University Of Arizona

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Existing heart disease and stroke prediction models (e.g., Framingham) tend to overestimate risk for Hispanics/Latinxs (H/L)s. This inaccuracy has significant economic and public health impacts associated with inaccurate surveillance, intervention targeting, and medical management. Model inaccuracies likely stem from pervasive underrepresentation of H/Ls in model development and validation efforts. Consequently, traditional risk factors for cardiovascular disease (CVD) may be specific to the populations upon whom they were derived, and not generalizable to H/Ls. In addition, there may be unique disease determinants for H/Ls that remain untested or unincorporated leading to error in prediction. Importantly, resilience factors such as culturally- moderated social capital may be critical to understanding risk in this population. Addressing these gaps will lead to better understanding of CVD risk with corresponding implications for targeted intervention strategies. This K99/R00 MOSAIC proposal will use secondary data to inform current 10-year CVD risk models using theory and data-driven methods to increase CVD prediction model accuracy in H/Ls. The proposed training plan establishes a solid foundation for a career investigating H/L CVD risk and resilience factors. The training plan leverages substantial resources at The University of Arizona and a mentoring team of senior content experts. The candidate will gain the following, 1) expertise in H/L CVD disparities, 2) advanced knowledge in CVD epidemiology, risk, and etiology and pathophysiology of atherosclerotic disease, 3) applied machine learning, cross-validation, and selection of risk prediction models, and 4) cultural factors and social capital influencing H/L CVD. The research proposal has three aims focused on evaluating and informing existing 10- year CVD prediction in H/Ls. Using secondary data from the Hispanic Community Health Study/Study of Latinos (HCHS/SOL), the candidate will (Aim 1 – K99) evaluate the prediction accuracy of current 10-year CVD risk models using a large H/L sample with significant representation of diverse H/Ls (HCHS/SOL). (Aim 2 – R00) the candidate will use available data to identify a group of target risk factors that improve risk prediction in H/Ls. (Aim 3 – R00) the candidate will test whether adding a social resilience component to CVD risk models will improve their prediction accuracy for this group. Machine learning will be used to identify valid predictors of 10-year CVD in Latinos. The social resilience component will capture the multi-dimensionality of social environments (e.g. spouse, family, neighborhood) using data reduction methods. The proposed research proposal adopts a holistic view of cardiovascular health to elucidate both risk and resilience factors in this growing ethnic group.

NIH Research Projects · FY 2025 · 2023-09

Abstract Presbycusis or age-related hearing loss (ARHL) represents the most prevalent sensory deficit. It begets social isolation and depression. We hypothesize that aging auditory neurons undergo cellular, and structural changes, resulting in demyelination and selective neuronal subtypes loss. The ensuing plasticity produces profound neural network re-wiring and aberrant functional plasticity along the auditory pathway. These predicted changes' progressive nature requires systematic analyses of the ARHL mechanism at different auditory pathway hubs, which can only be achieved through a joint collaborative effort. Motivated by this public health challenge, we have designed a collaborative multiscale study that addresses the aging auditory system's successive mechanisms. The clinical and translational outcomes of our findings promise to be vast. The overarching hypotheses are tested in three Projects, using resources and tools from four Cores. The investigative team consists of experts from genetic to physiology and imaging and analytical chemistry. Three projects (P1-3) are served by four Cores (A-D). Core A is for administrative oversight and organization of the Cores and Projects. The team includes Drs. Yamoah (Project 1), Xie (Project 2), Maria-Rubio/Williamson (Project 3), Yamoah (Core A), Yamoah/Lee (Core B), Fritzsch/Perkins (Core C), and Zhu (Core D). Together, they will determine the mechanisms of ARHL of sensory and neural etiology. The team has worked together synergistically and productively. The projects focus on critical centers of the auditory pathways recognized for the coding and processing of sound information. They include the primary auditory neurons (AN; P1), cochlear nuclei (CN; P2-3), superior olivary complex (SOC; P2-3), and auditory cortex (ACtx, P3). We use genetic, optogenetics, and pharmacogenetic mouse models (Core B). We employ structural analyses (Core C) and differential proteomic analyses of young-and aged-auditory neurons to uncover biomarkers (Core D). Integration of the program ensures outcomes that are overwhelmingly greater than the sum of the individual components. Identification of ARHL biomarkers is likely to pave the way for effective treatment strategies.

