University Of Colorado Denver

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY The human gut microbiome harbors microbes with the capacity to cause infection or drive pathogenic inflammation. Immune status often determines risk for microbiome-associated disease, which is typically attributed to immune impacts on microbial community composition. But pathogenic or commensal lifestyles can also be dynamically regulated within individual microbes, and there is far less understood about immune impacts on microbial inherent pathogenic potential. Candida albicans is a morphologically and transcriptionally dynamic commensal fungus that can cause life-threatening infections and exacerbate pathogenic inflammation. The ability for C. albicans to cause disease depends on its phenotypic state. Of particular importance is the formation of hyphae, which are elongated cells specialized for adherence and invasion, and promote disease in both infection and inflammatory settings. Immune status is crucial for determining risk for C. albicans-associated disease and both immune deficiencies and active inflammation are linked to C. albicans pathogenesis. However, the role of immune environment on in vivo C. albicans pathogenic potential is not well understood. Here, I will investigate the impact of two human relevant immune environments on C. albicans pathogenic potential. Project 1 will focus on IgA regulation of C. albicans biology. Anti-C. albicans IgA antibodies are found in the gut of most people, and I previously found that C. albicans hyphae and associated effectors are heavily targeted by IgA during colonization. In mouse models, IgA targeting is associated with reduced hyphae and reduced capacity to exacerbate colitis. Here, I will interrogate mechanisms by which IgA regulates C. albicans biology in vivo using a mouse colonization model that permits investigation of immediate regulatory impacts of IgA targeting on C. albicans biology. Using this model, I will interrogate IgA impacts on C. albicans morphology and gene expression, which will include single cell transcriptional profiling to investigate gene expression in individual IgA-targeted cells. The goal of Project 2 is to define the impacts of inflammation on C. albicans pathogenic potential. Evidence from human IBD studies and mouse models of colitis suggest that this fungus exploits inflammation to bloom and perpetuate disease. Here, I will use a mouse model of intestinal colitis to define inflammation-dependent impacts on C. albicans morphology and transcriptional profile, with the goal of defining C. albicans pathways responsible for disease exacerbation. This proposal will reveal fundamental mechanisms by which immune environment regulates C. albicans biology and advance our understanding of how C. albicans becomes pathogenic in certain people. Broadly, these efforts will provide a foundation for our long-term goal of identifying targeted therapeutic strategies to prevent commensal C. albicans reservoirs from causing disease.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY In the mouse retina, about 40 types of retinal ganglion cells (RGCs) communicate visual information to the rest of the brain. A great deal of processing takes place before RGCs send their output downstream. Some RGCs respond selectively to a narrow range of shapes, contrasts, and directions of motion or prefer localized stimuli that move differentially from their surroundings. These computations are supported by interactions between more than a hundred interneurons whose interactions give rise to the receptive fields (RFs) that describe the relationship between the stimulus to the response of the RGC. However, despite significant recent advances in the field, we still do not know what visual features are detected by the majority of RGC types. One obstacle to progress is current techniques to study RF composition, which either require prolonged recording sessions, challenging experimental techniques, or fail to detect crucial RF components. We are also limited in the conceptual understanding of how neural circuit organization translates to function and what RF motifs give rise to specific visual computations. In this proposal, we will take an innovative approach that combines machine learning techniques, biophysically realistic modeling, electrophysiology, and glutamate / calcium imaging to develop a comprehensive description of the visual abilities of multiple RGC types in complex visual scenes that is grounded in empirical data. The proposed research will substantially advance our understanding of basic and advanced response characteristics of visually active cells, opening new horizons in the examination of neuronal function in and beyond the retina.

NIH Research Projects · FY 2025 · 2023-09

Project Summary During early brain development, GABA is the main excitatory neurotransmitter due to a reverse chloride gradient mediated via the chloride co-transporter genes NKCC1 and KCC2. Signaling via the nicotinic receptor, a7nAChR, is a key driver of the GABA excitatory/inhibitory [E/I] shift necessary for normative neurodevelopment. Dysfunctional maturation of GABAergic neurotransmission and E/I balance in the brain is implicated in the pathogenesis of several neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASDs). Recently, our studies have identified a functional polymorphism in the Neuregulin 1 gene promoter, rs6994992, which is associated with elevated brain transcription of a novel NRG1 isoform, (NRG1-IV) and more critically lower levels of α7nAChR in the human brain. Signaling via the α7nAChR is a critical driver of the GABA excitatory/inhibitory (E/I) shift, mediated via the chloride transporters, NKCC1 and KCC2 and is also a known genetic risk factor for several NDDs, especially ASD, in the form of 15q13.3 microdeletion syndrome. In rodents, α7nAChR knockdown prevents the switch, resulting in aberrant maturation of GABAergic hyperpolarization and NRG1 is a key regulator of α7nAChRs; the specific role of NRG1-IV is unknown. In human studies, rs6994992 is associated with human cognition, sensory processing and anxiety behaviors, and data from our lab suggest attenuated sensory processing in babies carrying the T allele, and an interaction with maternal gestational dietary choline supplementation. Choline is a α7nAChR agonist, and a novel prenatal nutrient supplementation strategy shown to improve childhood behaviors and early brain development. In summary, the objective of this multidisciplinary proposal is the developmental characterization of a novel biological pathway linking NRG1, α7nAChR and NKCC1/KCC2 with regulation of E/I balance maturation, using a novel transgenic mouse (NRG1- IVtgNSE-tTA) genetically modified to express human NRG1-IV in brain and cutting-edge experimental approaches. We will test two main hypotheses.1). NRG1-IV overexpression contributes to atypical maturation of neocortical GABAergic signaling and E/I cortical balance, mediated via reduced α7nAChR and developmental expression of the chloride co-transporters NKCC1 and KCC2 and 2). Prenatal stimulation of α7nAChR, via dietary choline supplementation, will correct development of the switch, improve adult E/I cortical imbalance and neurobehavioral outcomes relevant to several NDDs, mechanistically via a7nAChR. This research will improve our knowledge of basic mechanisms of typical and atypical development related to a key signaling pathways involved in several NDDs, especially ASD, and identify new treatment/intervention approaches for people with developmental disabilities.

