University Of Connecticut Sch Of Med/Dnt

NIH Research Projects · FY 2025 · 2025-08

Particulate air pollution (PM2.5) is the 4th leading cause of morbidity and mortality. Attention from leading health organizations has recently turned to interventions to reduce exposure and prevent adverse health outcomes. As evidence has begun to mount for the efficacy of these machines, and will likely grow further in the coming years, it has, however, become apparent that for most people, including low-income populations in the US, cannot afford effective commercially available units. Low-cost commercial air purifiers are of low efficacy and often introduce new pollutants into the air. To our knowledge, there are no published intervention studies of Corsi-Rosenthal Boxes (C-R Boxes), the devices we propose to use in this study. We have a study team that is ideally suited for the proposed research. Ms. Creed has extensive experience building and deploying the C-R Boxes which became popular during the Covid pandemic. Dr. Brugge has three published RCTs of high-quality commercial air purifiers and another, full trial, nearing completion. Thus, he has knowledge and experience which qualifies him to lead this proposed study. Drs. Levy Zamora has extensive air monitoring expertise, will direct measurement of indoor and outdoor air pollution concentrations at each home. Dr. Eliasziw is a biostatistician with extensive experience analyzing randomized trials. We have three aims: 1)Conduct focus groups/interviews and BP measurements with participants who meet study inclusion criteria to refine our protocol; 2) Conduct a randomized crossover pilot trial with 65 participants to calculate preliminary effect size estimates to inform a larger crossover efficacy trial; and 3) Determine the feasibility of conducting a larger crossover efficacy multisite trial in the US. We will conduct our randomized cross over trial in three settings with low, medium, and high air pollution. Our low and medium air pollution locations will be in Hartford CT and our high pollution setting will be in Boston Chinatown, where we can reliably expect high pollution levels. Participants (N=65) will have dried blood spots collected and blood pressure measured at the start and end of each 4-week intervention session. Our randomized cross over design controls for time invariant confounding. Interviews and standardized questionnaires with study participants will provide feedback that will inform the decision as to whether to proceed with a full trial. We seek to meet targets for 80% recruitment and retention as well as 80% satisfaction as benchmarks for moving to a full trial. The findings for health end points will provide preliminary data to justify the potential future R01 proposal for a fully powered clinical trial. An expert elicitation process conducted with the research team in the final year will make the final decision about a future, full trial. The significance of this work is that showing efficacy of C-R Boxes for reducing exposure would lead to their widespread use and contribute to improving public health

NIH Research Projects · FY 2025 · 2025-08

Abstract The HIV/AIDS epidemic poses a major threat to public health, affecting 40 million people worldwide. Early diagnosis of acute HIV infection during the seroconversion window facilitates early intervention. Monitoring HIV viral load frequently during antiretroviral treatment (ART) is essential to confirm treatment effectiveness and detect viral rebound. HIV viral load testing, which quantifies HIV viral RNA (circulating virus) in plasma, is the gold standard and the most effective method for ART monitoring and acute HIV detection. However, standard HIV viral load testing methods rely on expensive equipment and well-trained personnel, limiting their use to centralized laboratories and hospital environments. Although several portable HIV RNA diagnostic platforms are commercially available, they still require relatively expensive (> $10K) benchtop instruments and trained technicians, rendering them unsuitable for point-of-care applications. Here, we propose to develop a rapid, affordable, and sensitive CRISPR cascade signal amplification approach for HIV viral load testing in clinical samples. We will investigate the allosteric regulation mechanism of CRISPR activation by HIV RNA with various secondary structures and sequences. Furthermore, we will develop a CRISPR cascade signal amplification assay for HIV detection at room temperature. To simplify the detection operation, we will incorporate the assay into a microfluidic platform to create an accessible diagnostic tool. We will validate the clinical application of this microfluidic diagnostic approach in collaboration with clinicians. If successful, this simple and cost-effective CRISPR cascade signal amplification method will enable acute HIV diagnosis and viral load testing at resource- limited settings.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD) are devastating progressive neurodegenerative diseases characterized by neuronal dysfunction and loss. Dysfunction of the vascular endothelium precedes and likely contributes to cognitive decline by impairing blood-brain barrier (BBB) integrity. However, the exact timing of endothelial dysfunction, neuroinflammation, and cognitive decline has not been determined, in part due to the challenges associated with reliably detecting and characterizing endothelial changes in vivo. Our lab identified a unique transcriptional state in capillary endothelium, marked by nuclear TDP-43 depletion, in human donors with ALS-FTD. TDP-43, encoded by the TARDBP gene, is an RNA-binding protein implicated in both familial and sporadic forms of ALS-FTD. Nuclear loss of TDP-43 leads to pathological protein aggregation. Mouse models of nuclear TDP-43 depletion show endothelial dysfunction and increased BBB permeability, with significant transcriptional overlap between these models and human patients. Understanding how vascular alterations, including endothelial dysfunction and BBB breakdown, evolve during disease progression remains a critical gap. To gain an understanding of molecular changes preceding vascular dysfunction, this project aims to leverage cerebrovascular endothelial extracellular vesicles (cEC-EVs) to sample the capillary brain endothelium, given their unique ability to preserve RNA and protein cargo. In Aim 1, we will identify markers of nuclear TDP-43 depletion in cEC-EVs using a brain endothelial cell-specific TDP-43 deletion mouse model combined with BirA-mediated biotinylation to selectively isolate brain-endothelial-derived secreted proteins and cEC-EVs. Complementary in vitro studies will validate EV content via RNA sequencing and proteomics. Aim 2 focuses on longitudinal characterization of EV composition in a TDP-43 knock-in mutation mouse model, which partially recapitulates molecular features of human ALS-FTD. These analyses will assess EV-derived RNA and protein markers as indicators of endothelial dysfunction across neurodegenerative disease progression. This research seeks to establish a novel approach for in vivo sampling of the BBB and to establish a timeline linking endothelial dysfunction to neuroinflammation and cognitive dysfunction. The approaches developed could offer valuable insights into vascular dysfunction in ALS-FTD and inform future diagnostic strategies.