NIH Research Projects · FY 2025 · 2023-09

Project Summary & Abstract Cirrhosis afflicts more than 4.5 million Americans and hepatopulmonary syndrome (HPS), the most common pulmonary complication of cirrhosis, occurs in up to 30% of patients and significantly increases mortality. No effective therapies exist due to our incomplete understanding of cellular mechanisms. Although classically recognized as alveolar microvascular remodeling causing hypoxemia, the poor correlation between hypoxemia, the degree of microvascular changes, and outcomes in HPS remain unexplained. To address this knowledge gap, we have identified novel abnormalities in the alveolus itself in experimental HPS, along with restrictive ventilatory defects and elevated circulating bile acids in human disease. Our hypothesis is that elevated circulating bile acid levels in cirrhosis, affect alveolar epithelial type 2 cells (AT2 cells), leading to impaired surfactant production and restrictive ventilatory defects which influence the progression and outcome of HPS. To elucidate the role of bile acids and AT2 cells in HPS, we propose to 1) assess the correlation between bile acid levels and the presence and severity of HPS 2) define the role of AT2 cells in HPS and 3) determine the mechanisms and consequences of bile acid medicated AT2 cell loss. Completion of this work will define the mechanisms and significance of AT2 cell dysfunction in HPS, identify novel therapeutic targets and form the foundation for a broader understanding of how chronic liver disease influences lung function.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Intimate partner violence (IPV) increases the risk of traumatic brain injury (TBI), because the physical assaults target the head, neck, and face. Women, more than men, across socioeconomic, racial, educational, regional, and other demographic variables are in harm’s way throughout their life and particularly during pregnancy. When one partner is pregnant, the frequency and intensity of physical assaults increase and remain focused on the head, neck, and face. But, the consequences of a TBI during pregnancy (gravida TBI; gTBI) on offspring neuro- development are unknown. Isolated TBI elevates stress and inflammation, which are known to divert fetal neuro- development with gestational exposure. The proposal goal is to provide proof-of-concept that gTBI can disturb neurodevelopment, thereby establishing gTBI as an environmental risk factor for developmental disorders. These studies cannot be performed in people or be derived from existing databases, thereby warranting laboratory studies. Preliminary data from this research team showed live births, low male weaning weight, distorted cortical circuity, reduced anxiety and depression, and a muted immune response principally in male gTBI offspring. These results encourage further investigation of TBI timing with respect to pregnancy, broader assessment of neuropsychiatric outcomes, enhanced neural circuit analyses, and molecular investigations of cell and synaptic change. The extent of neurodevelopment disruption is compared to a standard model of maternal immune activation (MIA) and respective controls. The central hypothesis of this proposal is that TBI during pregnancy leads to disrupted neurodevelopmental trajectory in the offspring that includes altered neurobehavioral performance, neurocircuit organization, and cell type-specific molecular disturbances. To test this hypothesis, a diffuse TBI will be delivered to timed-pregnant mice at 5 and 12 days post-coitum and then follow male and female offspring in terms of: [Aim 1] birth outcomes, offspring physiology, neurobehavioral phenotype; [Aim 2] neurocircuitry phenotype, and [Aim 3] synaptic protein expression and cortical cell type- specific gene expression (transcriptomics). Aim 1 will evaluate early post-natal behaviors; cognition, anxiety, depressive-like, and sensorimotor gating in young adult; and social behaviors in adult offspring. In Aim 2, cortical and hippocampal synaptic physiology and cortical connectivity will be evaluated by electrophysiology and laser scanning photostimulation, and aligned with quantitative neuronal morphology. In Aim 3, western blot quantification of synaptic proteins and cell type-specific transcriptomics inform circuit development and molecular trajectories. Impact: Successful completion of the proposed studies will provide the first proof-of-concept that consequences of TBI during pregnancy, often resulting from IPV, can distort developing brain circuity and determine a neurodevelopmental disorder behavioral phenotype.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Tenofovir prodrugs, tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide fumarate (TAF) are cornerstones of the first-line therapy in HIV/AIDS patients and there are at least 15 FDA-approved antiretroviral products that contain either TDF or TAF. However, due to their hydrophilic nature, low permeability, and premature hydrolysis or activation, TDF and TAF both have a considerably low oral bioavailability of 25% and 40% respectively. Given that these drugs need to be administered for the lifetime of HIV patients, strategies to improve oral bioavailability leading to optimal drug utilization and reduced therapeutic dose need to be developed. Transformation of ionizable, highly hydrophilic or hydrophobic drugs into ionic liquids (ILs), low- melting organic salts with a melting point < 100°C, has emerged as a novel and pharmaceutically viable approach to improving pharmaceutical processability, solubility, permeability, and oral bioavailability of drugs. Our preliminary data show that it is possible to transform ionizable hydrophobic drugs such as anthelmintic benzimidazoles, and hydrophilic ionizable drugs such as metformin hydrochloride into low-melting ILs using pharmaceutically acceptable fatty anion such as sodium docusate. Our preliminary further show that the developed ILs can be efficiently packaged into polymeric nanomicelles further leading to improved oral delivery and in vivo efficacy. Hence, we hypothesize that the transformation of TDF and TAF into amphiphilic ionic liquids (ILs) using generally regarded as safe (GRAS) fatty permeation enhancers and their subsequent incorporation into polymeric nanomicelles will improve oral bioavailability and in vivo antiviral efficacy. Our preliminary data show that TDF and TAF can be rapidly and efficiently converted to amphiphilic ILs using GRAS fatty permeation enhancers such as decanoic acid, undecylenic acid, oleic acid, and salcaprozic acid. Aim 1 will focus on the development, characterization, and pharmacokinetic evaluation of polymeric nanomicelles containing TDF-ILs or TAF ILs. Aim 2 will focus on the in vivo antiviral efficacy evaluation of oral polymeric nanomicelles containing TDF IL in humanized BLT mouse model of HIV infection compared to pure TDF or TAF to establish the proof of concept. The successful completion of this proposal is expected to lead to the development of clinically viable pharmaceutical formulations containing ILs of tenofovir prodrugs to achieve effective long-term management of HIV infection.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY The male ejaculate of internally fertilizing organisms is a diverse mixture of components including sperm and numerous other macromolecules that are transferred to females during mating. Research on male contributions to fertility has mainly focused on sperm, and, more recently on proteins present in the seminal fluid. While these studies have greatly advanced our understanding of postcopulatory interactions, the relatively high incidence of idiopathic infertility suggests that investigation of understudied molecules in the ejaculate may yield novel insights into novel factors mediating reproductive outcomes. We previously discovered the transfer of RNA from males to females during copulation in Drosophila arizonae. In this proposal, we provide evidence gathered with funding from our recent R21 award demonstrating translation of male RNA transcripts within cells of the D. arizonae female reproductive tract. Building off this work, the goals of the proposed research are to establish functional effects of seminal fluid RNAs on the female postmating response (Aim 1), investigate how they function in females (Aim 2), and identify the source and mechanisms by which they are packaged in males (Aim 3). We capitalize on unique aspects of our D. arizonae/D. mojavensis study system that combine to make a genetically tractable, manipulative experimental system that is particularly well suited to investigate the function of male RNA within the female reproductive tract. With this powerful system we combine proteomic analyses using our recently developed Variant Assisted SILAC Proteomic Analysis (VASPA) approach with detailed investigations of individual mdFTPs (male-derived female translated proteins). Given that RNA appears to be a conserved feature of male ejaculates, we expect our findings will have broad significance for better understanding postcopulatory interactions across a range of species, including humans.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY We are proposing a new approach to a hybrid imaging modality that has been called “b+g” or “pamma-positron” Imaging [Gri07] that promises to simultaneously overcome 1) the sensitivity limits of single-gamma-ray-photon emission imaging, 2) the challenge of distinguishing between two different positron-emitting isotopes, and 3) the physics-based spatial resolution limits inherent in radioisotope imaging based on detection of positron- annihilation photons alone [Lan14]. The intent is to significantly advance molecular imaging of the human brain by allowing visualization of smaller substructures, quantification of smaller amounts of radiotracer uptake, and simultaneous measurement of multiple dynamic and spatial uptake patterns in advanced multi-isotope studies of normal brain function. The required elements to make this feasible comprise i) a detector approach for annihilation and gamma-ray photons that can yield rich data for precise energy, position, and timing estimation for both photoelectric and Compton interactions, ii) processing algorithms in firmware and software to sort and make optimal use of the various combinations of signals that can occur with and without coincidence, iii) reconstruction algorithms based on likelihoods that incorporate probabilities of emission, detection, positron range, non-collinearity, and Compton kinematics, and iv) detection and compensation for attenuation and subject motion – effects that if not addressed will become limiting factors for resolution and image quality. In contrast to early efforts to accomplish b+g imaging with liquid xenon detectors [Gri07], scattering detectors as inserts into PET scanners [Yos20], or planar semiconductor detectors paired with scintillation cameras [Lan14], we propose instead to develop and demonstrate a single detector technology and associated data processing methods that can be used for both 511 keV annihilation photons and the higher-energy, singly- emitted gamma rays. Abbaszadeh (MPI) and Levin have pioneered an edge-on crossed-strip cadmium zinc telluride (CZT) detector approach to PET detectors that provides an ideal starting point [Abb16]. Among their attributes are high stopping power based on the edge-on geometry, 3D positioning that minimizes parallax, excellent energy resolution, and dynamic range up to 1.2 MeV in maximum photon energy deposited per interaction. Furthermore, when a photon undergoes an initial scatter followed by a photoelectric absorption, these modules yield data vectors that allow position and energy estimation for both interactions that can be analyzed with Compton kinematics [Abb17]. We will carry out a 2-year simulation and proof-of-principle phase (UG3) in which we demonstrate b+g detection with edge-on CZT modules and measure detector characteristics, develop simulations that support reconstructions, and demonstrate acquisitions with single and multiple isotopes. We will carry out a three-year UH3 phase to build a tomographic system with a field of view sufficient to investigate imaging of sophisticated dynamic phantoms and in vivo imaging of rodent brain, as a design study and precursor to a human brain system.