NIH Research Projects · FY 2025 · 2023-09

Project Summary The retina is a complex tissue composed of seven major cell types. Each of these cell types is needed for normal retinal function and therefore vision. All cells in the mammalian retina are formed during development and must last the lifetime of the animal. The production of three retinal cell types is dependent on the expression of Otx2, a homeodomain transcription factor. Otx2 is expressed during retinal development by precursors that give rise to five cell types, but it is only maintained by photoreceptors and bipolar cells into maturity. Loss-of- function studies show that mice lacking Otx2 cannot produce cone or rod photoreceptors, nor bipolar cell interneurons. Thus, cell fate decisions in the retina depend heavily on where and when Otx2 is expressed. To understand how Otx2 expression is regulated, we searched for its enhancers. Enhancers are non- coding regions of DNA that initiate and stabilize gene expression. Three potential enhancers of Otx2 were identified and shown to be expressed by OTX2+ cells. We next tested whether these enhancers were necessary for Otx2 expression. CRISPR-mediated deletion of one enhancer, DHS4, revealed a reduction in OTX2 expression embryonically yet the effect on postnatal OTX2 expression was modest. This suggested that other Otx2 enhancers are utilized in postnatal retinal development. To investigate this, I conducted CRISPR deletion experiments on the other two enhancers of Otx2, termed DHS2 and DHS15. Deletion of either enhancer showed a stronger reduction in OTX2 expression postnatally than DHS4. Interestingly, deleting both enhancers simultaneously did not have an additive effect on OTX2 reduction at later timepoints, suggesting that the complex landscape of Otx2 enhancers allows them to substitute for each other. My observations led me to hypothesize that a dynamic enhancer complex initiates and maintains Otx2 expression during retinal development. I will test this hypothesis in my proposal by completing two specific aims. In my first aim, I will employ a high-resolution chromosome conformation capture technique to reveal enhancer-promoter contacts at the Otx2 locus across retinal development. Additionally, this technique will reveal other potential enhancers of Otx2. In my second aim, I will test how this enhancer complex is disrupted when enhancers are perturbed. To do this, I will combine chromosome conformation capture with CRISPR-based enhancer perturbation techniques. This will allow me to discern enhancer dynamics and determine how enhancers compensate for each other to ensure Otx2 expression during retinal development. The completion of this proposal will improve our understanding of retinal development, complex gene regulatory mechanisms and provide me with the experience needed to continue onto a successful career leading my own academic lab.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT: Type 1 diabetes (T1D) is an increasing public health burden for which current therapies are focused on insulin replacement. Future T1D treatments will try to preserve endogenous pancreatic beta cell function in new onset T1D and replace beta cells via transplantation in long standing diabetes. Classically, T1D is viewed as an autoimmune disease but beta cell loss is also driven by genetic risk factors and metabolic stress. Much has been done to try to delay T1D progression from preclinical multiple autoantibody positivity to clinical disease, but progression is highly heterogenous. C-peptide preservation can improve diabetes control and decrease the risk for diabetes complications. There are a wide array of C-peptide measurements to examine endogenous beta cell function and stress and many genes have been associated with C-peptide preservation in T1D. Less is known about the influence of genetics on the preservation of endogenous beta cell function during the partial remission period (PRM) or “honeymoon” after diagnosis. These factors are also likely important for patients with chronic pancreatitis (CP) who have risk for islet loss but for whom genetic risk and metabolic stressors remain largely unexplored. Patients with CP have severe pain and progressive endocrine insufficiency due to persistent inflammation and hepatic insulin resistance. For patients with severe CP, total pancreatectomy with islet auto- transplantation (TPIAT) is an attempt to preserve a patient’s endogenous beta cell function while removing the source of their pain. Younger age and higher beta cell mass during transplantation are predictors for functional graft survival. Less is known about the role of beta cell genetics in these patients, which I will evaluate in this proposal. I am an emerging researcher with experience in basic science beta cells studies and clinical research defining heterogeneity in T1D. My goal in this career development award is to hone clinical research skills by studying the impact of genetics and markers of beta cell function in patients with newly diagnosed T1D and in a model of cell therapy (CP patients who have undergone TPIAT) with these aims: 1: Analyze if baseline markers of beta cell function and stress can predict PRM C-peptide preservation. 2: Evaluate if known pre-specified T1D SNPs are associated with PRM C-peptide preservation 3: Evaluate if pre-specified SNPs associated with C-peptide preservation predict insulin use and C-peptide in CP patients who have undergone TPIAT As part of this K23 award proposal, I outline educational, training, and scientific goals that will support my pathway to independence. I have assembled a diverse and broad mentorship/collaborator team to support this endeavor. This career development award will support my career goal to elucidate heterogeneity in beta cell (dys-)function in diabetes and to develop novel cell therapy interventions.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT ROCKY MOUNTAIN CANNABIS RESEARCH CENTER (RM-CRC) The United States has recently experienced enormous changes in the legal status and public acceptance of cannabis. Recreational and medical cannabis products that contain varying amounts of tetrahydrocannabinol (THC) and cannabidiol (CBD) are now widely available throughout North America. Despite these changes, research on the effects of THC and CBD has been lacking. The central goals of this Research Center of Excellence application are to evaluate, in three research projects that span emerging, middle-aged, and older adults, how CBD affects the use of and subjective response to THC, and whether these effects are mediated by the endocannabinoid system (ECS) and associated lipids. To that end, the three research projects within the RM-CRC utilize research strategies that capitalize on three key innovations. First, the projects will employ mobile pharmacology laboratories to collect biological samples and assess the acute effects of THC and CBD on key neurocognitive and behavioral outcomes, endocannabinoids, and other lipids that interact with the ECS. Second, under the auspices of an FDA IND we hold for hemp-derived CBD, projects will randomly assign participants to CBD or placebo and will parametrically manipulate either the dose of CBD or whether the CBD product contains THC, to determine how hemp-derived CBD products may impact the effects of THC. Finally, the projects rely on newly designed assays within our Cannabis Research Analytics Core (CRAC) to measure cannabinoids, metabolites, terpenes, endocannabinoids, and other lipid mediators in blood samples. The Data Harmonization and Analysis Core (DHAC) will deploy a single-solution system to support data collection, harmonization across projects, and sharing of the data with the larger scientific community directly and via NIH approved repositories. The DHAC will also harmonize previously collected data (from 7 R01s, n=1359) and conduct advanced machine learning analyses of the relationships between THC and CBD blood levels, endocannabinoids and other lipid mediators, and behavioral data. The results will inform center-wide analyses of the harmonized P50 project data. Thus, the CRAC and DHAC provide the RM-CRC with significant added value that will allow the Center to generate significant new knowledge with respect to how changes in the ECS and related lipids mediate the effects of THC and CBD across the lifespan. The RM-CRC is expected to be a significant resource to the field more broadly by providing other scientists access to data regarding CBD effects on psychiatric and health outcomes, cannabinoid, metabolite, and terpene blood levels, and 100+ endocannabinoids and related lipid mediators in approximately 2000 participants.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY (ABSTRACT) Our long-term goal is to elucidate the systems and circuits, within and across brain regions, responsible for decision making. The objective of this proposal is to examine the neural circuit basis for how, in the face of sensory uncertainty, perceptual decisions are adaptively biased to the most valuable option, as instructed by the recent history of choices and their outcomes. We address this question by focusing on the superior colliculus (SC), a key midbrain node in the network of brain regions responsible for selecting targets for movement (i.e., “spatial choice”), an important form of decision making amenable to circuit-level interrogation. The SC integrates input from numerous brain regions, has extensive intrinsic circuitry capable of integrating priors with sensory evidence as required by Bayesian frameworks, and outputs pre-motor orienting commands to downstream motor nuclei. Given its role as a functional hub for spatial choice, as well as its known cell types and circuitry, the SC is ideal for examining the neural circuit basis for adaptive decision making and how decisions are influenced by priors. In particular, the SC receives a robust input – of unknown function – from the mesencephalic pedunculopontine tegmental nucleus (PPTg), which we have shown represents recent spatial choices and outcomes. In a set of experiments in behaving mice, we test the overarching hypothesis that the SC adaptively biases spatial choice by integrating priors represented by PPTg input. We use a variant of an established spatial choice task for mice in which the dominant component of an odor mixture presented at a central port cues whether reward is available at the left or right reward port, and in which the left/right reward ratio changes across blocks of trials. On trials in which the odor mixture provides only weak evidence about reward location, decision making can be optimized by biasing choices towards the port yielding the larger reward. In Aim 1, we test the hypothesis that pre-motor SC output biases choice towards the most valuable target, by recording and perturbing the activity of genetically defined pre-motor SC neurons during the task. In Aim 2, we test the hypothesis that PPTg input transmits representations of priors to the SC that instruct adaptive choice bias in pre-motor SC neurons. We examine whether SC neurons – particularly inhibitory commissural neurons well positioned to mediating competition between left and right choices – represent choices and outcomes of previous trials, as we have seen in the PPTg. We then determine whether representations of priors and choice bias in the SC depend on PPTg input, by perturbing PPTg activity and recording SC activity during the task. If successful, the overall impact of our proposal will be the elucidation of a key circuit mechanism for how decisions are optimized by priors, a critical nervous system function. In addition, our proposal will enable future research into how genetically-defined and projection-specific neural circuits contribute to decision making, offering insight into basic brain function and neurological conditions in which decision making is impaired.