NIH Research Projects · FY 2025 · 2025-08

Abstract Syphilis rates in the United States and globally have skyrocketed since their nadir in the early 2000s, creating a pressing need for novel containment strategies rooted in improved understanding of the basic biology of Treponema pallidum (TPA). For three principal reasons, our understanding of syphilis pathogenesis has lagged behind most other bacterial infections. First and foremost was the inability to genetically manipulate TPA in vitro. Second was the limited knowledge regarding the genetic programs that enable syphilis spirochetes to persist in humans. Last was the poor understanding of TPA's outer membrane protein (OMP) repertoire, which constitutes the host-pathogen interface during human infection. This proposal leverages recent breakthroughs in basic syphilis research to investigate fundamental aspects of disease pathogenesis – nutrient acquisition and environmental sensing. Using the in vitro cultivation system developed by Edmondson and Norris, we generated data supporting physiologic/virulence-related functions for three of the five `atypical' FadL-like orthologs encoded by TPA. These findings led us to postulate that these OMPs form a new functional class of transporters that import poorly soluble nutrients required for growth of TPA, an extreme auxotroph. In Aim 1, we will phenotypically characterize TPA mutants containing insertions in one or more FadL loci to determine their respective contributions to growth in vitro, adherence to mammalian cells, and infectivity in the rabbit model. Our mining of the TPA genome also revealed regulatory pathways linking host-derived small molecules with the production of the cyclic nucleotide second messangers cAMP and c-di-GMP. In our working model, ligand binding by the periplasmic CACHE domain for TPA's adenylate cyclase TP0485 activates synthesis of cAMP by the protein's cytosolic cyclase domain. cAMP, in turn, enables DNA binding by TPA's canonical CRP transcription factor (TP0262), whose regulon includes members of the Tpr OMP family. We further postulate that cAMP serves as an activating ligand for the spirochete's cytoplasmic diguanylate cyclase (TP0981) GAF domain, leading to the production of c-di-GMP; c-di-GMP exerts its signaling function via TPA's sole PilZ protein (TP0086), an allosteric effector potentially mediating changes in transcription and/or motility potentially involved in persistence. In Aim 2, we will generate TPA strains defect in cyclic nucleotide signaling to assess their contributions to growth of TPA in vitro and infectivity in the rabbit model. Using complementary approaches, we will also examine crosstalk between the cAMP and c-d-GMP signaling pathways. The research described in this proposal will yield the first systems biology level understanding of physiologic, regulatory, and pathogenic processes that enable the stealth pathogen to establish persistence in individuals and flourish in at-risk populations.

NIH Research Projects · FY 2026 · 2025-08

Revised Abstract Section A significant minority of older adults display persistent cognitive impairments after the acute phase of a viral infection, referred to as Postviral Neurocognitive Dysfunction (PND). Underlying mechanisms remain poorly understood, though chronic neuroinflammation appears to reflect a key pathway. PND is debilitating, increases the risk for accelerated biological aging and dementia, and is associated with a substantial economic burden to society. Older adults are at heightened risk for PND given weakened immune systems, baseline age-related cognitive decline, and susceptibility to more severe acute viral illness. There is a critical lack of evidence-based treatments. The goal of this project is to determine the potential of a neuroplasticity-based computerized cognitive remediation (CCR) intervention for treating PND in older adults and probing underlying mechanisms. The proposed design is a randomized, two-arm, clinical trial pilot study. Older adults with PND (N = 75) will be assigned to a 6-week course of neuroplasticity-based CCR or an active, computer-based control condition. Specific aims are to: examine preliminary efficacy of CCR for improving cognitive performance and day-to-day functioning in older adults with PND (Aim 1); optimize and refine the CCR program for older adults with PND using iterative, data-driven, participatory design methodology (Aim 2); and determine if CCR reduces peripheral inflammation as a potential mechanism of clinical symptom relief (Exploratory Aim 3). The resultant data will guide a more definitive randomized controlled trial in PND via an R01. This K23 Career Development Award will provide the experience and training necessary for Dr. Lindbergh to achieve his long-term goal of becoming an independent investigator focused on developing neuroscience- informed behavioral treatments for late-life neurocognitive disorders. His plan for career development is organized around cultivating knowledge and skill in three primary areas: (1) development, administration, and evaluation of digital medicine technology; (2) clinical trial design, management, and data analysis; and (3) immunology, immune-inflammatory dysregulation, and blood-based inflammatory biomarkers. Dr. Lindbergh has assembled an outstanding mentorship team with expertise in each of these areas who will systematically evaluate his training progress and direct him through a robust curriculum of formal coursework, workshops, meetings, seminars, guided readings, and “hands-on” research experiences. The impressive infrastructure and multitude of resources available through the University of Connecticut Health Center—including the UConn Center on Aging, NIA-supported Claude D. Pepper Older Americans Independence Center, Psychiatry Department, Immunology Department, and Digital Media and Design Department—will provide the ideal scientific environment for Dr. Lindbergh to complete his training and transition to independence as a leading patient-oriented aging researcher.

NIH Research Projects · FY 2025 · 2025-07

PROJECT SUMMARY/ABSTRACT Neurodevelopmental disorders affecting the large and folded (gyrencephalic) human cerebral cortex require disease mechanisms that are robust in humans but insignificant in mice. For example, loss of ASPM (assembly factor for spindle microtubules) in humans causes severe microcephaly with 50-75% reduction in cortical volume, simplified gyri, epilepsy, and intellectual disability. In contrast, Aspm KO mice show <10% reduction. ASPM encodes a conserved centrosomal protein that regulates progenitor proliferation, delamination, and differentiation. Thus, it is unclear how loss of a neurodevelopmental gene such as ASPM causes remarkably severe phenotypes in the large, gyrencephalic human cortex compared to the mouse cortex. The ferret is a genetically and surgically tractable, gyrencephalic carnivore that bridges the gap between human brain disorders and mouse models. Its cortex shows transcriptional patterns similar to human cortex, and contains numerous outer radial glia, a progenitor cell type abundant in humans but rare in mice. Our germline Aspm KO ferrets show severe microcephaly with a 25-40% reduction in cortical volume and simplified gyri. Moreover, Aspm KO ferrets, but not mice, show premature progenitor delamination and death. Importantly, ASPM KO human cerebral organoids show the same progenitor defects as Aspm KO ferrets, as reported by the Vanderhaeghen lab (Benthem et al., 2023). However, the molecular mechanisms by which only human and ferret progenitors are uniquely susceptible to loss of ASPM are unknown. Our LONG-TERM GOAL is to elucidate the unique developmental mechanisms of the large and gyrencephalic human cortex in health and disease. ASPM microcephaly is a neurodevelopmental disorder for which human organoid, ferret, and mouse models are available, making it an ideal candidate for investigating gyrencephalic mammalian-specific mechanisms. Our CENTRAL HYPOTHESIS is that loss of ASPM triggers similar molecular changes in humans and ferrets, but not in mice, via gyrencephalic mammalian-specific ASPM interactors. To test the hypothesis, we propose three Specific Aims: (1) To systematically examine ferret and mouse cortical development in the presence and absence of ASPM using spatial transcriptomics; (2) To determine molecular mediators of progenitor delamination and death in ASPM KO human cerebral organoids and ferrets by comparing KO progenitors at one time point and similarly delaminating WT progenitors at a later time point in human organoids and ferrets using single-cell RNA sequencing; (3) To define the ASPM interactome in human, ferret, and mouse progenitors using proximity labeling and centrosome proteome analysis. The roles of newly identified mediators of progenitor defects will be tested in rescue experiments in ASPM KO human organoids and ferrets. With our strong expertise and cross- species approach using human organoids, ferret and mouse models, we are uniquely qualified to rigorously investigate gyrencephalic cortical development in health and disease.