NIH Research Projects · FY 2024 · 2023-08

SUMMARY We are proposing a new approach to a hybrid imaging modality that has been called “b+g” or “pamma-positron” Imaging [Gri07] that promises to simultaneously overcome 1) the sensitivity limits of single-gamma-ray-photon emission imaging, 2) the challenge of distinguishing between two different positron-emitting isotopes, and 3) the physics-based spatial resolution limits inherent in radioisotope imaging based on detection of positron- annihilation photons alone [Lan14]. The intent is to significantly advance molecular imaging of the human brain by allowing visualization of smaller substructures, quantification of smaller amounts of radiotracer uptake, and simultaneous measurement of multiple dynamic and spatial uptake patterns in advanced multi-isotope studies of normal brain function. The required elements to make this feasible comprise i) a detector approach for annihilation and gamma-ray photons that can yield rich data for precise energy, position, and timing estimation for both photoelectric and Compton interactions, ii) processing algorithms in firmware and software to sort and make optimal use of the various combinations of signals that can occur with and without coincidence, iii) reconstruction algorithms based on likelihoods that incorporate probabilities of emission, detection, positron range, non-collinearity, and Compton kinematics, and iv) detection and compensation for attenuation and subject motion – effects that if not addressed will become limiting factors for resolution and image quality. In contrast to early efforts to accomplish b+g imaging with liquid xenon detectors [Gri07], scattering detectors as inserts into PET scanners [Yos20], or planar semiconductor detectors paired with scintillation cameras [Lan14], we propose instead to develop and demonstrate a single detector technology and associated data processing methods that can be used for both 511 keV annihilation photons and the higher-energy, singly- emitted gamma rays. Abbaszadeh (MPI) and Levin have pioneered an edge-on crossed-strip cadmium zinc telluride (CZT) detector approach to PET detectors that provides an ideal starting point [Abb16]. Among their attributes are high stopping power based on the edge-on geometry, 3D positioning that minimizes parallax, excellent energy resolution, and dynamic range up to 1.2 MeV in maximum photon energy deposited per interaction. Furthermore, when a photon undergoes an initial scatter followed by a photoelectric absorption, these modules yield data vectors that allow position and energy estimation for both interactions that can be analyzed with Compton kinematics [Abb17]. We will carry out a 2-year simulation and proof-of-principle phase (UG3) in which we demonstrate b+g detection with edge-on CZT modules and measure detector characteristics, develop simulations that support reconstructions, and demonstrate acquisitions with single and multiple isotopes. We will carry out a three-year UH3 phase to build a tomographic system with a field of view sufficient to investigate imaging of sophisticated dynamic phantoms and in vivo imaging of rodent brain, as a design study and precursor to a human brain system.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Mosquito borne pathogens afflict millions of people worldwide and their prevalence is anticipated to increase as mosquitoes and their pathogens become resistant to currently available drugs and insecticides. While drugs can be used to treat some mosquito borne pathogens, a majority of transmission control comes through targeting the mosquito vector itself. While insecticides targeting both adult and larval stages are available, larvicides have the advantage of targeting a constrained target that is unable to readily evade treatment. Unfortunately, only a few larvicides are available for use and their modes of action are limited. Thus, there is a critical need for selective novel larvicides with additional modes of action. We have developed two novel delivery systems that target the uniquely high pH levels of the larval gut. We hypothesize that provisioning these compounds to immature mosquitoes will allow us to induce lethal effects in mosquitoes of medical importance. Through a recursive set of aims whereby compounds are readily synthesized, tested and evaluated we will test this hypothesis in two important mosquito vectors, the malaria vector Anopheles stephensi and the arboviral vector Aedes aegypti. In Specific Aim 1 we assess the activity of pH responsive compounds that are tuned to the unique larval midgut environments of Aedes aegypti and Anopheles stephensi mosquitoes. An initial set of compounds developed by our lab in the MaMa and PTBD classes will be provisioned to larvae and their efficacy will be assess through SDS-PAGE, imaging, and proteomic studies. In Specific Aim 2 we will determine the ability of pH sensitive compounds to negatively impact larval development and deliver larvicidal compounds specifically to the midgut of immature mosquitoes. Compounds developed to perturb the larval gut will be evaluated for any deleterious effects that they have on the mosquito. In depth studies of their metabolism, membrane integrity, and larval and adult fitness will be conducted. By the end of this study, we will have assessed the activity of two chemically distinct sets of compounds in the midgut of larval mosquitoes. The long-term goal is to develop flexible and highly mosquito-specific larvicidal compounds with the capacity to leverage unique modes of action.

NIH Research Projects · FY 2024 · 2023-08

Project Summary: Hypertrophic cardiomyopathy (HCM) is a complex genetic cardiac disorder that affects ~1/300 – 1/500 individuals worldwide. A common clinical manifestation of patients with HCM is an impairment in left ventricular relaxation (diastolic dysfunction). Beta-adrenergic stimulation is a key regulator of diastolic performance. During beta- adrenergic stimulation protein kinase A (PKA) mediates phosphorylation of a variety of sarcomeric targets, including cardiac troponin I (cTnI) at serine 23/24 (Ser23/24). This phosphorylation event results in a significant increase in relaxation (or de-activation) rates at the myofilament level. Previous work has shown that this observation is due to increases in calcium dissociation rate from the cardiac thin filament. Additionally, some thin filament HCM mutations have been shown to exhibit an impaired response to phosphorylation of Ser23/24 in ATPase assays and force-pCa measurements. While extensive work by several groups has investigated the structural basis for this increase in calcium dissociation rate, all previous studies, to the best of our knowledge, lack the key thin filament binding partners actin and tropomyosin, crucial components for allosteric regulation of relaxation. In this proposal we will perform TR-FRET experiments to assess both intramolecular and intermolecular interactions between the N-terminus of cTnI and C-terminus of cTnI and the N-terminus of cTnI and Site II of cTnC in the presence and absence of phosphorylation at Ser23/24. The experimental design will provide distances that we will employ in our atomistic thin filament model. We will then use stopped flow fluorescence anisotropy in order to probe transitions in dynamic behavior in the C-terminus of cTnI when Ser23/24 is phosphorylated as calcium dissociates from the cardiac thin filament. We hypothesize that phosphorylation of Ser23/24 will alter the rate at which these transitions occur and that these mechanisms may be altered by HCM causative mutations. To explore the possibility that the degree of observed diastolic impairment (and potentially the severity of the end HCM phenotype) may be mutation-specific, we propose to investigate the molecular effects of 3 independent known cTnI mutations at residue R145 in cTnI. This mutational hotspot includes HCM- linked mutations R145G, R145Q and the restrictive cardiomyopathy (RCM) mutation R145W. We will couple structural data from TR-FRET experiments to changes in calcium dissociation rate to investigate how structural changes impact function and if the diastolic dysfunction is additive in the presence of Ser23/24 phosphorylation. We will employ metadynamics simulations to obtain free energy changes and identify specific changes in interactions that occur from these mutations and phosphorylation as well as in the two calcium states (on and off). The in-vitro—in-silico coupled approaches proposed in this application will provide atomic level resolution of the structural changes that occur upon Ser23/24 phosphorylation in the WT state and in the context of known cTnI-linked HCM/RCM mutations, with a long-term goal of identifying targetable disease mechanisms.