NIH Research Projects · FY 2026 · 2023-09

Abstract/Summary Rheumatoid arthritis (RA), a common autoimmune rheumatic condition, has no cure and even with novel treatments, is associated with significant irreversible joint damage, physical disability, and numerous comorbidities. The appearance of serum anti-citrullinated protein antibodies (ACPAs) indicates RA-related autoimmunity and defines the start of the preclinical period of RA, which is the ideal time to identify relevant disease risk biomarkers and approaches for disease prevention. The etiology of RA has a genetic component, which may interact with environment in the development of disease. RA is characterized by excessive chronic inflammation, suggesting a failure in the ability to control/resolve inflammation. Mechanisms for both initiating and resolving inflammation are important for physiological homeostasis. Lipid mediators, products of the metabolism of omega-3 and omega-6 polyunsaturated fatty acids, are involved in both initiation and resolution of inflammation. We have shown that an elevated level of an individual omega-6 lipid mediator (5-HETE) increased risk of progression from RA-related autoimmunity to inflammatory arthritis. However, as lipid mediators share common pathways and enzymes, we propose that individual lipid mediators do not act in isolation and therefore should be analyzed in combination as a profile. We propose to conduct a study in three novel at-risk cohorts: the Targeting Immune Responses for Prevention of Rheumatoid Arthritis (TIP-RA) cohort of 81 ACPA+ individuals, the Studies of the Etiologies of RA (SERA) cohort of 79 ACPA+ individuals, and the StopRA cohort of 144 ACPA+ individuals in the preclinical period of RA that have been followed over time for the development of RA. We will create lipid mediator profiles (a composite score of combinations of highly correlated lipid mediators) indicating the ability to resolve inflammation by performing principal components analysis of the lipid mediators and look at the trajectories of these profiles over time. Aim 1 will determine if the association of these profiles with progression from ACPA+ to RA differs by genetic susceptibility to RA. We will also explore whether the association is mediated by cytokine profiles or trajectories. Aim 2 will explore the underlying mechanism by examining whether the lipid mediator profiles are associated with DNA methylation differences or trajectories. Aim 3 will examine inflammation resolution in the lung by identifying sputum lipid mediator profiles and cytokines associated with progression from RA-related autoimmunity to RA. Results from this work will provide the foundation for designing prevention studies by elucidating which combinations of lipid mediators play a role in inflammation resolution in preclinical RA. The inability to resolve inflammation can lead to chronic inflammation; a common pathogenic element of RA. Elucidating mechanisms as well as the site (ie lung) of inflammation resolution during the preclinical period of RA significantly contributes to the understanding of pathogenesis and development of innovative interventions for RA.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Craniofacial development is a complex process that requires various signaling pathways to mediate cross-talk between tissues that eventually differentiate into the cartilage and bone of the frontonasal skeleton. Defects in this process result in common craniofacial malformations, such as cleft lip and palate. Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in both human and mouse craniofacial development. PDGFRa has been shown to play a predominant role in NCC migration, contribute to proliferation of the facial mesenchyme at mid-gestation and promote osteoblast differentiation. Alternatively, PDGFRb primarily regulates proliferation of the facial mesenchyme past mid-gestation. Further, PDGFRa and PDGFRb have been shown to genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers, though the mechanism and function of signaling through these heterodimers remains unknown. We have used an innovative approach, bimolecular fluorescence complementation (BiFC), to explore individual, activated PDGFR dimers, which has revealed preliminary differences in dimer-specific activation, trafficking and downstream signaling dynamics. The goal of this proposal is to fully characterize these dynamics for PDGFRa/b heterodimers in vitro and in vivo, and to identify PDGFR dimer-specific interacting proteins that mediate differential trafficking of the various PDGFR dimers. First, PDGFRa/b heterodimers will be immunoprecipitated using a GFP-Trap nanobody in response to a timecourse of PDGF- BB ligand stimulation to examine the dimerization and autophosphorylation dynamics of PDGFRa/b heterodimers. Then, fluorescence microscopy experiments will be performed to examine co-localization of PDGFRa/b heterodimers with markers of various endosomal compartments in response to a timecourse of PDGF-BB ligand stimulation to examine the trafficking dynamics of PDGFRa/b heterodimers. These findings will be compared to our previous results for the PDGFR homodimers. Second, BiFC-tagged PDGFRa homodimers, PDGFRb homodimers and PDGFRa/b heterodimers will be purified using the GFP-Trap nanobody and analyzed by mass spectrometry to identify PDGFR dimer-specific interacting proteins. Novel proteins with demonstrated roles in receptor trafficking will be both overexpressed and repressed in the relevant PDGFR-BiFC stable cell line in the presence of PDGF ligand, and trafficking of the various PDGFR dimers will be assessed as above. Finally, Venus expression will be analyzed in craniofacial structures of E8.5- E16.5 embryos that are double-homozygous for PdgfraV1 and PdgfrbV2 BiFC knock-in alleles both in whole mount and in coronal frozen sections by fluorescence microscopy to examine the timing and localization of PDGFRa/b heterodimer formation during craniofacial development. The studies proposed here will determine how biological specificity is introduced downstream of individual PDGFR dimers and provide valuable insight into the mechanisms underlying mammalian craniofacial development.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract This proposal is a five-year research and training plan with a scientific focus on interactions between CD8 T cells and fibroblasts in synovial tissue from patients with rheumatoid arthritis (RA). We have found that the majority of CD8 T cells in RA synovium have an unusual phenotype characterized by low expression of classic cytotoxic proteins such as granzyme B, perforin, and granulysin. Instead, these cells express high amounts of granzyme K, which induces synovial fibroblasts to produce pro-inflammatory factors such as IL-6. Based on our preliminary data, we believe that granzyme K does more than simply activate synovial fibroblasts. We believe that granzyme K activates the complement system to induce so-called inflammatory priming of synovial fibroblasts, characterized by metabolic reprogramming and augmented activation to stimuli. The specific aims proposed here will investigate the interactions of CD8 T cells and synovial fibroblasts in three complementary ways. Aim 1 interrogates whether human granzyme K induces the metabolic reprograming associated with inflammatory priming and whether complement mediates the downstream effects of granzyme K. Aim 2 uses imaging mass cytometry, traditional immunofluorescence, and spatial transcriptomics to evaluate granzyme K+ CD8 T cells and their relationship with fibroblast subsets and complement deposition within the context of human synovial tissues. Aim 3 uses mouse models of inflammatory arthritis to determine the effects of granzyme K on clinical and cellular measures in inflammatory arthritis. This study combines mechanistic studies, patient-derived samples, mouse models, and cutting-edge transcriptomic technologies to provide the candidate new training in several key aspects of translational immunology. The candidates immediate career development goals are to gain experience with microscopy techniques, spatial transcriptomics, cellular metabolism, mouse models of autoimmune diseases, and bioinformatic analysis. A specific career development plan is described by both the candidate and her mentor Dr. Michael Brenner, MD, an expert in lymphocyte biologic and synovial inflammation. She also has the support of an Advisory Committee of experts in the areas in which she will build her skills. The candidate’s long-term goal is to attain a tenure-track faculty position pursuing research that integrates high-dimensional analysis of patient samples with detailed mechanistic studies to characterize cellular interactions within tissues in rheumatologic diseases, with the ultimate goal of identifying new cellular and molecular candidates for diagnosis and treatment of autoimmune diseases.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Influenza viruses remain a major global threat to human health, which is exacerbated by the lack of an effective vaccine. Humoral immunity does not target all parts of the virus evenly, a phenomenon known as immunodominance. Following seasonal vaccination, humans preferentially generate antibody responses against evolving epitopes of the hemagglutinin (HA) head domain rather than against broadly protective epitopes of the conserved stalk domain. In the absence of preexisting immunity against the HA head, as occurred with the 2009 pandemic H1N1 virus, humans can preferentially recall memory B cells against the stalk domain. However, naïve B cells against novel epitopes of the HA head are induced, populate the memory B cell pool, and are preferentially recalled following exposure to an antigenically similar virus. Therefore, immunodominance of the head domain and the preferential recruitment of naïve B cells against new epitopes remains a major obstacle for the generation of a broadly protective influenza vaccine. In order to generate broadly protective humoral immunity, a vaccine needs to be designed that induces robust and durable antibody responses against the stalk domain but prevents the recruitment of naïve B cells against neoepitopes. Immunodominance is in part dictated by which B cells can acquire antigen and efficiently present antigen to CD4 T cells. B cell avidity, the simultaneous binding of both binding-sites of an antibody with its epitope and the cross- linking of multiple B cell receptors on a B cells, plays a critical role in which B cell specificities are selected. B cells against the head domain have an avidity advantage, as these epitopes are more accessible than those of the stalk domain. We hypothesize that reducing B cell avidity for head epitopes will reduce competition for antigen, limit the induction of anti-head B cells, and preferentially select for B cells against the stalk domain. Using combinatorial mutagenesis and yeast-display, we will generate and select for a library of stable HAs with diverse head epitopes but an identical stalk domain, which we refer to as scrambled HA (Aim 1). By titrating the diversity of scrambled HAs, we will determine the effect of increasing head epitope diversity on which B and T follicular helper cell specificities are induced. We will perform these experiments in naïve and H1N1 immune mice to mimic immune histories in infants and adults, respectively (Aim 2). Moreover, we will immunize human ex vivo lymph node organoids generated from individuals of diverse immune histories to determine if our approach recalls memory B cells against the stalk domain (Aim 3). Together, this study will generate a vaccine that shifts immunodominance towards the stalk domain, which will be proven using multiple models of preexisting immunity. Although this proposal focuses on shifting immunodominance of anti-influenza humoral immunity, the approach taken provides a proof-of-concept that can be applied to other rapidly evolving pathogens for which immunodominance is a key barrier for successful vaccine generation, including HIV and coronaviruses.

