University Of Colorado Denver

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT Eosinophilic inflammation is a key feature of many human pathologies, and therapeutic targeting of eosinophils is of clinical interest, as illustrated by the new class of eosinophil-depleting drugs in development for allergic and inflammatory diseases. The full potential and consequences of these drugs are active areas of medical research, particularly as eosinophil depletion does not uniformly cause symptom reduction. Despite their post- mitotic state and short circulating lifespan, eosinophils can persist for days to weeks in certain tissue environments. Increasingly, there is evidence of a positive role of long-lived, “tissue-resident” eosinophils in tissue structure development and maintenance that counters the historic view of eosinophils as only having pro-inflammatory functions. Given the prevalence of eosinophilic inflammatory conditions and development of eosinophil-depleting drugs, there is a significant need to understand 1) which factors enable eosinophil differentiation, 2) how differentiated eosinophils interact with the surrounding tissue, and 3) what molecular events prolong survival of an otherwise short-lived post-mitotic cell. This project will elucidate the regulation and function of tissue-resident eosinophils at steady state and during allergic disease. We hypothesize that eosinophils respond to pro-survival signals in the epithelial environment by engaging cell cycle machinery to prevent apoptosis and support rapid gene expression during specialization. The three research areas will 1) target cell cycle machinery to manipulate eosinophil specialization in mucosal tissue, 2) delineate pre- and post-transcriptional events in eosinophil re-specialization, and 3) define and inhibit pathogenic functions of specialized eosinophils. These studies are technically and conceptually innovative in that they use high- sensitivity immunoassays, 3D organotypic tissue models, next-generation sequencing, and chromatin looping technologies to expand on paradigm-shifting evidence of a dynamic, long-lasting role of eosinophils in tissue. The technical approach is creative and designed to address eosinophil functions that are poorly understood but central to understanding and treating eosinophilic diseases, type 2 allergic diseases, autoimmune pathologies, and certain cancers. This research is ideally suited to the NIAID DP2 award due to its potentially transformative effect on the understanding of granulocyte biology. The applicant is well suited to lead the proposed work given her technical and analytical skill, research productivity, and success in creative problem-solving and collaborative project development. The DP2 award will augment the resources accompanying her newly independent position and allow Dr. Dunn to apply impactful new technologies to a clinically relevant but understudied area of eosinophil biology that has broader implications for the converging fields of clinical immunology, personalized medicine, and nuclear structure.