NIH Research Projects · FY 2026 · 2023-08

[Fasting hyperglycemia in diabetes mellitus (DM) is caused by unregulated hepatic gluconeogenesis (GNG), and the associated hyperinsulinemia promotes fatty acid (FA) and triglyceride (TAG) synthesis. Importantly the more common, insulin-resistant form of DM (T2DM) is less prevalent in women, even after controlling for obesity. Increased glucagon signaling, insulin resistance, and altered substrate availability have been offered as explanations, but the lack of correlation between hepatic gene expression and metabolic pathway function has hindered mechanistic progress. Understanding these limitations, our laboratories are uniquely positioned to study and integrate sex-specific differences in transcriptional regulation of GNG and lipogenic genes with direct measurement of metabolic pathways using 13C tracing and flux analyses. The overall hypothesis: glycerol metabolism is sex-dependent and crucial for both hepatic GNG and lipid metabolism. Several recent or new observations support this hypothesis: 1) Glycerol supplies the majority of the carbons for glucose and lactate synthesis in vivo. 2) Glycerol promotes its own conversion to glucose by simultaneously activating G6PC and inhibiting PCK1 expression. 3) E2 dramatically suppresses GK expression and glucose production from glycerol in primary hepatocytes (PHs) derived from ovariectomized mice. 4) Hepatic G3-P levels are low in liver- specific insulin receptor (Ir) and glycerol kinase (Gk) KO mice, demonstrating GK’s critical role in generating G3- P for GNG and TAG synthesis. 5) Liver-specific Gk KO eliminates glycerol as a GNG substrate, dramatically reducing total glucose production, hepatic expression of GNG and lipogenic genes and HFD-induced steatosis. 6) Glycerol metabolism increases the level of P300, a transcriptional co-activator and key regulator of hepatic function. Three specific aims are proposed: Aim 1 To characterize sex-dependent utilization of glycerol in obesity and T2DM mouse models. Employing obesity and T2DM models, estradiol (E2) depletion and replacement models, and a liver estrogen receptor KO model, we will study GNG substrate use in female and male mice using 13C labeled substrate infusions. Hypothesis: Glycerol use in GNG is sex-dependent and increased in obesity and T2DM. Aim 2 To determine the mechanism(s) by which glycerol regulates glucagon-stimulated GNG. Glycerol’s critical importance for glucose production in PHs suggests that mechanisms based on data obtained from PHs cultured in the absence of glycerol may be incorrect. Hypothesis: Glycerol augments glucagon- stimulated GNG by increasing G6pc gene expression while limiting PCK1 expression and substrates entering the lower GNG pathway. Aim 3 To determine how GK expression regulates hepatic GNG and lipid metabolism. We will study how insulin stimulates, and E2 suppresses GK expression, the former exacerbating and the latter mitigating hyperglycemia, elevated GNG, and hepatic steatosis. Hypothesis: Insulin and E2 regulate glycerol use as both a substrate for GNG and triglyceride synthesis and as a nutritional factor stimulating co-activator (P300) expression and regulating both GNG and lipogenesis transcription.]

NIH Research Projects · FY 2025 · 2023-08

Titin is the third myofilament of skeletal muscle where it spans the I-band and A-band regions of the sarcomere. Multiple titin mutations have been described that result in debilitating myopathies, highlighting titin's importance in skeletal muscle and the need to understand all of titin's functions fully. Our current understanding of titin is largely based on studying passive skeletal muscle and assuming that no established properties change when skeletal muscle is activated. However, recent studies suggest that titin's I-band segment interacts with the thin filament in contracting or diseased muscle, altering titin's extensibility from that in passive muscle and impacting passive force and thick filament activation. Possible thin filament interaction sites are the PEVK element and the N2A of skeletal muscle, the latter is part of a recently discovered novel stiffness regulation mechanism that involves MARP1, a stress response protein. Using mouse models aims 1 and 2 focus on the roles of the N2A and PEVK elements in regulating titin stiffness in skeletal muscle, including the effects of upregulating MARP. We also study the role of titin in activating the thick filament in skeletal muscle. Important work in the myosin field has shown that muscle activation requires thin filament activation (as is well-known) and thick filament activation mechanisms (a more recent discovery). In relaxed skeletal muscle, myosin is either in the super-relaxed (SRX) state or the disordered-relaxed (DRX) state. The conversion of SRX to DRX turns thick filaments ON, promoting contraction. Several mechanisms have been proposed to regulate the ON state of the skeletal muscle thick filament, including a mechano-sensing mechanism that involves thick filament strain. We have previously obtained evidence that titin-based passive force strains the skeletal muscle thick filament. Aim three will test the hypothesis that this converts SRX to DRX myosin in skeletal muscle and switches the thick filament from OFF to ON. High-resolution ATP turnover assays have revealed that although the SRX state occurs in each of the A- band regions of skeletal muscle (the D-zone, C-zone, and P-zone), the C-zone has the highest level. In addition to titin, the C-zone contains MyBP-C. Aim 4 will study the importance of each in SRX. It will also address the effect of locally perturbing titin strain (by deleting single C-zone domains) on SRX in skeletal muscle. This work has high novelty and addresses fundamental questions that have clinical relevance. All required models and tools are available, an experienced team of collaborators is in place, and extensive pilot data support the guiding hypotheses of the proposed research. This proposal is a significant step towards our long-term goal, which is to gain a detailed understanding of the roles of titin in both passive and active skeletal muscle and contribute to our understanding of the mechanistic basis of skeletal muscle disease.