NIH Research Projects · FY 2025 · 2023-08

Polycystic ovary syndrome (PCOS), one of the most common endocrinopathies in women, presents with anovulation in adolescence and reproductive dysfunction is worsened by excess weight. Females with PCOS also have lowered insulin sensitivity (IS), which integrates obesity and reproductive abnormalities. Obesity as well as excess testosterone and insulin are risk factors for endometrial carcinoma, secondary to the lack of ovulatory cycles with excess hormonal stimulation of the endometrium. Despite the high prevalence and gravity of comorbidities associated with PCOS, widely effective therapeutic options are lacking. Glucagon-like peptide-1 receptor agonists (GLP-1 RA) increase post-prandial insulin secretion and modulate gut and hypothalamic hormone responses to suppress appetite and cause weight loss. GLP-1 RA are approved to treat diabetes, and in higher doses, obesity. Limited data on older and less potent GLP-1 RA, such as exenatide or liraglutide, in women with PCOS are promising with improved menstrual frequency and lowered serum testosterone. Our initial results following 4 months of treatment with a more potent and oral formulation GLP- 1 RA, semaglutide, in adolescents with PCOS and obesity demonstrated lower post-prandial glucose, a shifted insulin curve to a more normal pattern with a resultant improved oral disposition index. The trial is too short of duration to assess reproductive dysfunction, although there is a trend for decreased serum testosterone and nearly half had improved menses frequency compared to the 4 months prior to the trial. Adolescents have lower IS than adults, and youth with diabetes or obesity respond less to GLP-1 RA than adults. Work is needed on the role of longer-term, more potent GLP-1 RA treatment in women with PCOS + obesity, especially across the reproductive lifespan. Further, there is a gap in understanding the underlying features predictive of GLP-1 RA response, limiting the ability to include GLP-1 RA in a personalized therapy plan for PCOS. Our overarching hypothesis is that weight loss and metabolic improvements are required to improve reproductive health in individuals with PCOS. The aims of this application will be performed in the context of a year-long clinical trial. After a 4-month observation period of either no medication or metformin treatment, we will treat females aged 12-35 years with obesity and PCOS for 10 months with semaglutide to induce weight loss and improve ovulation and lower testosterone. We aim to: SA1) Quantitate ovulation frequency before and after semaglutide in females with PCOS. SA2) Quantitate ovulation frequency following combination treatment with semaglutide + metformin. SA3) The ovulation response to semaglutide will relate to baseline characteristics and metabolic response to therapy. This study will define the reproductive impact of currently available medications on this common, high-risk disease, with the potential to immediately change health outcomes. We will also identify predictors of treatment response, as a step towards developing high-quality personalized treatment plans for those with PCOS + obesity.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Chronic Obstructive Pulmonary Disease (COPD) is a major cause of chronic respiratory symptoms, poor quality of life and death for millions of people in the US and worldwide. In vulnerable people, exposure to cigarette smoke over 2-3 decades leads to fixed, expiratory airflow obstruction, the sine qua non of COPD. Although 30-40% of smokers will eventually develop COPD, there are no clinical, physiologic, biologic or molecular measures that identify vulnerable people during the earliest phase of disease, i.e. before COPD can be diagnosed by impaired lung function (Early COPD). This inability to identify vulnerable people during early COPD drastically limits the ability of physicians to alter its course. This proposal will address this key research gap and meet a major goal of the COPD National Action Plan, by studying the role of airway basal progenitor dysfunction in detection, progression and pathogenesis of early COPD. Airway basal progenitor cells are essential for lung health and repair. By virtue of their ability to replicate (self-renewal) and differentiate into all cell types present in the epithelium (multipotentiality), airway progenitors can return an injured epithelium to normal structure and function. Preliminary data from older (~60 years old) people showed that bronchial and sinonasal basal progenitor function is reduced in smokers with COPD, smokers with low-normal lung function, and in younger smokers (~40 years old) without known lung disease. These findings imply that progenitor function has an important role in disease pathogenesis. Experiments using bulk and single cell RNA sequencing, metabolomics, and Seahorse measurement of glycolytic flux and mitochondrial respiration identified a strong signal for altered energy metabolism as a prime driver of progenitor dysfunction in older smokers with COPD or low-normal lung function, and in younger smokers with low progenitor self-renewal. Therefore, we hypothesize that sinonasal and bronchial progenitor dysfunction a marker for early and accelerated progression of COPD, and that progenitor dysfunction during early COPD is associated with altered cellular bio-energetics. We will test this hypothesis in 70 younger (age 30-50 years) smokers without known lung disease stratified by progenitor self-renewal for high and low progenitor function. An additional 20 never smokers without known lung disease and 20 smokers with COPD will be enrolled as controls. Aim 1 will use a cross-sectional design to determine whether bronchial and sinonasal progenitor dysfunction are markers of early COPD. Aim 2 will use a longitudinal design to determine whether bronchial and sinonasal progenitor dysfunction are markers for disease progression during early COPD. Aim 3 will determine whether bronchial and sinonasal progenitor dysfunction are associated with altered cellular bioenergetics. Successful completion of these aims could have a game-changing impact by focusing efforts on disease prevention before damage is irreversible, facilitating use of treatments that slow lung function decline, and by identifying early mechanisms of disease that together have the potential to save millions of lives.