NIH Research Projects · FY 2025 · 2024-08

B lymphocytes are critical players in the development of protective immunity, as well as in pathological states such as autoimmunity. Careful regulation of the strength and quality of biochemical signals that originate from their antigen receptors (BCR) is essential to prevent the unwanted activation of potentially harmful autoreactive B cells, and to generate optimally protective antibody responses. Inhibitory signaling mechanisms play a central role in regulating BCR signaling. Best characterized is the role of Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM)-containing inhibitory receptors that become activated upon co-aggregation with actively signaling BCRs. The BCR-associated Src-family kinase Lyn tyrosine phosphorylates their ITIM motifs, resulting in the recruitment of phosphatases such as the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 that actively suppress BCR signaling. SHIP-1 activity is increased in autoreactive B cells and plays a critical role in maintaining B cell tolerance by suppressing PI3K-dependent signaling. How SHIP-1 is activated in autoreactive B cells is still unclear. BCR stimulation by itself, without recruitment of known ITIM-containing inhibitory receptors, activates SHIP-1. In an unbiased screen, detecting tyrosine phosphorylated receptors that bind SHIP-1, we identified CD79A as containing the SHIP-1 docking site. CD79A and CD79B form a heterodimer that mediates signal transduction following BCR stimulation. Each chain contains a single Immunoreceptor Tyrosine-based Activation Motif (ITAM). Phosphorylation of the two conserved tyrosines in the ITAMs is the critical first step towards activating signaling. Previously, we showed that in autoreactive B cells CD79A ITAMs are predominantly monophosphorylated. Studies of cell lines expressing chimeric receptors suggest that monophosphorylation of the CD79A Y182 residue biases signaling towards inhibitory signaling. We hypothesize that CD79A ITAMs can act like a molecular switch wherein ITAM monophosphorylation tips the balance towards inhibitory signaling by preferentially recruiting the regulatory proteins Lyn and SHIP-1. To test this hypothesis, we have generated novel mouse models in which the ability of the CD79A ITAM to become tyrosine phosphorylated is variably restricted to monophosphorylation of the membrane proximal (Y182) or membrane distal (Y193) residue. In Aim 1 we will determine how individual CD79A ITAM tyrosines impact BCR signalosome composition and downstream signaling, both directly and indirectly via co- aggregated ITIM-containing receptors. In Aim 2 we will determine the impact of individual CD79A ITAM tyrosines on biological processes such as B cell development, B cell activation, antibody responses, and B cell tolerance. Specifically, we will determine if CD79A ITAM monophosphorylation of CD79A Y182 drives the anergic phenotype of autoreactive B cells. These studies will provide unique insights into the earliest activating and inhibitory events that shape B cell activation and tolerance. It will also lead to a better understanding of ITAM biology with direct translational implications for chimeric antigen receptor design.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Developing Interventions for The Protection of HIV-Exposed Uninfected Infants against Severe Infections HIV-exposed uninfected infants (HEU) have increased risk of severe infections, hospitalizations, and death in the first 1-2 years of life than HIV-unexposed infants (HU). This is likely due to decreased immune responses. The development of the infant immune system is guided by the gut microbiota, which use metabolites and other products as mediators. The immune system also affects the microbiome in this bidirectional relationship. We will compare the interactions of microbiome and immune responses in HEU and HU with the goal of identifying gut bacterial taxa and their metabolites responsible for the HEU immune defects that may be modulated with diet. In Aim 1, we will define immunogenomic and metabolomic profiles that explain the immune dysfunctions of HEU in the 1st year life. In 50 HEU and 50 HU at 6, 24 and 48 weeks of life, equally distributed between males and females, we will measure functional, phenotypic, transcriptomic, and epigenetic profiles and identify the genomic networks underlying the loss of NK and T cell effector function. We will integrate the immunogenomic with plasma metabolomic profiles to identify the metabolites associated with HEU immune dysfunction. We will then confirm the relationship between immunologic and metabolomic characteristics in vitro. In Aim 2, we will compare the gut microbiome composition and function in HEU and HU and identify bacterial taxa associated with the immunologic and metabolomic abnormalities of HEU. We will further confirm the relationship between microbiota and metabolome in vitro. We will also compare the composition of the respiratory microbiomes of HEU and HU. The respiratory microbiome may play an important role in the differential severity of respiratory tract infections of HEU and HU. Moreover, the respiratory microbiome is modulated by local and systemic immune responses resulting in a well-established correlation between gut and respiratory microbiota. In Aim 3, we will perform proof-of-concept studies of maternal and infant dietary interventions to modulate the infant gut microbiome. In the 1st study, we will randomize 24 pregnant people with HIV 1:1 to pasteurized fermented milk (PFM) supplementation during the last month of pregnancy or control. We will assess acceptability and compliance with the intervention and its effect on the neonatal gut microbiome and metabolome at 2-4 weeks of life. In the 2nd study, we will randomize 24 6-month-old HEU 1:1 to 4-week PFM supplementation or control and evaluate the same outcome measures as above at the end of the intervention. This highly impactful and innovative study will define the relationship of the infant gut microbiomes with immunogenomic, metabolomic, and respiratory microbiome profiles; will identify bacterial taxa that may increase the vulnerability of HEU to infections through decreased immune functions; and will test the ability to alter the HEU gut microbiome and metabolome through dietary interventions. Our findings will forge a path to design interventions to improve the HEU immune protection and decrease their risk of severe infections, hospitalizations, and death.