NIH Research Projects · FY 2025 · 2023-08

SUMMARY Identifying functional sequence variants and effector genes for disease-associated functional variants, understanding how the variants influence effector genes and disease, and utilizing this information to advance precision medicine are among the most important yet formidable challenges in current genetic research of common diseases. Sequence variants identified by genome-wide association studies (GWAS) can have substantial effects on gene expression, and identification of GWAS effector genes and the mechanisms involved has important clinical and scientific implications. Chronic kidney disease occurs in more than 10% of the general population. GWAS in up to 1 million people have identified more than 200 genomic loci associated with renal function, chronic kidney disease, or urinary albumin-to-creatinine ratio. Haplotypes in linkage disequilibrium (LD) at these loci contain more than 7,000 unique single nucleotide polymorphisms (SNPs). Very few studies have gone beyond associations to ascertain the effect of kidney-associated SNPs on gene expression or investigate the mechanisms involved. Such studies would have to overcome substantial challenges including the tissue-specific nature of the effect of regulatory SNPs on gene expression. Many LD regions are thousands of base pairs long and may contain dozens of SNPs. These long LD regions are particularly difficult to study. We have developed a unique approach and innovative methods for definitively and mechanistically linking haplotype variants, including long haplotypes, to genes in specific disease-relevant cell types. In RIGERR (Resources for Investigating Genetic and Epigenetic Regulation of Renal Disease), we propose to use this suite of sophisticated technologies to generate a large collection of innovative tools and resources to transform genetic and epigenetic research of kidney disease broadly. Aim 1 of RIGERR is to generate more than 100 lines of human induced pluripotent stem cells genetically engineered to contain readily editable or precisely reconstituted haplotypes or high-penetrance variants associated with kidney function or disease. These edited cell lines will be provided to the research community for a wide range of molecular, functional, or drug testing studies for kidney disease. In Aim 2, we will produce novel datasets and establish a workflow for utilizing these unprecedented resources to conduct mechanistic and in vivo studies of kidney function and disease, using albuminuria as an example. We have obtained exciting preliminary data that strongly support the conceptual and technical feasibility of RIGERR.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract: Aging-associated vascular and cerebrovascular dysfunction is prevalent in many connective tissue disorders. Marfan syndrome (MFS) is the most common monogenetic autosomal dominant disorder of connective tissue, characterized by mutations in the gene encoding for fibrillin-1 (Fbn1), with no gender or ethnic bias. Fbn1 protein provides structural support for muscles, bones, and blood vessels as well as a scaffold for elastin fiber maturation and to bind cytokines and prevent deleterious downstream signaling. MFS-associated Fbn1 mutation results in an increased risk of life-threatening problems involved in weakening of blood vessel walls that can lead to dilation, dissection, and rupture. The role of Fbn1 mutation on cerebrovascular function has barely begun to be addressed though MFS causes neurological deficits including headaches, migraines, cerebral aneurysms, stroke, and attention deficit hyperactivity disorder. Extracellular matrix (ECM) impairment, vascular wall weakening, and stiffening, blood brain barrier (BBB) permeability, and exacerbated cytokine production are hallmark alterations associated with cerebrovascular aging and are prevalent in MFS. These manifestations in aging and MFS occur due to increased transforming growth factor-beta (TGF-β) signaling. In mice, Fbn1 mutation induces vascular dysfunction by 6-month (6M) of age. This readily accessible model has been used in more than 290 studies, where only two have addressed the cerebrovasculature, demonstrating increased middle cerebral artery (MCA) wall/lumen hypertrophy, matrix metalloproteinases (MMPs) in the MCA, BBB permeability, and TGF-β cytokine and MMP production in the choroid plexus. These results and data from this lab suggest that increased cerebrovascular aging is occurring in this model similar to that of normal aging, but with an accelerated pace. This has led to the hypothesis that Fbn1 mutation accelerates aging-associated compromise in cerebrovascular function and neurobehavioral alterations. To test this, Fbn1+/- mice at 6M, and C57BL/6 mice at 6 (CTRL) and 12M (WT) will be evaluated for cerebrovascular alterations and neuropathology through these Aims: 1) measure the expression of TGF-β signaling molecules in plasma and the hippocampus in an Fbn1+/- mouse model. 2) evaluate cerebrovascular structure and function in an Fbn1+/- mouse model. 3) examine neuropathological morphology and function in an Fbn1+/- mouse model. A strong mentoring team supports this proposal and provides expertise and resources. Data from this lab support that Fbn1 mutation plays a critical role in accelerated cerebrovascular aging and neuropathology that increases the risk of vulnerability for more severe outcomes after neurological insult such as mild traumatic brain injury. Impact: This is the first investigation of Fbn1 mutation as a contributor to accelerated cerebrovascular aging and dysfunction, where the TGF-β signaling pathway may reveal mechanisms and therapeutic targets for prevention and protection against increasing vascular dysfunction and neuropathology in MFS, similar connective tissue disorders, and the aging population.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy featuring early metastasis, late onset of symptoms, and notorious resistance to existing therapies. A critically elusive aspect of this disease relates to the tumors which are often hypovascularized relative to other solid cancers, manifesting in poor perfusion and impaired drug delivery. In preliminary studies, we discovered that endoglin, normally an endothelial-specific TGF-beta coreceptor required for angiogenesis, is expressed as two variants in pancreatic cancer cells- the wildtype, which supports tumor-intrinsic growth and chemoresistance; and a novel splice variant with distinct structural features that gets secreted to inhibit tumor vascularization. To understand their interplay in the tumor microenvironment, we have generated a variety of cellular and pharmacologic reagents to interrogate the underlying mechanisms and their therapeutic potential. We propose to define novel paracrine mechanisms of TGF-beta signaling that suppress PDAC vascularization (Aim 1); and identify tumor-intrinsic endoglin pathways as critical therapeutic targets in PDAC (Aim 2); and determine the endoglin variants as distinct spatiotemporal targets during disease progression (Aim 3). Results from these studies will define TGF- beta-based mechanisms critical for PDAC tumor growth and vascularization and deliver clinically relevant data for improved patient-based therapeutics.

NIH Research Projects · FY 2025 · 2023-08

Background. Cardiac events are the leading cause of death among firefighters and occur while on-duty at an alarmingly high rate. Short, disrupted sleep is a major, modifiable risk factor for cardiovascular disease (CVD), and up to 40% of firefighters experience sufficiently severe sleep disruption to screen positive for sleep disorders. Cognitive behavioral therapy for insomnia (CBTi) is well-established as efficacious and effective within health care settings. However, the effectiveness of CBTi-informed sleep health coaching and sleep health promotion in workplace settings, and among firefighters specifically, is largely unknown. Theory-driven research is required to address this research-to-practice gap and also identify facilitators to and barriers of intervention implementation in this high-risk population. Goal. The primary objective of this proposal is to examine whether a scalable, CBTi-informed intervention, firefighter sleep health coaching (ffSHC), improves sleep disturbances when implemented in a real-world, high- demand work environment. Methods. This study proposes to conduct a stepped wedge cluster randomized controlled trial in order to examine the effectiveness of an evidence-based sleep health coaching intervention delivered to 400 career firefighters across 20 fire departments (Aim 1). The primary outcomes are (a) the pragmatic PROMIS Sleep Disturbances questionnaire and (b) the multidimensional sleep health index derived from actigraphy. Secondary and tertiary outcomes include: sleep related impairment, sleep continuity, blood pressure, heart rate, and long-term clinic-assessments of cholesterol, body mass index, and blood pressure. To advance implementation science, this project will employ the Integrated Promotion Action on Research Implementation in Health Services (iPARIHS) framework to guide a mixed methods formative evaluation (Aim 2a) of intervention adaptations and implementation strategies. A theory-based process evaluation (Aim 2b) is embedded within the clinical trial to explore combinations of factors that promote agency-level implementation and to inform future implementation research in public safety settings. Significance and Innovation. This research will accelerate the translation of sleep health intervention to workplace wellness and advance implementation science through the application of the iPARIHS framework. The use of sleep health coaches is innovative and models how programs may expand reach to address the global problem of insufficient sleep. The long-term goal of this research is to provide critical knowledge that facilitates the widespread implementation, dissemination, and sustained utilization of sleep health intervention to promote continuous, sufficient sleep and ultimately mitigate CVD risk. Achievement of study goals will improve the health and wellness of firefighters, a population whose health is vital to assuring public safety and supporting community resiliency.

