particularly clinical continuing care delivered after a treatment episode, are likely to play a key role in the long-term management of OUD for EAs. Although such services can be found across the spectrum of real-world substance use treatment, very little high-quality research has evaluated the efficacy or effectiveness of recovery support services or continuing care specific to the needs of EAs. Further, continuing care research has been largely researcher-driven and focused on abstinence, symptom reduction, and reducing cost, rather than fostering recovery capital and the full range of outcomes valued by EAs in recovery from OUD. Genuine partnerships and collaborations are needed to ensure research on continuing care reflects the values and needs of EAs taking MOUDs. We will advance this critical but under-researched area by building a multidisciplinary, community-engaged network: The Collaborative Hub for Emerging Adult Recovery Research (CHEARR). Our proposed team has extensive expertise in recovery support services, research with real-world substance use treatment organizations, and partnerships with agencies that serve EAs taking MOUDs. We propose three overarching goals that each provide actionable deliverables: (1) use a community-based participatory approach to develop the critical tools to conduct high-quality research in this area by creating and partnering with two community boards comprised of EAs who take or who have taken MOUD and recovery supports specialists who have expertise with EAs; (2) provide a hub of science on continuing care for EAs on multiple platforms to educate and engage the larger scientific community, communities impacted by OUD, and other key partner communities; and (3) create a trainee-to-investigator pipeline through a) a postdoctoral fellowship and student internship program and b) funding pilot studies that will produce preliminary data for NIH grant applications. In addition, the CHEARR team and community boards will partner to develop an EA-specific measure of recovery capital, a crucial tool that is currently missing from the scientific literature. Psychometric data on this measure will be collected as part of the pilot study program. Through these aims, CHEARR will foster rapid expansion of science and develop the infrastructure necessary to investigate continuing care services for EAs taking MOUD.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY The Candidate is a postdoctoral fellow committed to developing an academic research group focused on applying bioinformatics to understand the pathophysiology of alcohol-related liver diseases (ALD). His previous and current postdoctoral work has given him the unique skillset to answer questions involving gene expression/regulation in the immune system. The Career Development Plan describes 2 years of mentored research wherein the candidate will develop skills in clinical immunology, generate the sequencing data outlined in the proposal, and learn leadership skills to transition into independence. The next 3 years, after obtaining an independent faculty position, will be dedicated to developing new data analysis pipelines and establishing cell biological and bioinformatics tools to understand gene regulation in the innate immune system, which will allow the candidate to establish future projects in ALD immunology. Research Plan: ALD is a spectrum of disorders that affect a growing number of people worldwide. Alcoholic Hepatitis (AH) is a severe inflammatory disease that can increase the morbidity and severity of ALD disorders. In AH, the innate immune system is hypersensitive to microbial byproducts. Alcohol consumption causes gut-barrier disruption, leading to leakage of gut microbes into the portal circulation. The liver immune system is able to detect these microbes through pattern recognition receptors, including the Toll-like receptors (TLRs) and C-type Lectin receptors (CTLs). While TLRs have been well studied for their role in sensitizing the innate immune cells to microbial products, the CTLs have only recently been implicated in mouse models of ALD. CTLs are a large family of PRRs that sense a vast diversity of microbes, including bacteria, fungi, and viruses. Our work has found that many members of the CTL gene family are upregulated in the liver and peripheral blood mononuclear cells PBMCs of AH patients. CTLs were also upregulated in PBMCs in response to LPS/TLR4 signaling. We predict CTLs are upregulated in response to gut- derived LPS in order to increase sensing for other microbes that may be present in the blood, making this pathway a secondary innate immune surveillance pathway. In this proposal, we will test this hypothesis in three aims to address the functional role and regulation of this immune surveillance pathway, and the mechanism by which it is exacerbated in AH. First, we will use PBMCs isolated from AH patients to measure increased sensitivity to CTL agonists, as well as micro-/mycobiome sequencing to determine what microbial byproducts were in circulation in patients. Second, we will use single-cell RNA sequencing (scRNA-seq) to dissect the different monocyte subclasses, variation in CTL expression, and how different cell types respond to CTL agonists. Third, because CTL genes are clustered in the genome, we will use scRNA-seq and ATAC-seq (Assay for Transposase-Accessible Chromatin) to understand co-regulation of nearby genes in the genome. Altogether, this proposal will further our understanding of CTL mediated immune surveillance in host/microbial interactions during AH disease progression and potentially identify new therapeutic targets to decrease inflammation.