NIH Research Projects · FY 2024 · 2024-08

Project Title Long-term effects of universal health insurance in developing countries: Evidence from Mexico Project Summary/Abstract (30 lines of text) The implementation of government programs to increase access to health insurance has rapidly increased in developing countries in the last three decades. However, evidence on their long-term effects in a developing country is limited and provides mixed conclusions. Understanding the persistent impact of universal health insurance is particularly relevant as the hypothetical health improvements and reduced financial burden they may offer have the potential to shrink intergenerational transmission of poverty and improve social mobility of beneficiaries and their children. The efficacy of these programs remains unclear predominantly because of a lack of random variation in their implementation and the fact that in randomized controlled trials, control groups typically receive the benefits, at most, only a few years after the initial implementation. Long-term evaluation of these programs is further complicated by the substantial challenges of consistently tracking and following up baseline respondents, which can lead to bias-inducing, non-random attrition. This project contributes to filling the gap in what is understood about the broad consequences of increased access to health insurance in developing countries. Specifically, we provide new evidence from a population- representative sample on the long-term effects of Seguro Popular, a universal public health insurance program introduced in Mexico in 2002. Pairing the richness and innovative design of the Mexican Family Life Survey (MxFLS), a nationally representative, longitudinal survey with cutting-edge and rigorous non- experimental methods, represents a unique opportunity to make a substantial contribution to understanding the persistent impact of programs that increase access to health insurance in developing countries. The MxFLS collects comprehensive respondent data across time whereby every respondent surveyed in Mexico at baseline in 2002, and any of their children born after 2002, are target respondents for follow-up survey waves, including those who are domestic or international migrants. By integrating the MxFLS's ability to track economic and health outcomes of respondents over multiple years with the temporal and spatial variation created by Seguro Popular's rollout, as well as, the biological variation generated by the age individuals are first exposed to the program, this project will: 1) identify the causal effects of Seguro Popular on child height, which has been causally associated with indicators of well-being in later life, e.g., educational attainment, earnings, adult health, and mortality; 2) explore the dynamic impact across time of this health insurance policy to provide new insights into how supply and demand side constraints at the time of program implementation can alter its effectiveness; and 3) provide evidence on the impact of Seguro Popular on household expenditure, labor market, investment, risk, and migration behavior. In so doing, the project will have provided scientific evidence on the extent to which the provision of health insurance in a developing country setting improves the lifelong health and wellbeing of its population.

NIH Research Projects · FY 2025 · 2024-08

Abstract Short tandem repeats (STRs) are 1–6 bp repetitive and highly polymorphic DNA sequences. Expansions in dozens of STRs are associated with genetic disease. However, STRs are challenging to sequence and interpret, meaning that individuals with STR disease often go undiagnosed. In rare disease studies, it is now standard to prioritize candidate pathogenic SNVs, indels and SVs by excluding variants that have high allele frequencies in population-scale databases such as gnomAD. However, there is no such genome-wide database available for large STR expansions. I will produce a publicly available STR variation community resource, stratified by ancestry, to enable prioritization of candidate pathogenic STR expansions. Long-read sequencing technologies from PacBio and Nanopore have been heralded as the solution to accurately genotype long repeats because their reads can span the repetitive region. However, there are several challenges when genotyping STRS in long-reads that are not adequately addressed by existing approaches. I will develop a method to genotype STRs from long-read Oxford Nanopore sequencing data. It will discover informative reads using a combination of alignment and identifying repetitive regions in reads. It will then infer the genotype by integrating evidence from multiple reads, informed by my investigation of biases in these technologies. Drawing together new short and long-read computational approaches to calling STR expansions, and my population-scale STR catalog, with an emphasis on diverse and under-served populations, this proposal will establish a genetic diagnosis for hundreds of patients, while searching for new STR disease loci. I will analyze patient cohorts enriched for phenotypes associated with STRs from the UDN, University of Washington, Harry Perkins Institute of Medical Research and Children’s Mercy Hospital to solve cases and discover new disease- associated STRs in both short and long-read sequencing.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY/ABSTRACT Type 2 diabetes increases a person’s risk for cognitive impairment syndromes including, cognitive decline, mild cognitive impairment, and Alzheimer’s Disease and its related dementias. However, our ability to identify who among people living with type 2 diabetes is most likely to develop a cognitive impairment syndrome is limited because we have an incomplete understanding of the molecular mechanisms that lead to cognitive issues in people with type 2 diabetes. A better understanding of these molecular mechanisms can help to identify biomarkers that predict risk for cognitive impairment syndromes. Proteomic analyses provide an efficient and comprehensive approach to identifying molecular mechanisms of disease and are also recognized as an effective platform for biomarker discovery. However, to date, few human studies have applied proteomic approaches to investigate the mechanisms that drive development of cognitive impairment syndromes in people living with type 2 diabetes. Thus, additional proteomic studies are critically needed to illuminate the underlying mechanisms of cognitive impairment syndromes in people with type 2 diabetes, so to identify proteins that can be used as biomarkers of risk. The overarching goal of this pilot project is to use proteomic analyses to examine differences in plasma proteins and protein networks in people with type 2 diabetes, who do and do not develop mild cognitive impairment, as a first step toward identifying potential risk biomarkers for cognitive impairment syndromes in people with type 2 diabetes. First, to achieve this goal, our primary aim is to identify proteins associated with newly diagnosed mild cognitive impairment in people with type 2 diabetes (Aim 1). Additionally, we aim to provide further depth to our understanding of the molecular mechanisms driving the underlying neuropathology of mild cognitive impairment in people with type 2 diabetes. Thus, we will determine whether proteins associated with mild cognitive impairment in Aim 1 also relate to preclinical plasma-based biomarkers of neurodegenerative disease and Alzheimer’s disease neuropathology in people with type 2 diabetes (Aim 2). Results from this pilot study will reveal potential mechanisms by which people with type 2 diabetes develop cognitive impairment syndromes. Thus, providing important insights that could inform use of plasma- derived protein biomarkers for risk stratification of people with type 2 diabetes, and possible protein targets for development of novel therapeutics and treatment strategies to mitigate risk of cognitive impairment syndromes in people with type 2 diabetes.