NIH Research Projects · FY 2025 · 2025-07

Abstract It is now universally accepted that the alternative sigma factor RpoS serves as a master regulator for differential gene expression in Bb. Using a novel enrichment RNAseq technique, we established that the RpoS regulon changes dramatically as spirochetes undergo mammalian host adaptation and that the mammalian phase RpoS regulon includes virulence determinants required for early infection. The unexpected discovery that the BosR/RpoS regulon includes vlsE was a clear indicator that RpoS is needed not only to establish infection but also to maintain it. Using a rpoS mutant complemented with an IPTG-inducible rpoS allele, we established that Bb do not survive in mice once RpoS is turned OFF and that persistence involves elements within the RpoS regulatory network functionally unrelated to evasion of adaptive immunity. These findings set the stage for Aims to achieve our overarching objective – elucidating how RpoS-dependent gene regulation perpetuates the mammalian phase of the spirochete's enzootic cycle. In Aim 1, we use our IPTG-inducible system for controlling borrelial gene expression in vivo interrogation individual RpoS-regulated genes in specific milieus and time points throughout the course of murine infection. In Aim 2, we dissect the topological features and promoter elements used by RpoS, BosR and YebC to collaboratively control expression of vlsE and determine the relationship between expression of vlsE and switching in vivo. The proposed experiments will shed new light on how LD spirochetes persist in nature, the ecological substrate for human disease, as well as how they persist in human tissues, giving rise to the chronic, debilitating clinical manifestations that make them a major threat to health and wellbeing.

NIH Research Projects · FY 2025 · 2025-07

Summary: MicroRNAs (miRNAs or miRs) are short, non-coding RNAs emerging as promising tools for treating various diseases, including cancer and stroke. Their small size and similar sequences across species make them ideal targets for drug development. Recent FDA approvals for mRNA-targeting drugs, like Mipomersen for hypolipidemia and Nusinersen for spinal muscular atrophy, highlight progress in this field. However, miRNA- targeted therapies, especially for conditions like ischemic stroke, require more research. This proposal focuses on the miR-200/141 family, particularly miR-141-3p, which is significantly upregulated in stroke cases. Our recent studies show that stroke and factors altering stroke responses, such as social isolation, can modulate miRNAs from the miR-200/141 family. Specifically, miR-141-3p expression is highly upregulated. Treatments with commercial phosphorothioate (PS) or Peptide Nucleic Acid (PNA)-based miR-141-3p inhibitors have significantly reduced stroke injury, proving effective in both young and aged animals. Our preliminary data suggest that move advanced serine gamma PNA-based miR-141-3p inhibitors (sγPNA-141) are highly efficacious (>60% vs. PNA) in inhibiting miR-141-3p expression, reducing infarct injury, and improving long-term functional recovery after ischemic stroke. Elevated levels of miR-141-3p in human stroke patients make it a promising target for stroke therapy. Peptide nucleic acids (PNAs) are synthetic DNA mimics that bind to miRNA target sites, inhibiting their function. This proposal aims to develop new PNA analogs, such as gamma (γ)PNAs and several other analogues, with improved binding and solubility, thereby enhancing their effectiveness as miRNA inhibitors. The primary goal is to establish γPNAs-141 as effective treatments for ischemic stroke. The research includes three aims: 1. Synthesize and characterize newer and more potent γPNA-based miR-141-3p inhibitors (γPNA-141), using advanced γPNA-141 for better efficacy. We will perform quality control and assess binding affinity. 2.Identify the most effective γPNA-141 inhibitors and study their neuroprotective mechanisms in stroke models.3. Determine the optimal treatment window and assess the long-term recovery effects in aged mice after stroke.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY/ABSTRACT Osteoarthritis is a chronic inflammatory and degenerative disease of the joint affecting the cartilage, synovium and subchondral bone. Notch signal activation has been associated with the development of osteoarthritis, and inactivation of its canonical signaling prevents the osteoarthritis that follows destabilization of the medial meniscus (DMM). These findings indicate a role of Notch in the pathogenesis of osteoarthritis. However, the mechanisms responsible are not understood. We created a mouse model harboring a moderate gain-of-NOTCH2 function mutation and termed Notch2tm1.1Ecan. Notch2tm1.1Ecan mice are sensitized to the development of osteoarthritis following DMM surgeries, and chondrocytes from mutant mice are sensitized to the actions of tumor necrosis factor α (TNFα) and interleukin 1β (IL1β). The induction of interleukin (IL)6 and IL1β by TNFα and IL1β is amplified significantly in Notch2tm1.1Ecan chondrocytes, an effect that is independent of canonical signaling indicating the existence of novel signaling pathways operating in osteoarthritis. Our findings demonstrate a new role for Notch in the inflammatory response and osteoarthritis, but the cell and mechanisms responsible are not known. To explore the consequences of the NOTCH2 gain-of-function in specific articular cells, we created a Notch2 conditional by inversion (COIN) mouse model. The goal of our research is to establish the cell and mechanisms responsible for osteoarthritis and the enhanced inflammatory response to NOTCH2. Our specific aims are: Aim 1. To define the cell responsible for the NOTCH2-dependent osteoarthritis. To this end, we will utilize a conditional Notch2 mouse model to induce the NOTCH2 gain-of- function in specific articular cells; Aim 2. To establish the mechanisms responsible for NOTCH2 dependent osteoarthritis by defining transcriptome profiles and pathways responsible for osteoarthritis in the context of a NOTCH2 gain-of-function; and Aim 3. To establish the mechanisms responsible for NOTCH2-dependent inflammation by defining signaling pathways influenced by NOTCH2 to enhance the inflammatory response. The overarching goal of the proposed work is to discover novel mechanisms of Notch signaling operational in osteoarthritis and interrogate undiscovered Notch-related pathways and mechanisms responsible for an enhanced inflammatory response to Notch in cartilage.

NIH Research Projects · FY 2026 · 2025-05

ABSTRACT Osteogenesis imperfecta (OI) is a genetic disease that causes abnormal type I collagen production leading to bone fragility. Current pharmacological approaches result in an increase in the amount of defective matrix, without addressing the underlying collagen defect. An alternate approach is cell therapy. Cell therapy is based on transplanting stem/progenitor cells which can subsequently differentiate into osteoblasts and produce normal collagen. To date, preclinical and clinical studies attempting systemic transplantation of mesenchymal stem cells (MSC) in OI have had very limited success. We evaluated direct intra-bone transplantation of MSCs following total body irradiation as a therapeutic approach. Utilizing a set of stringent criteria for detection and evaluation of successful engraftment, we showed that donor MSC/progenitor cells are capable of differentiating into osteoblasts and osteocytes resulting in an increase in bone mass and improved mechanical properties of transplanted bone. Use of irradiation proved to be a critical for achieving successful engraftment. We hypothesize that by understanding mechanism of irradiation on local bone directed transplantation of MSCs we would be able to achieve a long- term successful therapy for OI. This approach of directed therapy to individual bones has the potential to improve bone growth and the outcome of the surgical procedures performed on the transplanted bones. Our study has three specific aims focusing on increasing a long-term functional engraftment of selected MSCs in the OI. Aim 1 we will evaluate different populations of cells that are accessible and can be utilized as progenitor sources for transplantation studies. In 2 aim we will determine the mechanisms by which irradiation improves engraftment. We will test the effects of irradiation on multiple lineages within the bone marrow-bone and endosteal niche. Effects on hematopoietic lineage, vascularization, mesenchymal progenitors will be examined. Understanding these mechanisms has the potential to facilitate development of optimal host preconditioning approaches for direct intra bone marrow transplantation. In Aim 3 we will test alternative methods to total body irradiation such as myeloablation. To avoid whole-body irradiation, cells will be transplanted into a femur following focal limb irradiation and effects of transplantation will be assessed with or without fracture. Our approach of targeted cellular therapy to individual bones has potential to improve the bone growth and the outcome of surgical procedures performed in OI patients. This work is prerequisite for success of any ex vivo gene-therapy attempts and our results will define a translational approach to improve treatment of OI.