NIH Research Projects · FY 2025 · 2023-08

Every year, nearly 25-50% of older adults fall. However, fall prevention efforts are not uniformly effective and factors contributing to fall risk are not completely understood. The vestibular system, part of the inner ear, is a known contributor to fall risk and recent evidence suggests that cognition may interact with vestibular function to influence falls. Patients with Alzheimer’s disease (AD) fall 2-3 times more than cognitively healthy adults and those with vestibular dysfunction exhibit changes in cognition. However, these relationships have not been fully elucidated. As the vestibular system is diverse, identifying which functional pathways (i.e., reflexive, perceptual, visual-vestibular integration) are related to both cognition and imbalance is vital for development of targeted interventions to reduce falls. The specific aims examine the extent to which vestibular function is associated with sensorimotor behaviors (balance and falls) in AD (Aim 1a) and the extent to which cognition is associated with sensorimotor behaviors in adults with vestibular loss (Aim 1b). Explicit comparisons between those with AD and vestibular loss will also be made (Aim 1c) in order to determine potential similarities and inform ongoing fall prevention interventions in these patient populations. Existing measures will be combined with novel laboratory techniques in order to directly inform clinical practice and elucidate fundamental mechanisms underlying fall risk. The K01 award period will enable the PI to build upon her existing clinical and research expertise in vestibular science and provide structured training in the following areas: a) aging, b) cognition and cognitive impairment, c) advanced techniques for assessment of balance and gait, and d) advanced techniques for measuring eye movements. Research and training experiences during this award period will help the PI become an independent researcher in vestibular physiology. The proposed work will take place at Ohio State University under the mentorship of Dr. Dan Merfeld and Dr. Yuri Agrawal in conjunction with subject area specific mentorship from leaders in their respective fields. The proposed training activities and research plan are consistent with the NIDCD’s strategic plan for research in balance, specifically addressing current understanding of self-motion perception in health and disease and factors contributing to imbalance and falls in older adults. The proposed training will be the foundation for future R01 applications using advanced vestibular assessment techniques to examine changes in sensory function related to diverse pathologies and relationships to postural control.

NIH Research Projects · FY 2025 · 2023-08

Abstract: More than one million cases of cutaneous squamous cell carcinoma (cSCC) are diagnosed annually in the US and approximately 4% of patients develop metastases and 2% die of cSCC; thus, a similar number of people die each year from cSCC as melanoma. Immune checkpoint inhibitors (ICI) are a new class of drugs that have transformed the therapy of multiple cancer types, but only half of cSCC patients respond to ICI treatment. ICI target receptors on T cells, such as PD-1, that are expressed after activation and function to turn off T cell responses. The response of cSCC patients to ICI demonstrates the ability of T cells to constrain cSCC growth. However, it remains unclear the extent to which CD8 and, in particular, CD4 T cells contribute to immune-mediated control of cSCC. While the focus of anti-tumor T cell responses has been on MHC class I neoantigens that elicit cytotoxic CD8 T cell responses, there is growing evidence that MHC class II neoantigens eliciting CD4 T cell responses are critical in constraining tumor growth and enhancing response to ICI. Thus, there is a critical need to understand the role of CD8 and CD4 T cells, especially the role of neoantigen-specific T cells, in controlling cSCC growth. We generated a novel physiologic cSCC transplantable model on the BALB/c background from a solar simulated light-induced invasive cSCC tumor. Preliminary data supports that T cells constrain the in vivo tumor growth in the cSCC model and that this model is sensitive to anti-PD-1 treatment. Using bioinformatic approaches with whole exome and RNA sequencing data, we have identified immunogenic MHC class I and II neoantigens predicted to elicit a T cell response based on the binding affinity and presentation of the neoantigen:MHC complex and neoantigen expression. Using melanoma patient data, our lab has previously demonstrated that these characteristics accurately predict the ability of a neoantigen to elicit a T cell response. The central hypothesis is that both neoantigen- specific CD8 and CD4 T cells contribute to immune-mediated control of cSCC growth and response to treatment with vaccination with immunogenic neoantigens alone or in combination with anti-PD-1. To address this hypothesis, we will determine the role of CD8 and CD4 T cells in controlling tumor growth, identify MHC class I and II neoantigens that elicit in vivo T cell responses, and evaluate the expression of functional and inhibitory neoantigen-specific CD8 and CD4 T cells throughout cSCC tumor growth. Then, we will vaccinate mice with dendritic cells loaded with irradiated tumor cells or immunogenic MHC class I and/or II neoantigens and compare the efficacy of these vaccination strategies in inducing CD8 and/or CD4 T cells to prevent cSCC growth and treat cSCC alone or in combination with anti-PD-1. We will demonstrate the requirement for CD8 and/or CD4 T cells through antibody depletion and adoptive transfer. The impact of this project is to 1) identify the contributions of neoantigen-specific CD8 and CD4 T cells in control of cSCC growth and 2) advance the application of personalized neoantigen vaccines to treat cSCC alone or in combination with anti-PD-1.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Postpartum hemorrhage is a complication of childbirth that affects 3-8% of births in the United States, each year this represents a minimum of 120,000 births. Postpartum hemorrhage is also a primary cause of maternal mortality worldwide. Rates of severe postpartum hemorrhage and hemorrhage requiring invasive treatments or blood transfusions are on the rise, particularly for labors that are induced. Severe hemorrhage is also more common among women of color, contributing to disparities in maternal health. Oxytocin is a naturally occurring hormone as well as a medication used to stimulate labor, prevent or treat postpartum hemorrhage. Oxytocin binds available oxytocin receptors in uterine muscle, stimulating contraction. While oxytocin is the first-line hemorrhage treatment, people who have been given oxytocin to stimulate contractions during labor are more likely to have a less effective uterine contraction response to oxytocin administered postpartum, leading to more bleeding and the need for other medical treatments or procedures. Currently, 4 of 10 people who hemorrhaged, did so despite not having been identified as high risk by current clinical prediction tools. This inaccuracy and the rising rates of hemorrhage indicate that more research is needed to help identify possible risks for this potentially life-threatening complication. Because people with ineffective labor contractions or a personal/family history (of hemorrhage) are more likely to have postpartum hemorrhage, the role of innate oxytocin function/ sensitivity is the primary focus of this investigation. As such, our lab has been researching biomarkers that can help identify people at risk for hemorrhage by testing how genetic and epigenetic variation of the oxytocin receptor gene is associated with oxytocin response and hemorrhage. In this proposal, we use a biosocial framework to test the central hypothesis that maternal variation in the oxytocin receptor gene can be useful for predicting pharmacologic oxytocin needs and hemorrhage. First, we will examine DNA methylation (epigenetic differences) from blood samples using banked data as well as prospectively collected non-invasive salivary samples in association with oxytocin needs in labor and postpartum hemorrhage. Social determinants of health will be examined in association with DNA methylation differences; evaluating the role of adverse environments in shaping the oxytocin receptor epigenotype. Furthermore, we will test how DNA methylation affects gene expression and the oxytocin receptor availability in uterine tissues. Second, we will examine genetic variants of the oxytocin receptor gene in association with the clinical endpoints with the aforementioned specimens and test pharmacologic response by measuring contractility of uterine muscle specimens. Given that clinicians have no method of predicting how well oxytocin will work in the emergency of postpartum hemorrhage, we aim to develop a clinically useful biomarker measuring intrinsic oxytocin sensitivity before labor or birth occurs. The long-range goal of this project is to use biomarker data to improve clinical decision- making, test personalized interventions and lower maternal morbidity.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY There has been a significant decline in cancer related mortality, partly due to the emergence of molecular targeted therapies. Unfortunately, the success of these drugs including tyrosine kinase inhibitors (TKIs) has been tempered by a concomitant rise in the prevalence of cancer therapies-related cardiotoxicity. Ponatinib, a currently FDA-approved third-generation TKI, is used to treat chronic myeloid leukemia (CML) patients carrying the gatekeeper mutation breakpoint cluster region-Abelson (BCR-ABL) T315I. Despite its effectiveness, a considerable number of patients receiving ponatinib suffers from various cardiac complications. Several studies have linked ponatinib-induced cardiotoxicity to impaired pro-survival signaling pathways leading to cell death. However, the molecular signaling pathways leading to these events remain obscure and a better understanding of how cardiomyocytes respond to ponatinib may provide new insights into novel mitigation therapies. The heart must adapt to stress conditions that occur as a result of intracellular or extracellular factors. The integrated stress response (ISR) is one of the circuits responding to stress and serving to restore proteostasis by regulating protein synthesis, although prolonged ISR activation leads to cell death. Whether the ISR is activated and plays a protective or detrimental role in ponatinib-induced cardiotoxicity are largely unknown and may represent an amenable therapeutic target which will be the focus of my current proposal. My preliminary data suggests that ponatinib causes mitochondrial dysfunction in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Interestingly, mitochondrial damage appears to trigger activation of the ISR and is mediated by a kinase called general control non-repressed 2 (GCN2). I also found that inhibition of the ISR using a novel small molecule called ISR inhibitor (ISRIB) successfully blunted the cardiotoxic effects of ponatinib both in vitro and in vivo. Hence, the central hypothesis of my proposal is that the ISR pathway which is activated upon sensing mitochondrial damage plays a pivotal role in mediating ponatinib-induced cardiotoxicity. Aim 1 will investigate whether activation of GCN2 couples mitochondrial damage to ISR activation upon impaired mitochondrial reactive oxygen species (ROS) and adenosine triphosphate (ATP) level. Aim 2 will assess whether ponatinib induces apoptosis and cardiac dysfunction through the GCN2/eIF2α/ATF4 axis. Lastly, aim 3 will explore whether pharmacological suppression of the ISR even after the onset of ponatinib-induced cardiotoxicity remains cardioprotective without compromising the efficacy of ponatinib against tumor cells. Taken together, at the conclusion of these studies, we will have significantly expanded our knowledge by which how ponatinib-induced mitochondrial dysfunction is sensed to trigger the ISR; whether this activation contributes to cardiac pathology; and if crosstalk between these two pathways can be targeted as a therapeutic avenue to mitigate ponatinib- induced cardiotoxicity clinically.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY Noise trauma can lead to loss of parvalbumin-positive inhibitory interneurons in the auditory cortex, which is associated with audiotory processing deficit and tinnitus in rodent models. The mechanisms underlying noise- induced PV neuron loss are unknown. We propose to systematically characterize noise-induced PV neuron deficits and examine the hypothesis that differential activation of TNFR1 and TNFR2 in cortical PV neurons determines the fate of the PV neurons following noise trauma, with TNFR1 biasing for, and TNFR2 biasing against, neuronal loss and dysfunction. Specific Aim 1. Examine cell death as a mechanism of noise-induced PV neuron loss. PV-Cre-tdTomato mice will undergo a procedure to induce noise trauma. Auditory cortical tissue will be collected at four time points (1, 3, 5 and 10 d later) to cover the entire cell death process. Sections of cortical tissue will be double stained for PV and cell death markers (TUNEL staining for apoptosis, and antibody staining of p-RIP3 for necroptosis). Colocalization of tdTomato (driven by a constitutively active promoter to mark all PV neurons even if they stop expressing PV), PV and the cell death markers will be analyzed to quantify PV neuron death vs. lack of PV. Specific Aim 2. Characterize input and output synapses of the surviving cortical PV neurons following noise trauma. PV-Cre-tdTomato and PV-Cre-ChR2-tdTomato mice will undergo the noise trauma procedure. Ten days later, acute auditory cortical slices will be prepared. Patch clamp recording of PV neurons in PV-Cre- tdTomato slices will be made, and excitatory and inhibitory synaptic inputs on PV neurons will be examined. In addition, optogenetically activated PV neuron output onto Layer2/3 pyramidal neurons will be examined. The input-output function, short-term plasticity, and synaptic depletion/recovery dynamics will be characterized. All results will be compared between noise trauma and sham trauma groups. Specific Aim 3. investigate the roles of TNFR1 and TNFR2 in noise-induced PV neuron loss and synaptic dysfunction. We will knock down TNFR1 or TNFR2 in cortical PV neurons of PV-Cre-tdTomato and PV-Cre-ChR2-tdTomato mice using our custom-made viral vectors for Cre-dependent expression of TNFR1 or TNFR2 shRNA. The viral vector expresses GFP to allow comparison between transfected and untransfected PV neurons. We will examine effects of TNFR1 or TNFR2 knockdown on noise-induced PV neuron loss (as in Aim 1), synaptic dysfunction (as in Aim 2) and behavioral deficits (as in Masri et al., 20218).