NIH Research Projects · FY 2025 · 2024-08

Project Summary In the brain, neurons are connected by intermingled excitatory (E) and inhibitory (I) synapses. The correct E/I balance is essential for proper brain function. Recent in vivo studies demonstrate that excitatory and inhibitory synapses communicate with each other locally and inhibitory synapses could actively regulate the structure and function of excitatory synapses. In vivo optogenetic stimulation and suppression of inhibitory activity decreases and increases the stability of excitatory synapses, respectively. Our preliminary two-photon imaging and chemogenetic data further show that PFC interneuron activity may affect excitatory synaptic plasticity throughout brain development. These data strongly suggest that inhibitory synapses heterosynaptically regulate excitatory synapses. However, there is much yet unknown about the cellular and synaptic mechanisms by which inhibitory GABAergic synapses locally regulate excitatory glutamatergic synapse formation and elimination during brain development. Using advanced optical techniques, this R21 grant proposal addresses key questions of fundamental significance; 1) how do inhibitory synapses heterosynaptically regulate excitatory synapse structure and function throughout brain development in an age-dependent manner? And 2) what signaling mechanisms underlie the cross-talk between inhibitory and excitatory synapses during development and plasticity of neural circuits? Our work will provide a powerful experimental framework for understanding how the brain achieves and maintains E/I balance, and how this balance is altered in neurodevelopmental disorders arising from E/I imbalance such as autism and schizophrenia.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Striatal dopamine serves a critical role in motivation, reward learning, and decision making. Aberrations in dopamine transmission are thought to underlie a variety of neuropsychiatric diseases and substance abuse disorders. Yet, despite dopamine’s prominent role in behavior and disease, mechanistic understanding of how transmission occurs at the subcellular level has remained limited due to methodological restrictions. Structural data shows that dopamine neurons form extensively arborized axons with sparse release sites (varicosities) that are often unassociated with postsynaptic receptors. Combined with early methods of measuring dopamine, these findings have led to the belief that dopamine signals primarily through ‘spillover’ transmission, characterized by slow changes (hundreds of milliseconds or more) in low concentrations of dopamine (<1 µM) over a broad radius (~10 µm). However, accumulating evidence has recently challenged this notion. Imaging of fluorescent dopamine sensors has revealed the presence of highly localized dopamine ‘hotspots.’ Further, electrophysiological recordings show that endogenous dopamine release rapidly modulates postsynaptic targets and is only precluded by concentrations far higher than those observed in spillover release. These data suggest that dopamine signaling can also occur through tightly coupled ‘synaptic’ transmission, characterized by rapid changes (milliseconds) in high concentrations of dopamine (10-100 µM) that are spatially restricted (~ 3 µm). The stark difference in spatiotemporal structure between spillover and synaptic transmission suggests that these two modes mediate distinct functions. Yet, little is known about how spillover and synaptic dopamine transmission are regulated or how these two forms of transmission inform healthy and diseased behaviors. This study will implement an innovative combination of synaptic electrophysiology and behavioral assays with a battery of perturbations to dopamine release to determine: (1) whether spillover and synaptic transmission occur through independent or intertwined mechanisms of release, and (2) whether these two forms of transmission serve distinct functions in reward behaviors. Together, these experiments will test the hypothesis that synaptic and spillover dopamine release are two independently regulated forms of transmission with distinct behavioral functions. By illuminating the microarchitecture of dopamine release, this work will lay a new framework for understanding how dopamine transmission shapes reward behaviors critical to both health and addiction.