NIH Research Projects · FY 2025 · 2025-05

Project Summary/Abstract Parkinson's disease (PD), Parkinson’s disease dementia (PDD), and dementia with Lewy bodies (DLB) are the common neurodegenerative disorders severely affecting the aging population worldwide. PD is recognized as the most common neurodegenerative movement disorder. Up to 80% of individuals with PD develop cognitive impairment which progresses into overt dementia, leading to a diagnosis of PDD. DLB accounts for roughly 5% of dementia cases in elderly people and is associated with severe and widespread pathological findings of Lewy bodies in the brain, followed by parkinsonism in over 85% of cases. Most patients with PD, PDD and DLB do not carry mutations in known disease-causing genes. Recently, loss-of-function variants in the low- density lipoprotein receptor-related protein 10 (LRP10) gene have been associated with PD, PDD, and DLB. How its loss-of-function is mechanistically involved in the pathogenesis of PD, PDD and DLB and what is the physiological and pathological role of LRP10 remain largely unknown. No LRP10 animal models have been developed and characterized. We propose to generate and characterize the first LRP10 knockout (KO) mouse model. Our preliminary data shows that LRP10 KO mice exhibit both locomotor and cognitive function deficits and develop a-Synuclein (aSyn) and tau pathology. Proteomic analysis and immunoprecipitation (IP) pulldown revealed potential LRP10 targets/ interactors. Based on these observations, our hypothesis is that the loss of LRP10 function causes PD and dementia, which acts through LRP10-specific targets. To test this hypothesis, we will examine neuroinflammation, synaptic dysfunction, neurodegeneration, and associated cellular and behavioral deficits with aging in LRP10 KO mice and determine the potential underlying molecular mechanisms. We anticipate that our work will provide insights in LRP10 physiological functions and pathological roles, and further provide a common mechanism for the Lewy body disorders (PD, PDD and DLB) in general.

NIH Research Projects · FY 2026 · 2025-04

ABSTRACT Synaptic adhesion molecules (SAMs) control the structural and functional properties of synapses and thereby shape neural circuit development and function. Genetic variants of SAMs are linked to various neurological disorders, but the mechanisms by which they exert such pathogenic effects remain elusive. The central goal of this research plan is to determine the mechanism by which the secreted synaptic protein C1QL3 promotes excitatory synaptogenesis. It was previously shown that C1QL3-deficient mice exhibited fewer excitatory synapses and multiple behavioral abnormalities. In preliminary work, we have demonstrated that C1QL3 mediates the formation of a cell-cell adhesion complex via interactions with both neuronal pentraxins (NPTX1/R) and adhesion G protein-coupled receptor B3 (ADGRB3). Furthermore, in a screen for novel C1QL3 binding partners, we identified a candidate matricellular protein involved in the regulation of matrix metalloproteinase (MMP) activity and showed that its overexpression increases excitatory synapse density in cultured neurons. These results suggest an MMP-mediated regulatory mechanism for the action of the C1QL3 adhesion complex. Our overarching hypothesis is that C1QL3 and MMPs co-modulate synaptic plasticity by regulating a novel cell- cell adhesion complex at excitatory synapses. To characterize C1QL3 interactions with its binding partners and to advance our understanding of these interactions in governing C1QL3 functions, we have developed the following specific aims. 1. To elucidate the structural basis for the transsynaptic interactions of C1QL3 with both ADGRB3 and NPTX1/R using NMR spectroscopy, mutagenesis, and cell-based assays. We hypothesize that such interactions promote synapse formation and maintenance. We have obtained a 1H-15N HSQC spectrum of the binding domain of C1QL3 and will carry out chemical shift perturbation (CSP) titrations with the corresponding interacting domains of NPTX1 and ADGRB3. We will identify each binding interface, and characterize mutations that will weaken these interactions. Mutant C1QL3 proteins will be tested in vitro for their ability to promote both the formation of the adhesion complex and cell-cell adhesion. We will look for correlations between the structural and physiological effects of the mutations. 2. To elucidate the mechanism by which MMPs regulates C1QL3- mediated synapse homeostasis. We hypothesize C1QL3 interacts with a novel protein that interferes with the proteolytic cleavage of the C1QL3 adhesion complex by MMPs. CSPs will be used to identify specific C1QL3 residues involved in interactions with this protein. We will investigate, as in Aim 1, the effects of mutations on these interactions in cellular contexts. This exploratory proposal will provide insights into the assembly and regulation of a novel transsynaptic adhesion complex and enhance our understanding of how this complex controls synapse structure and function. 1

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY / ABSTRACT Osteoarthritis (OA) is the most prevalent joint disease and a leading cause of chronic pain and disability, affecting approximately 32.5 million Americans. In the absence of disease-modifying therapy, there is an urgent need for effective, widely available approaches to aid in the prevention and management of this common condition. As an important lifestyle factor, alcohol use has been associated with over 60 acute and chronic health conditions controversially. Previous studies have examined the cross-sectional association of alcohol consumption with the prevalence of knee OA, but the evidence from well-designed cohort studies or randomized clinical trials remains limited. Recently, using the data from Osteoarthritis Initiative (OAI), a multi-center prospective cohort study of knee OA, we observed a prospective association between excessive alcohol intake and a higher risk of knee OA. However, self-report alcohol assessment methods may be subject to recall bias and measurement error. Inaccurate measurement of alcohol exposure may make it difficult to detect moderate associations with disease risk. Biomarkers of alcohol intake taking into account the bioavailability and metabolism may better represent long-term exposures. Recently, metabolomics, by measuring a large number of downstream components (metabolites), provides the most integrated profile of biological status reflecting environmental and genetic interactions, and therefore may more precisely define alcohol and dietary exposures and provide better estimates of disease risk in epidemiologic studies. To date, intervention studies have identified a variety of metabolites related to alcohol consumption, and 15 of them have been well-validated by multiple studies. However, no study has ever assessed the association between alcohol-related metabolites and the future risk of knee OA. Using the existing data from a well-defined case-cohort study including 603 participants (237 incident knee OA cases and 366 non-cases) in the OAI cohort, we propose to examine whether individual metabolites related to alcohol intake predict the subsequent incidence of radiographic and symptomatic knee OA. Additionally, we will derive a composite score to measure the metabolomic signature of overall alcohol consumption accounting for the relative importance of individual metabolites and examine the association with the knee OA risk. An external sample from the Multicenter Osteoarthritis Study (MOST) will be used to validate the findings. Then we will examine whether alcohol-related metabolites and the metabolomic score influence imaging markers of knee OA (e.g. cartilage damage, bone marrow lesion, effusion). Finally, we will examine whether imaging markers of knee OA mediate the associations of alcohol-related metabolites and the composite score with the risk of knee OA. Using advanced metabolomic techniques, this innovative study will examine whether alcohol consumption is a modifiable risk factor for knee OA and offer the potential to identify OA prevention strategies, therefore it will have large potential public health implications. In addition, this study will provide preliminary data for future R01 grants to examine the underlying biological pathways in a multi-omics study (such as genomics, metabolomics, proteomics, etc.).