NIH Research Projects · FY 2024 · 2023-07

PROJECT SUMMARY The prevalence of type 2 diabetes (T2D) is steadily increasing, highlighting a critical need to understand the etiology of this condition. In line with the dramatic rise in T2D, chronic insecticide and herbicide use has also increased, with RoundUp being the most applied herbicide in the US. As glyphosate, the active ingredient in RoundUp, targets the shikimate pathway in found in plants, but not mammals, glyphosate is proposed to be safe for human use. However, our preliminary data show that, even at a dose equivalent to the US Acceptable Daily Intake, chronic glyphosate exposure impairs oral glucose tolerance in mice. Unlike mammalian cells, several species of gut bacteria utilize the shikimate pathway, and data from our lab and others indicates that chronic glyphosate exposure alters the gut microbiome. It is now well-known that the gut microbiome impacts host health, mediated at least in part by bacterial modification of host endogenous compounds, including bile acids. Primary bile acids, produced in the liver, are biotransformed into secondary bile acid species by gut bacteria and act as signaling molecules involved in glucose homeostasis. My preliminary data shows that chronic glyphosate exposure in mice is associated with a decrease in secondary bile acids, likely occurring due to gut microbiome shifts. As secondary bile acids primarily agonize the G-protein coupled bile acid receptor 1 (Gpbar1, also known as TGR5), and TGR5 activation is beneficial for glucose tolerance, it is plausible that glyphosate-mediated shifts in the gut microbiome impact glucose homeostasis via modification of bile acids and TGR5 signaling. This hypothesis will be tested in the following Aims: 1) Determine the impact of glyphosate exposure on glucose tolerance and the gut microbiome and determine if the gut microbiome is necessary for the effects; 2) Determine how glyphosate alters enterohepatic bile acid homeostasis and if TGR5 mediates the effects of glyphosate on glucose tolerance. This fellowship will provide training in transgenic mouse colony maintenance, shotgun metagenomic sequencing and analyses, and bile acid quantification, as well as opportunities for collaboration with experienced scientists in the field and professional development through conference attendance and presentations. The lab of Dr. Frank Duca and the University of Arizona provide an excellent environment for this research, with access to the Microbiome Core at the Steele Children’s Research Center, the University of Arizona Gnotobiotic Facility, as well as knowledge from researchers in the fields of pharmacology and toxicology and metabolism.