NIH Research Projects · FY 2024 · 2024-08

Abstract The overall goal of this project is to address the critical unmet need for longitudinal data from clinical outcome assessments (COAs) and plasma biomarkers for the rare syndrome of posterior cortical atrophy (PCA) to enable clinical trial readiness with currently available candidate therapeutics. PCA was described in 1988 in five patients with progressive dementia that began with higher-order visual dysfunction and relative sparing of `memory, insight, and judgment' with a predominance of atrophy in the parieto-occipital regions. We now understand that Alzheimer's disease (AD) is the underlying pathology in up to 100% of patients and onset usually occurs at a younger age, between age 50 and 64 years. The visual-predominant phenotype and lack of longitudinal data combine to impede clinical trial design for PCA. Notably, widely used COAs for AD clinical trials rely on changes in memory and domains that are relatively spared in PCA, and assessments of higher-order visual impairment are very limited in COAs in AD trials. For these reasons, PCA presentations of AD do not meet the inclusion criteria for traditional AD clinical trials. To overcome barriers to inclusion and to clinical trial design, we will enroll 25 people with PCA, defined by 2017 diagnostic criteria, and longitudinally assess, over one year the following outcomes 1) one patient-reported and one informant-reported PCA-specific COA, 2) a novel PCA COA staging method using a probabilistic ordering of decline on 13 tasks that are inclusive of higher-order visual functions, 3) AD-COAs widely used in AD clinical trials, and 4) plasma biomarkers of neurodegeneration and AD pathology. In Aims 1 and 2, we will measure the responsiveness of COAs to PCA progression and the reliability and internal consistency of an informant-based PCA-specific COA. The convergent validity and responsiveness of the novel PCA staging COA will be assessed in Aim 2. Lastly, we will evaluate the responsiveness of plasma biomarkers to clinical changes in exploratory Aim 3 in anticipation of the future use of biomarkers as surrogate markers of treatment impact in a clinical trial. The establishment of these longitudinal benchmark measures using COAs and plasma biomarkers will serve as an essential foundation for enabling PCA clinical trial readiness with promising and currently available candidate therapeutics.

NIH Research Projects · FY 2025 · 2024-08

Abstract New therapeutic options are needed to treat people infected with multi-drug resistant HIV-1 phenotypes. Here we propose to both investigate and exploit the non-catalytic, essential function of HIV-1 integrase (IN) during virion morphogenesis as a novel therapeutic target. Our highly collaborative research team has already made important contributions toward these goals. Specifically, our studies have revealed that IN binds the viral RNA genome (vRNA) in virions and that these interactions enable formation of properly matured, infectious HIV-1 particles. Moreover, we found that IN tetramers bind with high affinity to select vRNA elements. These findings have begun to uncover mechanistic details behind the non-catalytic function of IN. We have been also investigating the mode of action of a promising class of novel antiretroviral agents termed allosteric HIV-1 inhibitors or ALLINIs. Our studies have demonstrated that ALLINIs inhibit IN-vRNA interactions in virions by inducing aberrant IN multimerization. Consequently, ALLINI treatments yield inactive virus particles with ribonucleoprotein complexes mislocalized outside of the protective capsid. Our more recent studies have focused on characterizing the antiviral mechanism of action of a lead clinical candidate ALLINI pirmitegravir (PIR, STP0404) discovered by ST Pharm, Republic of Korea. This highly safe and potent HIV-1 inhibitor has recently advanced into Phase 2 clinical trials in the USA. Here we propose to build on our highly successful research and have set out the following two multidisciplinary and complementary specific aims. In aim1, we will continue our investigation into the non-catalytic role of IN during virion morphogenesis. For this, we will use biochemical assays to delineate sequence and structural features of vRNA preferentially recognized by HIV-1 IN. Furthermore, we will determine cryo-EM structures of IN tetramers bound to cognate vRNA segments. In addition, we’ll develop a novel and powerful in vitro model system for assembly of mature virus like particles by using IN mediated encapsulation of vRNA inside capsid like particles. Taken together, these studies will markedly advance our understanding of the non-catalytic function of IN during virion morphogenesis and the underlying mechanisms for assembly of mature virions. In Aim 2, we will continue our investigation into a highly promising investigational drug PIR. Specifically, we will delineate the mechanism for how PIR impairs IN-vRNA interactions. Furthermore, our structural, biochemistry and virology experiments will uncover the underlying mechanisms for drug-resistance to PIR. In turn, the findings from these studies will inform our medicinal chemistry efforts to rationally develop improved chemotypes with an enhanced barrier to resistance. Collectively, the two specific aims will elucidate key aspects of the HIV-1 IN biology and will exploit these insights to advance novel therapies to treat people living with HIV-1. 1