NIH Research Projects · FY 2026 · 2025-02

ABSTRACT Ubiquitin-Proteasome Pathway (UPP) is a tightly regulated machinery that controls the degradation and turnover of 80-90% of proteins in human cells. Its malfunction is linked to a plethora of human diseases, from neurodevelopmental disorders and viral infections to cancer. The overarching goal of our research program is to develop a detailed understanding of how UPP components recognize their substrates and execute their function, which is of paramount importance for the development of new therapeutic strategies in the future. This MIRA proposal aims to extend our successful UPP research program with a vision to structurally and functionally characterize critical ubiquitinating and de-ubiquitinating enzymes implicated in human diseases. Previously, we have explored protein regulators of the p53 pathway most commonly affected in cancer with a focus on the deubiquitinating enzyme USP7. As a result of this work, we have shown that FL-USP7 is a dynamic enzyme that adopts multiple conformations with transient inter-domain interactions in solution, characterized conformational dynamics of the catalytic domain of USP7 and showed that its active site switches between inactive and low- populated “excited” active states. Together, our data suggest that structural plasticity might be a key feature of USP7 required for the regulation of its activity and might even represent a common mechanism shared by other deubiquitinating enzymes with conserved catalytic sites. This proposal will take advantage of our recent discovery of USP7 mutants with enhanced enzymatic activity identified among patients suffering from Hao-Fountain syndrome – a novel and rare neurodevelopmental disease caused by mutations of the Usp7 gene. Hyperactive mutants are rare in enzymes and provide a unique opportunity to investigate mechanisms of enzymatic activity regulation. In Project 1 we will address the question of the role of conformational dynamics in the regulation of USP7 activity by comparing the dynamics and structures of the hyperactive mutants to those of the WT USP7. A combination of enzymatic assays, NMR relaxation experiments, and X-ray crystallography will be used. Project 2 will focus on the structural characterization of a complex between USP7 and its substrate implicated in Hao-Fountain syndrome development, MAGE-L2 using sequence analyses, quantitative binding assayas, NMR, and X-ray crystallography. In Project 3 cell-based assays and proteomic analyses of Hao- Fountain patient cells will be used to identify major cellular substrates/pathways affected by USP7 malfunction. The anticipated results will provide fundamental molecular mechanisms of action of clinically relevant deubiquitinating enzyme and will help guide future efforts to develop novel therapeutic strategies to treat human diseases associated with UPP malfunction.

NIH Research Projects · FY 2026 · 2025-02

ABSTRACT Atherosclerosis is a cardiovascular disease that underlies 50% of all deaths in Western societies. This disease is characterized by chronic inflammation and lipid-laden plaque buildup on the interior of major arteries, and the risk factors for atherosclerosis can be due to genetic and/or environmental factors. The major cell type involved in atherogenesis is macrophages, which are necessary for the uptake and clearance of inflammatory lipoprotein molecules. Dysfunctional lipoprotein metabolism by macrophages can cause macrophages to become trapped within the intimal wall of arterial vessels, leading to a positive inflammatory cascade of macrophage trapping, macrophage death, and increased immune recruitment and elevated inflammation in the atherosclerotic tissues. “Transient receptor potential protein, melastatin family member 7” (TRPM7) is a part of the larger “TRP” family of channels. This family of channels is highly implicated in cellular sensing of ligands and extracellular stressors. TRPM7 is a unique channel due to its functional kinase domain. Thus, TRPM7 is often termed as a “channel- enzyme”, or “chanzyme”. Using a translationally relevant atherosclerosis model, we have recently found that TRPM7, specifically the TRPM7 channel domain, is involved in atherogenesis, and that Trpm7 deletion is sufficient to reduce atherosclerotic phenotypes. Furthermore, my pilot studies suggest a pro-inflammatory role of TRPM7 channel function in macrophages in vitro. Accordingly, in Aim 1, we will generate macrophage-specific TRPM7 deletion mice and subject them to a translationally significant atherosclerosis model involving a high-fat, Western diet. We will determine how the in vivo deletion of macrophage TRPM7 affects atherosclerotic phenotypes, including plaque size, plaque stability, immune cell infiltration and recruitment, and inflammatory status. This model will provide valuable insight into the in vivo role of macrophage TRPM7 in the development of atherosclerosis. Aim 2 will determine the role of TRPM7 signaling in inflammatory pathways in macrophages and aid us in understanding how ion channels can modulate cellular responses in response to extracellular stimuli. Additionally, the use of primary human macrophages will help us to identify a valuable translational impact of TRPM7-mediated inflammatory signaling in a clinically relevant manner. The long-term goal of this exciting project is to uncover a specific, cell-specific target for the treatment of atherosclerotic individuals. This highly translational project will provide me with exciting opportunities to conduct multidisciplinary investigations. The training goals developed by my sponsor and me will lead to novel discoveries and will contribute greatly to my career goal of becoming an independent research scientist. Collectively, this current proposal will foster and support me to take full advantage of the intellectual community and resources that are available to me via the UConn Health network.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT A variant of unknown/uncertain significance (VUS) is a change in an individual’s genetic sequence that has an unknown effect on the individual’s health. It is estimated that several hundred thousand individuals in the US carry VUSs in the gene encoding cardiac troponin T, TNNT2, an essential component of the thin filament of the sarcomere—the basic contractile unit of the cardiomyocyte. Because pathogenic TNNT2 variants cause at least two forms of heart failure with distinct prognoses and treatment responses, this VUS pathogenicity knowledge gap has tremendous consequences in the clinic. Previous functional studies of a small number of pathogenic TNNT2 variants have supported a myofilament tension-based pathogenesis model whereby TNNT2 variants that increase myofilament tension through increasing thin filament calcium (Ca2+) sensitivity cause hypertrophic cardiomyopathy (HCM), a disorder with impaired cardiac relaxation that is the most common cause of sudden cardiac death in young athletes. In contrast, TNNT2 variants that decrease myofilament tension through decreasing Ca2+ sensitivity cause dilated cardiomyopathy (DCM), a disorder with impaired cardiac contraction that has a mortality rate of 50% at five years due to progressive heart failure. The central goal of our proposal is to reclassify the pathogenicity of all TNNT2 VUSs and identify their pathophysiology, which would more precisely inform heart failure risk type and treatment responses in the clinic. In Aim 1, we will study the 251 TNNT2 VUSs from ClinVar, an extensive database of genomic variation in humans linked to disease status. We will use a recently developed experimental platform designed to comprehensively interrogate the function of TNNT2 variants within the context of human cardiomyocytes (iPS-CMs) differentiated from induced pluripotent stem (iPS) cells and validated using 3-dimensional cardiac microtissue assays exhibiting advanced cellular maturation, multicellular architecture, and biophysical inputs. In Aim 2, we will perform a deep mutational scan of all possible missense TNNT2 variants using iPS-CMs expressing a transcriptional reporter assay, flow cytometry, and next-generation sequencing that we previously validated to predict the pathogenicity and pathophysiology of gold-standard TNNT2 variants with >91% accuracy. Finally, in Aim 3, we will interrogate the pathogenicity in vivo of two VUSs that function like pathogenic variants in vitro, using C57BL6/J inbred mice and myotropic adeno-associated viruses (MyoAAVs) delivering physiological troponin T levels. Our study will provide essential pathogenicity and pathophysiology determinations for VUSs in an important heart failure gene, establish new insights into troponin T functions, and provide a blueprint to resolve VUSs in other heart failure genes.