NIH Research Projects · FY 2026 · 2023-07

SUMMARY Non-alcoholic fatty liver disease (NAFLD) is the most common cause of abnormal liver function tests in the US and its progressive form, termed non-alcoholic steatohepatitis (NASH), will soon be the leading indication for liver transplantation. There are currently no effective medications to treat NASH, no biomarkers to determine disease progression or risk of post-transplant recurrence and no effective platforms for high-throughput drug screening. Although NASH is related to obesity and diabetes, the pathogenic factors that cause disease progression to NASH/Cirrhosis are poorly understood. Compared to an invasive liver biopsy, peripheral blood mononuclear cells (PBMCs) can be easily obtained from patients with NASH and end-stage NASH/Cirrhosis patients requiring liver transplantation and re-programmed into induced pluripotent stem cells (iPSCs). These iPSCs may then be differentiated into iPSC-hepatocytes, which are human liver-like cells that can be cultured in ex vivo bioengineered systems tailored to normal and cirrhotic liver stiffness, enabling an investigation that is independent of the compounding metabolic and environmental factors that complicate analysis within human or animal systems. In this proposal, we will utilize an iPSC-hepatocyte platform to determine the effects of extracellular matrix (ECM) stiffness and unfolded protein response (UPR) cell signaling on hepatic lipid metabolism. We will initially unwind the impact and interplay between matrix stiffness and patient-specific propensity for NASH in patient-derived iPSCs and analyze the impact on lipid metabolism and lipidomics (Aim 1). ER stress and the unfolded protein response (UPR) has been shown to be important in the pathogenesis of NASH. Thus, we will use iPSC-hepatocytes to develop a platform for determining the interaction between UPR signaling and lipid metabolism relevant to NASH (Aim 2). iPSC-hepatocytes will be treated with ER stress reducing compounds including the chemical chaperone tauroursodeoxycholic acid (TUDC) or FXR/bile acid agonists, and the effects on cell differentiation, gene expression and lipid metabolism will investigated. Finally, iPSC-hepatocytes will be used to study the cell signaling and pathogenic mechanisms of NASH in iPSCs from patients with rapidly progressive NASH/Cirrhosis that require liver transplantation. We will develop an iPSC- hepatocyte platform identifying matrix and UPR factors responsible for NASH using iPSC-hepatocytes from NASH/Cirrhosis patients listed for liver transplantation (Aim 3). This MPI proposal leverages the collaboration between three PIs at two institutions with extensive experience investigating 1) iPSCs, bioengineering matrices, ECM biology, and transplant surgery, 2) hepatic lipid metabolism, cell signaling and transplant hepatology, and 3) lipidomics and metabolomics. The development and optimization of these iPSC-hepatocyte platforms will have important implications for determining the pathophysiology of NASH, developing biomarkers to determine risk for NASH progression and for use in drug development and personalized drug screening.

NIH Research Projects · FY 2025 · 2023-07

Project Summary/Abstract The purpose of the University of Arizona’s (UA) Industrial Hygiene (IH) Program is to provide master’s level graduate training in the core occupational safety and health area of IH. The Program began in 1978 and is housed in the Mel and Enid Zuckerman College of Public Health (MEZCOPH). There are three tracks: MPH, MS, and PhD; NIOSH support is requested only for the master’s degree programs. The Program is guided by 14 (11 regular and three adjunct) faculty members and an Industrial Advisory Committee made up of OSH/IH professionals from the region. There are currently ten masters’ students and twelve doctoral students in the program. The purpose of this NIOSH Training Project Grant funding application is to financially support IH master’s students. A unique strength of the UA IH program is our focus on mining health and safety and relationship with the recently formed School of Mining and Mineral Resources (SMMR). Arizona and the Southwest have one of the richest endowments of copper and related commodities on the planet. The mineral resource development industry has a long history of occupational exposures that lead to workplace injury and illness. Solving these problems and providing a healthy and safe workplace is the role of the professional IH. The UA IH program is the only program of its kind in the Southwest so it must provide the bulk of the new IHs for the region. The master's’ programs require forty-two credit hours to graduate, usually accomplished in two years. Comprehensive coursework is provided in the key areas of IH including air monitoring theory and practice, occupational safety, physical exposures, toxicology, and environmental health. Program graduates find employment as OSH professionals in industry, government, and academia. With education obtained in the program, and depending on their previous background, they can expect to sit for the CIH and/or CSP exam early in their career. The program goals for the next budget period are: 1) to continue to provide MPH and MS level IH training to increase the number of trained IHs, with a particular focus on the Southwest; 2) to continue to provide training specific to exposures in the mining industry with opportunities for student internships and research projects; 3) to maintain CEPH accreditation; 4) and to expand training, internship and student research opportunities focused on microbiological exposures. Diversity is a priority for the program, and the University will continue to support this through its emphasis on diversity recruiting throughout the southwest.

NIH Research Projects · FY 2025 · 2023-07

PROJECT ABSTRACT There are 13 cancer subtypes that are linked to obesity and these account for 40% of all cancers diagnosed in the United States annually. American Indian/Alaska Native (AI/AN) populations are 1.6 times more likely to be obese than the general population. Many factors influence this elevated risk including poverty, nontraditional foods, related social determinants of health, and physical inactivity. Furthermore, AI/AN people have the worst cancer survival rates of any US racial group. Obesity-related inflammation likely drives cancer risk and adverse outcomes, and yet, this is a reversible process. AI/AN are known to be underrepresented in clinical trials and research, and even more importantly, they have never been included in an intervention designed to modulate inflammation prior to cancer surgery. A small handful of studies in non-AI/AN featuring lifestyle interventions following diagnosis of obesity-related cancer and implemented during the short window of opportunity (WOO) before cancer surgery, a strategy known as prehabilitation (prehab), have shown some significant findings. These include alterations in the expression levels of inflammatory markers in serum and the tumor microenvironment (TME), factors that may influence carcinogenesis. Given the higher prevalence of obesity and worse cancer outcomes, the AI/AN population may have the greatest gains from this line of research. We hypothesize that a prehab intervention modeled after published literature then adapted with community collaboration for AI cancer patients will be feasible, acceptable, and successful at modulating inflammatory biomarkers. The proposed project is to (1) complete the adaptation of a prehab intervention using the candidate’s preliminary research, (2) implement the prehab translational clinical trial for AI patients with obesity-related solid tumor cancer preparing for surgery, and (3) measure inflammatory biomarkers pre and post-intervention to assess responsiveness. Study outcomes include pre and post comparisons of serum biomarkers (insulin, leptin, hsCRP, IL-6, TNFα, cortisol AM, prealbumin); tissue biomarkers (Ki67, insulin receptor, TNFα, NFκB, NOS2, cleaved caspase-3); anthropometric measurements (blood pressure, weight, waist circumference); lifestyle behavioral measures (validated diet/exercise tools: REAPS, IPAQ, 6MWT, STS). The proposed training plan builds on the applicant’s background in surgical oncology, public health, and AI/AN cancer disparities to include new training in (a) clinical trial design and implementation, (b) biomarker measurement and (c) professional development. The rigorous research and training strategies will promote the candidate’s goal of successfully transforming into an independent, funded translational clinical trialist working on behalf of AI/AN to narrow cancer disparities. This will be achieved with the support of a highly experienced mentorship team and the well-resourced training environment of The University of Arizona, an AI/AN-serving institution and home to the only NCI-designated Comprehensive Cancer Center in the state of Arizona.