NIH Research Projects · FY 2025 · 2023-08

Summary Antibiotic resistance (AR) is a high-priority urgent threat. AR pathogens such as the ESKAPE group cause millions of infections and hundreds of thousands of deaths. While current strategies such as genetic and drug screens have helped identify genes and mutations critical for AR in specific pathogens, there is a broad lack of methods to help understand AR’s origin and continuous adaptation. AR can arise in a pathogen via a variety of molecular changes, including acquiring protein domains, individual genes, or metabolic capabilities. Hence, predicting and overcoming AR in emerging pathogens or discovering new AR mechanisms requires a holistic understanding of AR evolution across multiple molecular scales. However, leveraging these diverse datasets is challenging because original databases are siloed from each other. Further, the different data types are hard to integrate in a biologically-meaningful way across scales. In this project, we describe a computational discovery framework combining evolutionary analyses and machine learning to integrate AR data across multiple scales to gain mechanistic insights into AR molecular features in ESKAPE pathogens and predict AR in new (re)emerging genomes. We will implement our approach as open FAIR data repositories, open software, and web platforms for the computational, experimental, and clinical AR communities. We will work closely with AR collaborators, end-users, and the open software community during and following the project duration to ensure the release of accessible, user-friendly, interactive platforms. Finally, in the post-award expansion phase, we will work with NIAID-funded bioinformatics consortia for downstream integration of data and methods and long-term sustainability. The framework will develop in this project will be broadly applicable to advance understanding of AR in understudied and emerging pathogens (beyond ESKAPE) towards ending the arms race between microbes and drugs by creating better treatment outcomes.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY The purpose of this grant submission is to request funds to partially support the 2023 Annual Colorado Immunology and Microbiology Conference (CIMC). This will be the 22nd meeting of this conference, which has been highly successful since it was first held in 2000. The meeting will be held the Steamboat Grand Hotel in Steamboat Springs, CO on August 30th – September 1st, 2023. The Chairs for the 2023 meeting will be Drs. Kelly Doran and Roberta Pelanda who will organize the meeting along with additional members of the organizing committee: Drs. David Beckham, Jena Guthmiller, and James Scott-Brown. CIMC program encompasses a wide range of exciting topics in Immunology and Microbiology. Topics will include cancer immunology, diabetes, autoimmunity, respiratory immunology, microbial pathogenesis, host-pathogen interactions, inflammation, and therapeutics. The overall goal of the CIMC is to bring the world’s leading scientists and the most promising young investigators and trainees together in a collegial and supportive environment. The specific goals are to: 1) provide a forum for the presentation of unpublished, cutting-edge research on Immunology and Microbiology and foster formal and informal discussions on how these results advance the field, 2) include investigators and clinicians focusing on diverse topics and define the impact on human disease and health 3) expand the boundaries of the work of the scientists within this field, and 4) ensure development and integration of young investigators, including women and those in under-represented groups into the broader research community in a meaningful and interactive way. The collegial atmosphere will include organized discussion sessions during talks and poster sessions, and opportunities for informal gatherings in the afternoons and evenings, providing an ideal setting for scientists from different disciplines to exchange ideas and foster cross-disciplinary collaborations both among themselves and the next generation of investigators. Thus, the CIMC will provide a unique and fundamentally important opportunity to bring together leading investigators in a format designed for the free exchange of research results and ideas covering diverse aspects of Immunology and Microbiology and relationship to disease. The CIMC will promote diversity, equity and inclusion and the development of promising young investigators in the field and their integration into the larger research community. In the short term, this will significantly enhance the ability of investigators to address critical problems in infectious and immune related diseases, while in the long term it will ensure the continued success of their efforts and those of the next generation of investigators.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT My career focuses on research, clinical, and translational efforts to improve lifestyle treatment for obesity. Obesity is a major contributor to cardiovascular disease (CVD), accelerated by the current epidemic of highly palatable energy dense food, reduced physical activity, and insufficient sleep. While weight loss can reduce CVDrisk, sustaining weight loss is a major challenge for most individuals with obesity. It is imperative to identify innovative and evidence-based strategies to promote greater and more durable weight loss. My collaborative research program focuses on understanding predictors of successful weight loss maintenance and translating those findings into strategies to improve lifestyle weight management programs. Specifically, I develop and evaluate novel weight loss interventions, focusing on alterations of the timing or pattern of energy intake and physical activity (PA). My studies involve rigorous, objective measures of free-living energy expenditure and energy intake, and multi-disciplinary collaborations to comprehensively assess potential biologic, behavioral, psychosocial, and environmental predictors of treatment response. Collectively, these studies provide a framework and wealth of data for trainees to perform hypothesis-driven patient-oriented research (POR). I am deeply committed to mentoring the next generation of innovative, collaborative clinical researchers to reduce obesity and cardiometabolic disease risk. I have 8 years of mentoring experience supporting new and early- stage clinical investigators in POR (undergraduate, graduate, and medical students; MD and PhD post-doctoral research fellows; and junior faculty). My mentees have an excellent record of publication productivity, NIH grant funding, successful career transitions, and retention in academic medicine. The K24 Mid-Career Investigator Award will provide dedicated time for me to deliver focused mentorship to junior clinical investigators of exceptional potential and capitalize on the outstanding mentoring, research, and training resources of our campus. I will pursue leadership and administrative training, as well as develop multidisciplinary collaborations to enhance my knowledge and skills in sleep/circadian physiology and qualitative methodology to extend the breadth and reach of my obesity treatment POR program. I have assembled an experienced team of senior clinical researchers with extensive mentorship experience and leadership roles in clinical and research training programs on campus to serve as a K24 Advisory Committee to guide me during the K24 award period. My specific aims are to 1) Mentor junior clinical investigators to create a pipeline of well-trained patient-oriented researchers focused on obesity, nutrition, PA, and sleep, and 2) Build and enhance my collaborative research program designed to rigorously evaluate strategies involving alterations of the timing or pattern of behaviors (diet, PA, and sleep) to develop more effective lifestyle interventions to treat obesity, reduce cardiometabolic disease, and improve health. As a dedicated physician-scientist, I am well-positioned to enhance the quality and breadth of POR in weight management and CVD risk reduction and become a national leader in this field.