NIH Research Projects · FY 2025 · 2024-08

SUMMARY Preeclampsia (PE) remains a poorly understood, multiorgan vascular disease that is responsible for the death of up to 50,000 women and 1 million infants per year. Defective placentation and an abnormal immune microenvironment at the maternal-fetal interface are central to the etiology of PE. Specifically, insufficient extravillous trophoblast (EVT) invasion and, in turn, incomplete remodeling of the uterine spiral arteries, limits placental perfusion and provokes the systemic maternal vascular endothelial dysfunction hallmark of PE. The nuclear factor kappa B (NF-κB) family of transcription factors is a master regulator of inflammatory responses that promote EVT invasion. Yet, excessive NF-kB activation in the vascular endothelium can impair uterine vasodilation and reduce uteroplacental blood flow. We recently identified a novel NF-κB variant (rs230511) that is associated with PE. Here we will combine robust human physiological studies and CRISPR-Cas9 gene-editing to establish the functional importance of this NF-κB variant for inflammatory responses in pregnancy, and the role of NF-κB more generally in PE. We hypothesize that rs230511 increases PE risk by enhancing inflammatory cytokine responses that, in turn, impair uteroplacental vascular function and perfusion. We will study a Bolivian high-altitude (≥ 8,250 ft) population, where the incidence of PE is three-fold greater than at sea level. Aim 1 will establish whether rs230511 is associated with impaired spiral artery remodeling, elevated circulating inflammatory cytokine levels, lower uteroplacental perfusion, and reduced fetal growth. Using DNA isolated from 400 Andean maternal-infant pairs residing in La Paz, Bolivia, we will (1) determine maternal and infant rs230511 genotypes; (2) quantify the extent of spiral artery remodeling, (3) determine maternal and umbilical venous inflammatory cytokine levels, uteroplacental blood flow and fetal growth; and (4) contrast these primary variables by rs230511 status in maternal-infant pairs. To account for the effects of admixture, we will estimate individual admixture using genotype data obtained by the Illumina Multi-Ethnic Genotyping Array. Aim 2 will determine whether rs230511 disrupts inflammatory responses in human vascular endothelial cells (HUVECs). We will use the CRISPR-Cas9 system to create a targeted rs230511 conversion (C→T) in HUVECs and measure the effects on transcriptional responses of NF-κB target gene networks to TNFα exposure by RNASeq, and inflammatory cytokine secretion using a high-sensitivity multiplex assay. Our findings will be applicable for identifying novel pathways to improve reproductive outcomes in other populations given that hypertensive disorders of pregnancy are HA are equivalent to those at LA in terms of risk factors, physiological manifestations and consequences for mother and child, and the genetic variant of interest is not confined to HA populations. By advancing the understanding of maternal and fetal genetics for the regulation of inflammatory responses during pregnancy and PE, our project will help guide future mechanistic studies, aid the development of preventive strategies to improve reproductive outcomes and identify women at an increased risk of PE.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Shigella are bacterial pathogens that are a major source of moderate-to-severe diarrhea, and globally, a leading cause of long-term disability and disease in children. Illness necessitates Shigella invade epithelial cells lining the colon and then spread between the cells of the epithelial monolayer. This type of spread enables bacteria to avoid immune detection while acquiring nutrients and increasing the size of their replicative niche, thus intercellular spread is essential for severe disease. To spread, Shigella repurposes the actin cytoskeleton to acquire actin-based motility within cells. Shigella that move to the plasma membrane deform it into structures known as protrusions. These protrusions are essential for spread, because they enable bacteria to push into adjacent, uninfected cells. While these macroscopic steps are known, the molecular mechanisms that drive them are much less understood. Here, I propose to study the molecular mechanisms for how synaptopodin (Synpo) promotes Shigella flexneri intercellular spread. Our data demonstrate that Synpo is required for disease caused by S. flexneri, as it is essential for the bacteria to spread between cells. We show that Synpo enables the formation of longer actin fibers within the actin tail and enhances the efficiency of protrusion formation. It is unclear how Synpo promotes these processes, my preliminary data show S. flexneri infection alters the phosphorylation state of Synpo within defined or predicted protein interaction domains. I hypothesize that the phosphorylation state of Synpo is altered during infection such that Synpo forms a complex with distinct proteins during infection, enabling the recruitment and function of these proteins at sites of bacterial protrusion formation and sites where bacteria polymerize actin. To test aspects of this hypothesis, I propose the following aims: Aim 1. To define whether phosphorylation of Synpo is necessary for S. flexneri intercellular spread. Aim 2. To define whether Synpo promotes localization of host proteins to S. flexneri during infection. The completion of these aims is likely to define new mechanisms about how Synpo functions in cells and about how bacteria spread intercellularly. These approaches will further define the role of post-translational modifications of Synpo, identify proteins that interact with Synpo, and determine how Synpo is localized to particular positions within the cell. Further, many pathogens that cause diarrhea require mechanisms similar to Shigella, suggesting that my studies are likely to yield general insights into how pathogens cause diarrheal disease.