NIH Research Projects · FY 2025 · 2024-09

Abstract Our objective is to build a data repository focused on collecting and sharing bone phenotyping data performed on rodent animal models. Over the last ten years, our group has been actively involved in the bone phenotyping of over 200 mutant mouse lines. Working through the organization, storage, and viewing of datasets generated from this project made us recognize the extreme value that a skeletal repository would have for the entire bone community. We now propose to dramatically expand this effort and build a computational infrastructure, global in-scale, to collect and share bone phenotyping data generated by national and international researchers across the field. While significant limitations exist in the skeletal phenotyping of human patients, rodents remain the animal model of choice to study skeletal biology and model human skeletal diseases, where extensive phenotyping is routinely performed. Because the experimental animals are genetically homogeneous, the data captured is quantitative and, when coupled with experimental metadata, provides valuable insight into the mechanistic regulation of bone tissue. The magnitude of experimental questions being tested in rodent animal models to determine a skeletal phenotype is highly relevant to human disease, making for a rich data source that that if properly archived and shared can be exploited to understand mechanisms of skeletal regulation for rodents and humans. Unfortunately, existing rodent databases that collect genotype-phenotype data are extremely broad in scope and are not built to ingest bone phenotyping data. Taken together, this project addresses an unmet need in the skeletal field and will provide a highly valued resource that investigators can computationally mine to develop superior therapeutic approaches to treat skeletal disease. Here we have assembled a talented multidisciplinary team of bone biologists and computer scientists reinforced by the resources provided by UConn’s High Performance Computing Facility and Digital Experience Group to build a sustainable skeletal phenotyping repository. Our resource development plan has three major goals: 1) Develop a data ingestion method rich in the acquisition of metadata along with raw experimental data. 2) Develop a database system that incorporates the use of identifiers and ontologies to maximize data access and interoperability with outside databases. 3) Develop a web portal that maximizes FAIR (Findability, Accessibility, Interoperability, and Reuse) principles and becomes the go-to resource for investigators to enhance their research. A vital component of this resource plan also involves the use of scientific emissaries based in North America, Europe, and Asia that will engage with members of the bone community and be responsive to their needs.