NIH Research Projects · FY 2025 · 2023-08

Project Summary The proper functioning of learning, memory, and cognition requires the activity-dependent strengthening of excitatory synapses in the hippocampus via a process known as long-term potentiation (LTP). LTP can be impaired in ex vivo hippocampal slices by incubation with the peptide amyloid-β (Aβ); increased concentrations of this peptide are highly associated with early synaptic deficits in Alzheimer’s disease (AD), a progressive neurodegenerative disease. LTP is known to require the Ca2+/calmodulin-dependent protein kinase II (CaMKII), and specifically its localization to excitatory synapses, driven by direct binding of CaMKII to the NMDA-type glutamate receptor GluN2B. This localization of CaMKII to excitatory synapses is impaired by incubation with Aβ, revealing a potential mechanism underlying Aβ-induced synaptic deficits. Interestingly, the impairments of LTP and CaMKII movement caused by exogenous Aβ incubation are alleviated by loss of the amyloid precursor protein (APP). While the proteolytic cleavage that APP undergoes to form Aβ is well-characterized, this apparent downstream role as a mediator of Aβ-induced impairment remains largely unexplored. Importantly, individuals with Down syndrome (DS), a genetic developmental disorder, express increased levels of APP due to triplication of the APP gene. As loss of endogenous APP “desensitizes” neurons to the synaptic deficits caused by Aβ, it may conversely be true that these increased APP levels “sensitize” neurons to the effects of Aβ. Initial results indicate that APP is not only necessary to mediate CaMKII impairments caused by exogenous Aβ, but also sufficient to impair CaMKII-GluN2B binding in heterologous cells, further implicating APP as a direct mediator of downstream Aβ-induced CaMKII impairments. Thus, this proposal will investigate various aspects of APP’s role as a mediator of Aβ, including whether neurons expressing higher levels of APP are more sensitive to Aβ-induced synaptic impairments, which domain(s) of the APP protein are necessary to mediate these impairments, and which specific mechanism(s) downstream of APP and Aβ are driving impairments in LTP-related CaMKII movement. To answer these questions, we will be utilizing several different genetic mouse lines, including a model of DS, various mutant constructs of APP, and a recently developed photoactivatable CaMKII. The results of the experiments outlined in this proposal will provide valuable insight into the role of APP in driving synaptic impairment (underlying hippocampal memory deficits) caused by Aβ in both AD and DS.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Although total knee arthroplasty (TKA) is effective at reducing pain from end-stage osteoarthritis (OA), it is an acute trauma to the joint which exacerbates the underlying weakness and disability common with OA, leading to long-term atrophy and further disability. Attenuation of this acute loss of strength and function can lead to improved long-term outcomes. Key to attenuating this acute loss is effectively managing postoperative knee swelling. Knee swelling after TKA is profound and is theorized to be a major driver of this acute strength and functional loss. This early postoperative strength loss is driven by a deficit in voluntary activation that acutely limits rehabilitation potential and leads to long-term atrophy and disability. Traditional interventions such as elastic compression stockings (e.g., thromboembolism-deterrent [TED] hose) have minimal effectiveness in reducing swelling after TKA. We have demonstrated in a preliminary study that an inelastic, adjustable compression garment can decrease early postoperative swelling by 50%. However, there is a need to formally evaluate the preliminary effects of this garment on all outcomes to inform sample size calculations for a larger trial. Additionally, as a majority of research to date on quadriceps activation and swelling has relied on laboratory induced swelling, this trial will be uniquely positioned to explore in-vivo mechanistic relationships between changes in swelling and quadriceps activation. Therefore, we propose a randomized controlled study of 58 older adult participants undergoing TKA to determine if an inelastic adjustable compression garment (INCOM) improves postoperative swelling more than elastic TED hose (CONTROL). Both groups will wear the assigned garments for the first 3 weeks after TKA and participate in a standardized rehabilitation program after TKA. Outcomes will be assessed preoperatively and postoperatively at weeks 1, 3 (end of garment use),12 and 26. This study will enable us to determine (AIM 1) if INCOM results in improved surgical limb swelling control after TKA (primary outcome) compared to CONTROL and (AIM 2) to explore the preliminary efficacy of INCOM on the outcomes of quadriceps strength, pain, range of motion, physical function, and patient reported outcomes after TKA compared to CONTROL. Additionally, we will explore (EXPLORATORY AIM) the mediating role of swelling on outcomes after TKA. This study has high potential to prevent acute strength and functional losses in the short-term and improve long-term health of the millions of older adults who will undergo TKA annually. This study will uniquely provide insights into 1) causal relationships between swelling and strength/functional outcomes and 2) mechanisms for quadriceps activation deficits. Study findings will also provide insights into the management of numerous other patient populations with significant quadriceps activation deficits (knee OA) or extremity swelling (post-surgical or trauma).