NIH Research Projects · FY 2024 · 2024-08

The sense of taste is mediated by taste buds on the tongue. Each bud is a heterogeneous collection of taste receptor cells (TRCs), and all TRCs, which transduce sweet, bitter, salt, sour and umami tastes, are continually renewed from adult stem cells outside of taste buds. However, taste function can be perturbed by numerous insults, including drugs and viral infection that affect TRC renewal. Ex vivo organoids are now a conventional method for study of cellular and molecular mechanisms of adult epithelial homeostasis, e.g., intestine. We and others have recently established this technology for the taste system, where isolated stem cells from the circumvallate taste papilla (CVP) of adult mice are cultured to generate TRC-containing lingual organoids; this model is used increasingly to study the impact of drugs and pathogens on taste homeostasis. While informative, however, results in mice may not precisely reflect how taste is perturbed in patients. Therefore, here we proposed to establish human TRC-replete lingual organoid lines from CVPs obtained postmortem from organ donors as a first step in developing human in vitro models. Our strategy overcomes several logistical and technical barriers to production of lingual organoids in humans. 1. We now have a pipeline to obtain CVP biopsies through a non-profit organ and tissue procurement organization. 2. We are expert at mouse lingual organoids and will apply our skill to human tissue. 3. We will work closely with our Organoid and Tissue Modelling Core, who have already created organoid lines from human postmortem donor pancreas and intestine. Aim 1. Validate and characterize CVP samples in male and female human donor tissue. Humans have multiple, easily visible CVPs. Initially, PSR staff will process harvested biopsies to verify CVPs are reliably obtained. Subsequently, TRC and stem cell marker gene expression will be assessed via q-PCR, in situ hybridization (ISH) and/or immunofluorescence (IF). We will perform single cell RNA-sequencing (scRNA- seq) of fresh male and female biopsies to transcriptionally profile CVP epithelial cell populations, identify differentially expressed genes in human TRCs and stem cells, and construct lineage models. Aim 2. Establish human lingual organoids from CVP biopsies. Initially, small tissue pieces of CVPs will be cultured under our mouse lingual organoid protocol. Organoids will be assessed for TRC and stem cell marker gene expression via qPCR, ISH and IF as in Aim 1. The organoid protocol will be modified as necessary; permutations may include differences in treatment of starting material, plating density, culture media components, and duration of culture. Once TRC-replete organoids are produced reliably, organoid lines from at least 2 female and 2 male donors will be maintained through serial passage and storage of frozen stocks. Additionally, we will perform scRNA-seq of 1 male and 1 female organoid line for bioinformatic comparison with CVP datasets obtained in Aim 1.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Craniosynostosis is the premature fusion of one or more cranial sutures. The growth at the cranial plates normally separated by the fused suture is arrested, resulting in compensatory overgrowth parallel to the fused suture. This abnormal development causes head malformations and can lead to increased intracranial pressure and developmental complications. Patients with this condition normally undergo surgical treatment to remove the growth constraints and create more aesthetically normative head shapes. However, the traditional evaluation of these patients after treatment has been based on subjective clinical expertise. During the last decade, three- dimensional (3D) photogrammetry has gained popularity to evaluate craniofacial anomalies, but existing methods to analyze this data have failed in their clinical translation due to the use of inefficient and inaccurate processing techniques. Hence, there is a lack of quantitative evidence to evaluate and compare the continuous process of head shape normalization between different surgical treatments. This proposal aims to develop new geometric deep learning methods to enable the fully automated, real-time evaluation of head shapes using 3D photogrammetry at the clinic and will address the lack of quantitative evidence in the objective assessment of surgical outcomes. The first aim of this proposal is to characterize the normalization of head shape after corrective surgery for different treatment approaches. A statistical model of head shape normalization for each surgical approach will be created to quantify novel metrics of head shape anomaly and probabilistic risk of craniosynostosis. This model will be the first to incorporate the effects of age at surgery, sex, and the pre-surgical severity of head shape anomalies on the normalization of head shape. The second aim of this proposal is to develop a personalized geometric deep learning framework to determine the optimal surgical approach for each patient with craniosynostosis. This aim will incorporate a novel context-encoding geometric deep learning method to estimate the expected post-surgical head shape normalization for each potential surgical treatment and identify the optimal surgical treatment for every patient based on objective retrospective data. The results from these aims will enable the data-driven, personalized, and objective assessment of surgical treatment for craniosynostosis that can be used to optimize patient management. This proposal includes a comprehensive training plan consisting of mentored computational training in the development of deep learning methods, mentored clinical and translational research training at Children’s Hospital Colorado, and didactic coursework in statistical and machine learning methods. This proposal is uniquely positioned to be successful given the multidisciplinary environment at the University of Colorado Anschutz Medical Campus with resources in translational research at Children’s Hospital Colorado and machine learning expertise within the Medical Image Phenotyping lab through the Department of Biostatistics and Informatics.