NIH Research Projects · FY 2023 · 2022-09

Abstr act Certain invasion-related maternal-fetal diseases (IMFDs) occur due to either insufficient or excessive placental invasion into the endometrium. These are serious conditions, sometimes requiring surgical interventions including hysterectomies, and leading to symptoms including fetal growth restriction and preeclampsia. Previous work from the mentor (Kshitiz, UConn Health) and collaborators (Profs. Levchenko and Gunter, Yale) studying the evolutionary history of diverse placental phenotypes has established the central role of the endometrial stromal fibroblasts (ESFs) in controlling the extent of the invasion. The proposed training and research plan will allow me to study the molecular basis of the endometrial stromal control of placental invasion, including the effect of ESF-trophoblast signaling on this regulation, as I gain the training and experience needed to launch my independent research career. During the K99 phase, my previous training and experience in bioinformatics and computational biology will be augmented by training from my mentor in the systems biology approach, constituting a closed loop methodology combining phenotypic assays, theoretical modeling, experimental validation, hypothesis refinement feeding back into experimental investigations. Exploiting the apposite model of the regulation of placental invasion in eutherian mammals to understand IMFDs, I have since mathematically mapped and experimentally validated the genomic basis of this variation in depth of placental invasion through specific regulatory molecules such as GATA2 and TFDP1. Further, I found evidence that stromal invasability genes could be conserved across tissue types, with congruent effects between placental invasion in ESFs and melanoma invasion into skin fibroblasts. This opens avenues for delineating the molecular mechanisms of ESF-trophoblast signaling effects on the stromal regulation of invasion, with likely parallel mechanisms underlying dysregulated invasion in IMFDs. Using bioinformatics, bioengineered assays, mathematical modeling I found and validated the effect of IL11s secreted by extravillous trophoblasts (EVTs) on the decidual ESF invasability and MMP1 production through SOCS3. I will also explore how different subpopulations within the human endometrium interact with the invading EVTs, and the downstream signaling effect of this interaction. All molecular components identified by these methods will be validated on a bioengineered in-vitro stromal invasion assay, functionally advanced by me, to map the stromal genotype to specific invasion related sub-characteristics. Another microfabricated technology platform, that I co-developed with the mentor will be augmented to infer the sequential EVT-ESF paracrine cross-talk . During my R00 phase I will build informatics-mathematical models to predict IMFD outcomes by integrating models from my two K99 aims with deep analysis of patient sample ESF sequencing data

NIH Research Projects · FY 2024 · 2022-08

Project Summary/Abstract The liver is now recognized as an immunological organ with unique properties. Its immune response is tightly controlled to ensure immune tolerance to microbial, dietary, and metabolic products flowing from gut to liver through the portal vein. However, certain risk factors induce hepatic immune dysregulation, resulting in the development of liver disease. A high-fat and high-sugar diet (HFS), a typical Western-type diet (WD), is identified as a major risk factor contributing to the development of nonalcoholic fatty liver disease (NAFLD), ranging from simple steatosis to the advanced form of non-alcoholic steatohepatitis (NASH). Given dietary changes worldwide, NAFLD is rapidly becoming the leading cause of liver disease affecting 25% of the population worldwide. Mounting evidence indicates that the HFS and gut microbiota interaction generates a spectrum of dietary and microbial components and outcome metabolites that can induce inappropriate hepatic immune activation, suggesting a key role of the Diet/Gut/Liver/Immune axis in NASH . However, the underlying mechanisms are poorly understood. Furthermore, very little is known about the specific microbes and metabolites that regulate intrahepatic immunity. To address these major knowledge gaps, the investigators have developed a NASH model by feeding wild-type mice with a choline-low HFS (CL-HFS) (0.05% choline) which closely approximates a typical WD in composition. This model is characterized by gut dysbiosis, metabolic disarray, abnormal hepatic immune response, and liver-resident macrophage (MΦ) and hepatic stellate cell (HSC) activation, reflecting typical pathologic properties in human NASH patients. Using the model, the investigators demonstrate that selective suppression of gut microbiota preventively and therapeutically inhibits CL-HFS-induced NASH. Metagenomic and metabolomic analyses in combination with in vitro and in vivo experiments identified Blautia producta (B. producta) and its product 2-oleoyglycerol (2-OG) as an unrecognized bacterium and metabolite contributing to CL-HFS-induced abnormal hepatic immune response. Of particular clinical relevance, enrichment of gut Blautia and high levels of hepatic 2-OG are found in human NASH patients. Mechanistic studies suggest that 2-OG primes MΦs via G protein-coupled receptor 119 signaling, subsequently activating HSCs. These exciting results support the hypothesis: CL-HFS, B. producta, and 2-OG, by activating MΦs through GPR119 signaling pathways, cause hepatic pathogenesis and HSC activation. This hypothesis will be tested in the following Aims: Aim 1: Determine MΦ as a cellular basis of CL- HFS-induced NASH pathogenesis mediating crosstalk between gut microbiota, HFS, and liver; Aim 2: Determine GPR119 as a molecular basis of MΦ mediating hepatic pathogenesis induced by CL-HFS, B. product, and 2-OG. This study will dissect the underlying cellular and molecular mechanisms to advance the understanding of the role of the Diet-Gut-Liver axis in hepatic immunity, which will advance the development of dietary and microbial interventions that therapeutically suppress this global health threat.