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT We are entering an era of precision medicine for cancer screening in which screening initiation and maintenance is based on personal risk estimates that incorporate multiple risk factors, with the goal of maximizing screening benefits and minimizing the harms. One increasingly common clinical application involves using cancer risk prediction models to inform which patients are screened, how often they are screened, and at which ages screening occurs (i.e., risk-based screening). For breast cancer screening with mammography, the U.S. Preventive Services Task Force recommends informed choice about when to begin regular screening for women age 40-49. Making an informed choice means determining whether, for an individual woman, the likelihood of receiving a screening benefit outweighs the likelihood of harms such as false positive results and overdiagnosis. A critical component of informed choice is a woman’s personal cancer risk, because the expected benefit of screening depends on a woman’s personal risk of breast cancer, with women at lower risk being less likely to benefit and more likely to be harmed than women at higher risk. Hence, incorporating informed choice into the mammography screening decision-making process means that women are asked to make decisions about screening based on their personalized breast cancer risk estimates. However, a critical barrier to implementing guideline-recommended informed choice in mammograpy screening is that many people reject that their risk estimate is true for them personally, a phenomenon we refer to as risk rejection. While previous research has focused on risk comprehension as a high-impact target for improving medical decisions, we argue that risk rejection is also a high impact target for improving health decision making because people who reject their risk are unlikely to make health decisions based on that risk estimate, even when they comprehend the estimate. An estimated 1-in-4 women believe that their breast cancer risk estimate is not accurate for them, and little is known about why, or the implications for screening decisions. In Aim 1, we will use a longitudinal survey to understand the scientific nature of risk rejection. Aim 1a will identify key demographic and psychological predictors of risk rejection. In particular, we will test 4 theory-informed hypotheses about why people reject their risk: motivated reasoning, prior knowledge, distrust, and lack of perceived personal relevance. Aim 1b will identify the impact of risk rejection on risk-concordant screening behavior. In Aim 2, we will conduct interviews and use experiments to identify communication strategies to reduce risk rejection. These studies will provide a comprehensive understanding of a phenomenon – risk rejection – which, if not addressed, will impede the implementation of informed patient choice in breast cancer screening and personalized, risk-based medicine more broadly.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY This K24 Mid-Career Mentoring Award supports my further career development and 50% protected time for mentoring and research. My patient-oriented research has bridged the intersection between HIV and geriatric medicine, through which I have developed a research program to describe the epidemiology and underlying mechanisms of HIV and aging, and introduced interventions focused on the successful aging of people with HIV (and other infectious diseases). Through the K24 award, I propose to take my research from the bench and the bedside into the clinic and community by gaining training in dissemination/implementation and community-based research principles (Training Aim 1) and health equity and inclusion in research, mentorship, and leadership (Training Aim 2). To provide hands-on training for these career development aims, I will focus on improving methods for measurement of muscle mass and quality in physical activity interventions (Aim 1). In Aim 2, I will work to improve methods for pragmatic and feasible interventions through a systematic review of existing interventions, input from key informants, and an environmental scan, with a focus on what is feasible and acceptable. We will conduct a systematic review and individual or group interviews, and used mixed methods to incorporate these findings with data from the parent R01, HEALTH. Lastly, I will use knowledge from Aims 1 and 2 to enhance existing physical activity programs for greater dissemination and implementation (Aim 3). I will be supported by mentors with expertise in dissemination/ implementation and health equity, in addition to three ongoing mentors with cross-disciplinary research programs, outstanding mentorship accolades, and leadership positions within their institutions. Lastly, my institution has provided me with the additional support, most critically in time protected from additional clinical or administrative duties. In summary, the proposed K24 will leverage my current R01 to serve as a training platform for trainees and incorporate my career development objectives, ultimately advancing the science of interventions to preserve physical function in older people with HIV and support patient-oriented researchers.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY The acute respiratory distress syndrome (ARDS) is characterized by severe lung inflammation and carries a mortality of 30-40%. Some patients recover quickly while others improve slowly and often develop lung fibrosis. The factors that drive these disparate outcomes are unclear. Pulmonary macrophages play key roles in the pathogenesis of ARDS by releasing inflammatory cytokines, recruiting neutrophils, and releasing cytotoxic and apoptotic factors which damage the lung epithelium. I hypothesize that inflammatory programming of pulmonary macrophages and continuous recruitment of monocytes with inflammatory programming into the lung drive prolonged inflammation and transient fibrosis in acute lung injury. Aim 1 of this proposal will use an existing single-cell RNA sequencing (scRNAseq) dataset generated by the Janssen laboratory to test the hypothesis that specific recruited monocyte-derived macrophage subsets from a mouse model of prolonged inflammation will exhibit transcription profiles consistent with perpetuating inflammation and fibrosis. This transcription profile would include expression of pro-inflammatory cytokines, chemokines, and cytotoxic factors. The transcriptional programming of recruited macrophages in a prolonged inflammation model will be compared to recruited macrophages from a model of self-limited inflammation. Aim 2 will use a compartment and lineage tracing mouse model I have optimized to test the hypothesis that prolonged lung inflammation is characterized by continuous recruitment of monocytes into the airspace and pulmonary interstitium that mature into pro-inflammatory macrophages, with little proliferation in situ whereas self-limited inflammation is characterized by an early, single wave of recruitment. Understanding the transcriptional profiles of recruited macrophage populations will provide insight into the pathophysiology of limited versus prolonged lung inflammation. Defining the kinetics of monocyte recruitment to the airspace and interstitium in limited versus prolonged inflammation will inform both the pathophysiology of slowly resolving lung inflammation and the timeline for interventions.

NIH Research Projects · FY 2024 · 2023-08

My long-term career goal is to become a productive and impactful independent investigator in the field of craniofacial development, by conducting cutting-edge, multidisciplinary research to better understand the genetic, molecular, and cellular mechanisms of craniofacial development and disorders. The goal of my research is to improve human health by elucidating disease mechanisms and contributing towards efforts to develop therapeutic strategies. This application will provide the framework to achieve these goals through a coordinated career development plan that utilizes the expertise of a diverse team of mentors and advisors, and the supplemental training opportunities provided by coursework and conferences. These factors are woven into my research strategy addressing important questions in craniofacial development and mechanisms of disorders and expands my training into new areas that are essential for my transition to an independent researcher career. This includes becoming an expert in genetic and transgenic models of zebrafish, genome- wide sequencing approaches like single cell ATAC-seq and ChIP-seq, and human craniofacial anatomy. My mentor team, led by Drs. Clouthier and Nichols, will provide valuable guidance throughout the training plan, including the academic job search and career transition process. Moreover, this training will be carried out at in a stellar research environment in the Department of Craniofacial Biology, and the University of Colorado Anschutz Medical Campus, that collectively have the resources, core facilities, and faculty members needed to ensure the success of this career development plan. This application will address important yet unstudied events in intracellular signaling downstream of the Endothelin receptor type A (EDNRA) that establishes patterning domains along the dorsal-ventral (D-V) axis of pharyngeal arch 1 (PA1). EDNRA signaling is required for lower jaw development and is disrupted in a human craniofacial disorder called Auriculocondylar syndrome (ARCND). EDNRA can signal through all four classes of G proteins, though how dynamic use of different G proteins by EDNRA to pattern different domains of PA1 is unknown and extremely difficult to study. This proposal will use three aims to test the hypothesis that the Gq/11 class exclusively patterns the intermediate domain of PA1, and that Gq/11 regulates patterning gene expression by inducing genome-wide changes to chromatin accessibility. Aim 1 will use genetic and transgenic approaches to determine whether Gq/11 is necessary and sufficient for intermediate domain patterning. Aim 2 will use multimodal single-cell analysis to define cis-regulatory elements and gene regulatory networks controlled by Gq/11. Aim 3 will use zebrafish models of ARCND to understand how disease alleles impact EDNRA-Gq/11 signaling. Furthermore, this information will be used to better understand human cases of ARCND. Collectively, this comprehensive research training and career development plan will ensure my successful transition from a mentored postdoc to a well-prepared independent researcher.