NIH Research Projects · FY 2025 · 2024-09

ABSTRACT Current data continue to reflect that the likelihood of being infected with HIV is elevated among certain groups who have historically been less well represented in research, including youth ages 13-24, young people of color (POC), and sexual and gender minority (SGM) youth. During the past 13 years, our team has completed 6 sizable HIV/STI behavioral prevention intervention studies with youth: 3 large-scale NIH-funded randomized controlled trials (RCTs) and 3 smaller single-arm projects targeting reducing HIV/STI and syndemic health risk behavior, generating a collective total of N=1,136 independent youth who were successfully enrolled in and delivered HIV/STI and/or syndemic health risk prevention programming (>91% fully completing interventions) and followed at 1-, 3-, 6-, and/or 12-months post intervention delivery (overall retention rates of >92% across waves). When examined individually, each study revealed that youth significantly improved post-intervention, but effect sizes were small. Yet, even among disparate intervention modalities delivered with high fidelity – such as those targeting introspective examination of the capacity for change (motivational interviewing; MI), education-based approaches, reward-based interventions (behavioral skills training; BST), and mindfulness- based approaches, the differences between intervention types have been minimal. One consistent theme relevant to youth intervention outcome emerged: the role of common factors, and specifically, youth:provider relationship. For this proposal, we engage experts in clinical (MPI Feldstein Ewing) and quantitative (Co-I Bryan) evaluations of HIV/STI and syndemic risk reduction with youth, advanced longitudinal models of youth alcohol and cannabis use and their intersection with HIV/STI risk (MPI: Chung), and longitudinal mixed-effects machine learning and patient-provider relationships (Co-I Yang, Consultant Choudhary). Together, we aim to collaboratively harmonize our 6 sizable HIV/STI and syndemic behavioral intervention studies to interrogate these data using cutting-edge analytic methods and robust machine learning model testing. This innovative approach will unearth crucial relations in youth HIV/STI and syndemic intervention response, in a diverse sample of youth at very high risk for HIV (M = 17 years of age, range 13-24 at baseline; 43% female; 21% SGM; 54% Hispanic; 9% African American; 7% Native American/Alaska Native). These data are crucial to informing next step HIV/STI and syndemic intervention programming, as they will illuminate what works (and does not) in this age group. In terms of study goals, this R01 will use an atheoretical data-driven approach to harness youth prevention intervention data to drive a developmentally- specific model of youth HIV/STI and syndemic prevention intervention response. Via machine learning, we will investigate associations between youth:provider relationship and intervention outcomes (primary outcome: HIV/STI risk behaviors) among adolescents engaged in HIV/STI risk behaviors.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Chemical modifications of RNA have been seen to have numerous biological consequences including regulation of development. Ribosomal RNA is heavily modified by 2’O methylation (2’O-Me). There is increasing evidence that regulation of rRNA modification impacts developmental programs including neuronal differentiation. Fibrillarin (FBL) is known to be the canonical 2’O methyltransferase that applies 2’O-Me to ribosomal RNA. While total loss of FBL is lethal as it is critical to ribosome biogenesis, modulation of FBL expression can regulate stem cell differentiation. Overexpression of FBL extends mouse embryonic stem cell pluripotency, while partial knockdown induces neural stem cells marker expression. Additionally, altering FBL expression can cause changes in 2’O-Me of rRNA. Changes in 2’O-Me of rRNA have been seen to alter translational levels of select mRNAs and modulate cap-independent translation. The role of additional methyltransferases in shaping ribosomal function in neurogenesis is unknown. FBL has a mammalian-specific paralog FBLL1. The function of FBLL1 is unknown, though its structural similarity to FBL suggests that it is also a methyltransferase. Its role in neuron differentiation has not been studied. Unlike FBL which is expressed throughout all adult tissue types, FBLL1 is specifically expressed in the brain and testes. Within the brain, FBLL1 is expressed exclusively in neurons. While FBL expression decreases, FBLL1 expression increases through neuronal differentiation. We hypothesize that FBLL1 may act as an additional RNA methyltransferase that applies distinct 2’O-Me to ribosomes to shape translation through neuronal differentiation. Indeed, our preliminary results show that, when ectopically expressed in HEK293 cells, FBLL1 binds 18S rRNA and localizes to the nucleolus, the site of rRNA modification. In neurons, FBLL1 localizes to the nucleolus. Additionally, we observed reduction in 2’O-Me of specific sites on rRNA with genetic loss of FBLL1 in neurons. The goal of this project is to understand the function of FBLL1 and its impact on translational regulation through neuronal differentiation through two Specific Aims. Aim 1 is to characterize the protein and RNA binding partners of FBLL1 that it may use to function in neurons. Aim 2 is to determine the role of FBLL1 in regulation of rRNA 2’O methylation and effects on ribosome function. We will examine how translational changes induced by FBLL1 promote gene expression needed for neurogenesis. This work may reveal an enzyme that creates cell type-specific rRNA modifications to fine tune translation in neuronal development.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Mitochondria play essential roles in eukaryotic cells. However, they are often subject to stress originated from various sources, both intrinsic and extrinsic. In response to such stress, cells activate a diverse array of stress response mechanisms collectively known as the mitochondrial stress response, aimed at restoring mitochondrial homeostasis and preserving overall cellular fitness. Our recent work has uncovered a key mitochondrial stress response pathway involving the integrated stress response (ISR), a cellular strategy for coping with diverse stress conditions. Despite distinct triggering mechanisms, all the ISR pathways converge on the phosphorylation of eIF2a by four stress-specific kinases (PKR, GCN2, HRI and PERK). This phosphorylation event leads to an attenuation of global protein synthesis while upregulating the expression of stress response transcription factor like ATF4. Our research has revealed an OMA1-DELE1-HRI signaling cascade as a distinct link between mitochondrial stress and the ISR, offering a unique therapeutic target for manipulating the ISR in disorders associated with mitochondrial dysfunction. However, we currently lack a comprehensive understanding of how cells integrate mitochondrial stress programs to maintain a homeostatic mitochondrial network. The long-term goal of our lab is to comprehensively elucidate molecular and cellular mechanisms of the mitochondrial stress response spanning from the molecular mechanisms of stress sensing and transmitting to the ultimate outcomes. Recent findings by us and others highlighted critical roles of protein trafficking in sensing and relaying mitochondrial stress. By leveraging reporter-based assays, CRISPR screens, reconstituted cell-free assays and proteomics approaches, a primary objective of our lab is to elucidate the molecular mechanisms governing protein dynamics during mitochondrial stress. While emerging as a primary mitochondrial stress response under various stress conditions in a wide range of cell types, there remains a significant gap in understanding how the OMA1-DELE1-HRI-mediated ISR integrates with other mitochondrial stress response programs to maintain mitochondrial homeostasis. Another key objective of our lab is to systematically elucidate the intricate mechanisms involved in maintaining a homeostatic mitochondrial network. Considering that mitochondrial dysfunction is a major hallmark of numerous diseases such as primary mitochondrial diseases and neurodegenerative diseases, our mechanistic investigations into the mitochondrial stress response are anticipated to significantly advance our understanding of these pathological conditions and will potentially identify novel therapeutic targets to mitigate their impacts.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT GlycoRNAs are newly discovered biopolymers that are comprised of small non-coding RNAs and a sialic- acid containing N-glycan. These RNAs decorate the mammalian cell surface and exhibit double-stranded regions that are capable of detection by anti-dsRNA antibodies. Currently, there are no known functions linked to glycoRNAs. However, diverse pathogens employ glycosidases to breakdown host cell surface glycans to enable efficient intracellular invasion. Further, intracellular infection results in dramatic transcriptional changes and increased deployment of glycosylated molecules on the cell surface to aid in host defense. To this point, we hypothesize that the abundance and diversity of glycoRNA is altered upon intracellular infection. Our preliminary data show that infection of HeLa or THP-1 cells via Salmonella Typhimurium (STm) or Herpes Simplex Virus-1 (HSV-1) results in a dramatic increase in cell surface dsRNA, suggesting a change in glycoRNA abundance. Building on this data, we aim to investigate this phenomenon both in vitro and in vivo using a sepsis model. Here, we will use click-chemistry to isolate glycoRNA from splenocytes of C57BL/6J and Toll-like receptor 4 knockout mice injected with lipopolysaccharide or STm. This will allow us to examine how pathogens influence the expression of glycoRNA at the physiological level. Aim 1 will explore and characterize these changes through flow cytometry and RNA sequencing by employing click-chemistry isolation of glycoRNA. In immunology, the “guard hypothesis” postulates that host-derived sentinel molecules can monitor (guard) for pathogen invasion by being targeted by virulence factors. Damage or perturbations to these guarded molecules then activate potent immune responses. We hypothesis that glycoRNAs may function in part as guard molecules. We found that treating total small RNA fractions from HeLa cells with the N-glycan-specific glycosidase PNGase F resulted in greater innate immune activation than untreated (glycosylated) RNA. This effect was abrogated by treatment of this RNA fraction with RNase, suggesting that glycoRNAs are endogenous RNAs guarded by their associated glycan which prevents recognition as immunostimulatory self-RNA at steady state. To further characterize this mechanism, we will define the RNA sensors that detect glycoRNA in the absence of N-glycans and downstream signaling pathways responsible for initiating an innate immune response. Lastly, we will identify the secondary structures of glycoRNA that confer cellular sensitivity. Thus, Aim 2 will investigate the mechanism by which N-glycans shield endogenous RNA from triggering an innate immune response, and our hypothesis that N-glycans serve as a determinant of self- or non-self nucleic acid. Our proposed experiments will enhance our knowledge of these new cell surface molecules and expand our understanding of host-pathogen interactions. This fellowship and funding will enable me to explore a newly discovered field while taking full advantage of the mentorship and training opportunities offered by UConn Health and the Jackson Laboratory, directly helping to advance my career as an independent researcher.

NIH Research Projects · FY 2024 · 2024-08

ABSTRACT Hao-Fountain syndrome is a newly identified rare pediatric neurodevelopmental disorder caused by deletions and mutations of the USP7 gene. The molecular mechanisms and pathophysiology of the disease remain poorly understood, preventing the development of potential treatments for Hao-Fountain patients. USP7 is a deubiquitinating enzyme implicated in the maintenance of protein homeostasis, stem cell identity, self-renewal, and control of dendritic branching in primary neurons. A model system has not yet been created but is indispensable for understanding the disease and for the evaluation of potential personalized therapies for Hao- Fountain Syndrome. In this proposal, we will create the first human-based cellular model of Hao-Fountain to test the effect of disease-linked mutations on the enzymatic function of USP7 (Aim 1), and on neuron differentiation potential and morphology (Aim 2). The patient-derived induced pluripotent stem cells will be differentiated into cortical neurons to create the Hao-Fountain model system that will be shared with the greater community to accelerate the understanding of this newly discovered and devastating disease.