NIH Research Projects · FY 2024 · 2022-08

Project Summary My long-term goal is to identify how individuals can mitigate exposures to the deleterious health effects of air pollution through practical lifestyle adjustments. My primary project objective is to investigate how an individual's choices influence personal exposures to traffic-related air pollutants (TRAP) and the corresponding acute health effects. It has been reported that traffic pollutants may cause up to half of all air pollution related mortalities. Despite the burden from such widespread, involuntary exposures, few studies have examined the magnitude of personal exposures due to commuting exposures. A commuter's exposure is dependent on the mode of transport, time of day, route, and fuel type. Public transportation, bicycling, and walking have been promoted as ways to reduce air pollution by reducing the vehicle fleet, yet few studies have examined how exposures would be modified due to a change in the mode of transportation or the subsequent health effects. In this study, 65 participants will be asked to utilize two modes of transportation (car and either bicycle, rail, or bus) during their typical commute over 4 days (two days per mode). During each 24-hr period, the participants will be examined for changes in oxidative stress biomarkers and acute cardiovascular and respiratory endpoints and 7+ pollutants and noise levels will be quantified in real-time. I hypothesize that there will be a significant association between exposure levels and acute health endpoints. An examination will be conducted into whether this association is modified by mode of transport. I will also examine the influence of the time of day an individual chooses to commute to work and exercise outdoors in a second cohort of 75 participants. Since TRAP tends to be most concentrated during two relatively short periods of time (morning and evening rush hours), I hypothesize that altering the time of exposure will notably change personal exposures. This project will bring in a key components of the personal exposure paradigm, an individual's decision- induced reductions in exposure. We will identify modifiable factors/personal choices that can reduce exposures with the objective of identifying which reductions in exposure can lead to meaningful health benefits. This knowledge will enable individuals to make lifestyle choices that can reduce their exposure independent of the ambient air quality or regulatory changes. This will be particularly beneficial in the study location (Baltimore), where the combustion-related mortality rate is highest in the U.S. It is expected that the information obtained in this K99/R00 will be applicable to other metropolitan areas with similar infrastructure. Through this research, my didactic coursework, and the guidance of my mentoring team (comprised of a pulmonologist, a biostatistician, an environmental epidemiologist, and an exposure scientist), I will acquire critical skills required to be a successful, independent researcher in environmental health sciences.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Phosphorylation is one of the most ubiquitous, reversible posttranslational modifications in cells. The enzymes responsible for controlling the phosphorylation state of the cell are kinases, which catalyze the transfer of the γ-phosphate moiety of ATP to substrates, and phosphatases, which catalyze the reverse hydrolysis reaction, the removal of the phosphate moiety from phosphorylated substrates. Thus, phosphatases dynamically reverse the effects of kinases. Because phosphorylation is critical for all biological processes from cell growth to differentiation to development, the location and duration of the reciprocal actions of kinases and phosphatases must be exquisitely regulated both temporally and spatially within the cell. Consequently, when this tight regulation is disrupted, dysregulation of phosphorylation signaling ensues and the consequence is most often disease. Deletion of either one of two PP1 regulators—SDS22 (PPP1R7) or Inhibitor-3 (I3; PPP1R11 or Ypi1 in yeast)—is lethal in yeast (essential genes), highlighting their biological significance. However, since their discovery, different biological roles have been assigned to SDS22 and I3, including roles in mitosis (SDS22), E3 ligase functionality (I3), PP1 biogenesis, among others. Thus, while it is clear that SDS22 and I3 are essential PP1 regulators, their true biological function(s) and especially their mechanism(s) of action are still unknown. This has hindered progress in understanding their roles in PP1 biology. In cells, these proteins form both heterodimeric (SDS22:PP1 and I3:PP1) and a heterotrimeric (SDS22:I3:PP1; SIP) PP1 complex. The structure and function(s) of the individual dimeric complexes, if and how the structure and function(s) of the trimeric complex differs from those of the dimeric complexes and the role(s) of each complex in PP1 holoenzyme formation are major questions in the field. Further, additional data suggest that dissociation of the SIP complex requires the AAA+ ATPase p37/p97. However, the molecular details of SIP complex dissociation have also remained elusive. The presented research project uses a powerful integrated approach that combines structural biology with biochemical and cell biology experiments to obtain novel insights into the molecular mechanisms used by these regulators to control PP1 activity and direct PP1 holoenzyme assembly. Because PP1 holoenzymes have critical roles in human diseases, the proposed work will provide novel strategies for selectively inhibiting PP1 activity by targeting the PP1 holoenzyme formation and subunit exchange, which is essential for understanding how distinct PPPs contribute to disease.