NIH Research Projects · FY 2025 · 2023-08

Common mental disorders (CMD) of depression and anxiety are prevalent and largely untreated among Kenyan pregnant and postpartum women living with HIV (PPWH). CMD lead to poor maternal and child health outcomes and contribute to lack of HIV care engagement and virologic failure in PPWH. While efficacious treatments for CMD exist, scaling treatment within routine health care in low- and middle-income (LMIC) settings will require stakeholder engagement and both effectiveness and implementation data to inform scale up and sustainability. Our team has integrated other efficacious interventions into antenatal (ANC) and HIV care in Kenya. We now propose to integrate proven mental health services using a collaborative care model, combined with a low intensity evidence-based intervention (problem solving therapy), while targeting known social determinants of HIV-related health for PPWH (stigma and IPV). Building on the current multidisciplinary approach for HIV care in Kenya, our proposed Collaborative Care Model (CCM) will utilize existing peer mentor mothers, non-specialist behavioral care managers, and psychiatric nurses; and will incorporate a consultant psychiatrist into the ANC/HIV care team. Guided by the EPIS (Exploration-Preparation-Implementation-Sustainment) framework, the overall study goal is to integrate collaborative care for perinatal CMD within routine ANC/HIV services in Kenya, assess the costs and cost-effectiveness of this approach, and work with policy and decision makers to determine key considerations for scale-up. Specifically, in Aim 1, we will identify contextual barriers and facilitators to refine an optimal integration model for delivering collaborative care model using multi- method data collection (focus groups with providers, in-depth interviews with key informants, and a clinic readiness checklist). A workshop with our Advisory Board comprising both county and national level stakeholders, will allow us to translate findings into a locally relevant CCM. In Aim 2 we will test CCM PPWH health during depression retention Aim 3, we will refine CCM implementation strategies through cost-effectiveness and dissemination research. We will carry out costing and cost-effectiveness analysis and invite policy and decision-makers to participate in a nominal group technique process to elucidate factors for further scale up and sustainment of the CCM approach. Findings from this study will developing a scalable model adaptable to other LMIC settings, contributing to global HIV and maternal health goals while and addressing the burden of untreated CMD. in antenatal care for in a hybrid type 2 implementation-effectiveness trial using a stepped wedge design at 15 primary care facilities in southwestern Kenya . We will i ntroduce CCM care for PPWH diagnosed with CMD antenatal care. The co-primary health outcomes at 12 months postpartum will be (1) recovery from or anxiety symptoms in PPWH, and (2) proportion PPWH with sustained viral suppression and in HIV care. Key implementation outcomes are feasibility and acceptability. Finally, in

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY / ABSTRACT The objective of this proposal is to identify new approaches for targeting clonal hematopoiesis (CH). CH is characterized by selective expansion of hematopoietic stem and progenitor cell (HSPC) clones harboring mutations in genes such as TET2 and DNMT3A. These HSPC in turn produce pathogenic mutant myeloid cells that can contribute to several aging-related co-morbidities including cardiovascular disease (CVD) and all-cause mortality. An expanded mutant HSPC pool may also contribute to increased risk of hematological malignancies. CH prevalence is significantly elevated in the elderly and individuals with prior genotoxic exposures, smoking history, and/or chronic inflammatory disease. These conditions are associated with chronically perturbed physiological homeostasis, characterized by hyper-inflammation. Understanding the mechanism(s) promoting the selective expansion of mutant HSPC is crucial for prioritizing therapeutic targets that can suppress CH. A central premise of our application is that that CH arises from a targetable interplay between inflammatory signals and altered metabolic programming that supports the energetic needs and thereby the preferential expansion of CH HSPC. Using the mouse as a model representing key features of human CH, our preliminary data show that CH HSPC exhibit increased levels of the transcription factor Hif-1α aberrant glycolytic metabolism and increased ATP production relative to wild-type HSPC. We find that inflammatory cytokines, particularly IL-1β strongly potentiates CH HSPC expansion, glycolytic metabolism and Hif-1α activity. Strikingly, treatment of CH mice with OLT-1177, an NLRP3 inflammasome inhibitor that prevents cleavage and activation of IL-1β potently suppresses CH. We hypothesize that CH is the result of an interdependent mechanism in which NLRP3-mediated IL-1β production potentiates Hif-1α and downstream glycolytic activity to support CH HSPC expansion. We propose that NLRP3 inhibition disrupts this circuit, limiting expansion of CH HSPC. To address the mechanism, we propose two Specific Aims: 1) we will characterize the metabolic features of Tet2Δ/Δ, Tet2+/Δ and Dnmt3aR878H/+ CH HSPC using in vivo mass spectrometry- and flow cytometry-based analyses of metabolism and glucose flux. We will also identify the extent to which CH HSPC rely upon Hif-1α and glycolysis for their energetic needs; 2) we will evaluate the requirement for NLRP3 in promoting aberrant glycolytic activity and/or preferential expansion of CH HSPC. We will use molecular genetics approaches to assess the role and mechanism of Hif-1α and NLRP3 in regulating CH HSPC metabolism and promoting their expansion in vivo. Using our non-conditioned adoptive BM transfer mouse model of CH, we will validate our mechanism using pharmacological inhibition of NLRP3 with OLT-1177 and establish whether NLRP3 blockade suppresses HSPC expansion, aberrant metabolic activity and accumulation of inflammatory immune cells in the heart and other tissues. This work has the potential to re-frame CH as a therapeutically targetable metabolic phenotype, significantly improving health among groups with high prevalence of CH such as the elderly.

NIH Research Projects · FY 2024 · 2023-08

Project summary The identification of intracranial hypertension (ICH) is essential during pediatric neurological and neurosurgical evaluation. If untreated, it may lead to brain injury, developmental delay or even death. However, current non- invasive evaluation of ICH is based on clinical symptoms that have poor correlation with it, and imaging findings are highly variable and subjective. Hence, non-invasive evaluation of ICH is challenging and often inconclusive. Additionally, invasive intracranial pressure monitors can help identify ICH but they are usually avoided because they often offer inconclusive results and carry important risks of hemorrhage and infections. Consequently, ICH remains underdiagnosed in the population. In this project, we aim to create non-invasive methods to automatically identify the presence of ICH in pediatric patients using CT images of the head. The main hypothesis of this project is that an elevated intracranial pressure results into a decrease of bone mineralization, which can be quantified from CT images. This hypothesis is supported by clinical reports of patients with elevated pressure or brain growth constraints showing thinner and less dense cranial bones, and of patient with intracranial hypotension presenting an abnormally thick cranial bone. During this project, we will: (1) create a sex- and age-specific normative statistical model of cranial bone thickness and density between birth and 10 years using a large retrospective CT image dataset of subjects without cranial pathology; (2) quantify bone abnormalities in patients with ICH and create machine learning models to identify them using a retrospective dataset of patients who required surgical treatment for pressure release; and (3) perform a pilot study to identify and quantify local signs of elevated pressure in patients with cranial growth constraints caused by craniosynostosis. Upon completion of this project, we will have established normative references of cranial bone mineralization, identified and quantified bone mineralization changes associated with ICH, and created automatic tools to identify them. In addition, if our exploratory study in patients with craniosynostosis succeeds, it will prompt a larger clinical study of the presence of ICH in these patients that could translate in a substantial improvement of their clinical management through timely interventions, especially in older children and relapsing patients.