NIH Research Projects · FY 2025 · 2024-08

Project Summary This submission is in response to PAS-23-086, Small R01s for Clinical Trials Targeting Diseases within the Mission of NIDDK. Hypothyroidism is a common condition associated with excess of cardiovascular risk and poor quality of life which are not completely abrogated by treatment with levothyroxine (synthetic T4, LT4), potentially because of the inability to compensate for the loss of T3 secreted by the thyroid gland. Experimental data in animal models indicate that only combination LT4 and liothyronine (synthetic T3, LT3) can restore normal concentrations of thyroid hormones across the tissues target of the hormonal action. Clinical trials based on LT4/LT3 combination therapy and desiccated thyroid extracts (containing T3) have provided conflicting data, but the plurality of the results indicates a preference toward T3-containing therapies when compared to LT4 alone. Conversely, the short half-life of T3 poses concern of cardiovascular toxicity due to fluctuating levels of T3, particularly when LT3 is prescribed in single dose. No study has systematically assessed the optimal dose and frequency of LT3 administration in LT4/LT3 combination therapy. There is an unmet need to define a safe, effective, and feasible regimen to be applied in large trials aimed at assessing LT4/LT3 therapy efficacy and safety. We have conducted clinical studies aimed at characterizing the pharmacokinetics and pharmacodynamics of LT3 and, more recently, we completed a R21-sponsored LT4/LT3 combination therapy clinical trial in patients undergoing total thyroidectomy. The data suggest that LT4/LT3 combination therapy is effective in normalizing thyroid hormone levels and in preventing the rise in serum cholesterol and weight when compared to LT4 alone. Moreover, our results from a prior study appear to negate a clinical role of rapid T3 action, supporting the use of LT3 in single administration. These original findings serve as foundation for the current proposal. Hypothesis: A once daily administration of LT4/LT3 combination therapy will be (i) effective yet safe, (ii) comparable to a twice daily LT4/LT3 administration regimen, and (iii) superior to LT4 therapy in improving clinically relevant indices of thyroid hormone action. To test this hypothesis, we propose a randomized, three-arm, double-blind, controlled, escalating dose parallel pilot study in which 90 patients with hypothyroidism (30 each group) will be randomized to six months of treatment with LT4, LT4/LT3 with LT3 once daily, or LT4/LT3 with LT3 twice daily. This novel and rigorous study based on our original observations will fill the knowledge gap of effects and dosing of LT4/LT3 combination therapy, laying the foundation for large multicenter trial(s) able to demonstrate the effectiveness (or lack thereof) of LT4/LT3 combination therapy, fulfilling the aims of PAS-23- 086.

NIH Research Projects · FY 2024 · 2024-08

Project Summary/Abstract The proposed effort is focused on integrating community voices into existing organizational efforts to advance linguistic inclusion in clinical research. Census data indicate that close to 1 in 5 people in the United States (US) speak a language other than English at home. This number is significantly higher in urban areas, but rural areas and midsized communities are also seeing a growing increase in the number of people who speak languages other than English. Despite the growth in linguistic diversity in the US, linguistic diversity in clinical trial and research participation overall remains limited. Linguistic inclusion is paramount to bringing more treatments to all people more quickly. Inclusive samples reduce the ever-present bias towards English- speaking individuals that inhibits generalizability to the broader population; whilst failure to include, fuels homogeneity in trial participation and further stymies translation. Through our work with the Boston University Clinical Translational Science Institute we have launched efforts to advance organizational changes to support linguistic inclusion. This study builds on those efforts and is specifically designed to increase participation Spanish-speaking patient participation in the design of our linguistic inclusion efforts. Through participatory planning, we will engage patients with staff and researcher champions in the development of patient-informed culturally and linguistically appropriate outreach and educational materials and researcher education. Participatory planning can facilitate engagement and ensure that services reflect the needs of those on the receiving end. As such, our specific aims are threefold. We will 1) Explore Spanish-speaking patients’ perspectives, preferences, unmet needs when considering joining clinical trials, and barriers to participation; 2) employ a participatory planning process to design a) a messaging for a campaign focused on clinical trials participation for Spanish-speaking patients; b) a patient-informed educational manual; and c) a “From Their Voices” video for investigators that will be available in the IRB website; and 3) disseminate campaign messaging and educational manual and video among Spanish-speaking patients, BUMC researchers and more broadly. Solutions that lead to diverse linguistic representation among research participants are critical for identifying and increasing access to specialized novel treatments that are effective for diverse racial, ethnic, and cultural groups, and may, thus, serve to help reduce health disparities.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Biological sex in placental mammals is determined by their X and Y chromosomes, which, though once a pair, diverged after the Y was confined to the male germline, and progressively lost most of its ancestral genes. To maintain dosage parity of X-linked genes with XY males, XX females evolved a mechanism to randomly silence one X in each cell during early development, a process termed X chromosome inactivation (XCI). However, some genes evolved to escape XCI and resist attrition on the Y. These “escapee” genes are expressed from two copies in males and females alike, because development is sensitive to their dosage. Indeed, lack of the second sex chromosome (monosomy-X) causes miscarriage in many placental mammals. Another group of genes is specific to the X, but nonetheless escapes XCI, and thereby contributes to sex-divergent gene expression. The relevance of genes escaping XCI to human health and development is evidenced by sex differences in disease, monosomy-X leading all causes of miscarriage, and live-born Turner syndrome (TS), which predisposes TS females to fatal cardiac events due to altered metabolism and vascular malformations. Yet, it remains unknown how and which escapees contribute to this outsized health burden, because far fewer genes escape XCI in the pre-eminent mouse model, which therefore tolerates monosomy-X without miscarriage and little developmental impact overall. To address this gap, we generated monosomy-X alongside isogenic euploid human induced pluripotent stem cells (hiPSCs) from male and female samples mosaic for the second sex chromosome. We exploited this unique resource to demonstrate impaired placental gene expression in a trophoblast model of monosomy-X, and herein propose to extend these studies to link specific X/Y gene pairs and downstream pathways to this miscarriage-relevant phenotype. Our deep characterization of euploid XX hPSCs also revealed that escapee genes likely seed X reactivation when XCI master regulator XIST is repressed. Here, we propose to use novel hPSC lines we since established to elucidate the mechanisms of XCI, escape and reactivation, all of which have remained largely enigmatic in humans. Our sophisticated epigenetic tools enable de novo establishment and maintenance of XCI in female hPSCs, which also will benefit the generation of validated and stable hiPSC lines for in vitro modeling of X-linked disease and sex differences. We anticipate this research program to ultimately yield mechanistic insights that may inform new means for reactivation of functional alleles in heterozygous females manifesting X-linked recessive and dominant disorders, and provide molecular-genetic approaches towards dissecting escapee gene contributions to TS and human sex differences.