NIH Research Projects · FY 2025 · 2024-08

HIV infection and unintended pregnancy are highly prevalent, life-altering events among adolescent girls and young women in low- and middle-income countries. Due to a number of biological, social, and structural factors, adolescent girls and young women in sub-Saharan Africa remain a sub-population highly susceptible to HIV infection. Further, the majority of pregnancies among adolescent girls and young women occur in low- and middle-income countries with the highest rates found in sub-Saharan Africa. In Zambia, female university students are a neglected, high-risk sub-population for both HIV infection and unintended pregnancy. Oral pre- exposure prophylaxis and modern contraception are key biomedical prevention tools but increasing the uptake among young women in sub-Saharan Africa has proven challenging. Based on preliminary research, we propose to design a novel peer navigator pre-exposure prophylaxis/contraception mobile health intervention targeting high-risk female university students in Zambia. The peer-navigator intervention will be facilitated by a tablet-based platform to assess HIV and unintended pregnancy risk, provide education and information, link high-risk young women to sexual and reproductive services, assess preferences for peer support, track the use of health services, and provide ongoing support for pre-exposure prophylaxis and contraception uptake and persistence through regular communication. Using a mixed methods phased approach guided by integrated behavioral and mobile health theories, we will first adapt an existing mobile application using Human-Centered Design and qualitative methods to create a new tablet-based application to serve as a platform for peer navigators to promote and track pre-exposure prophylaxis and contraception use among high-risk female students (Aim 1, R21 phase). After several iterations have been evaluated by stakeholders, we will test the application and peer-navigator intervention package in a small prospective single-arm trial, collecting data on the functionality of the application and the acceptably/appropriateness of the peer-navigator intervention (Aim 2, R21 phase). After refining the application and intervention package, we will then thoroughly test the mHealth intervention through a single-site hybrid type 1 effectiveness-implementation randomized controlled trial to 1) determine the effectiveness of the intervention to increase pre-exposure prophylaxis and modern contraceptive use, 2) establish underlying behavioral mechanisms, and 3) evaluate implementation-related factors that will enhance the likelihood for the intervention to be implemented, sustained, and scaled (Aim 3, R33 phase). If proven effective, the peer- navigator mobile health intervention will provide a scalable model to fill-in persistent gaps in HIV prevention and reproductive health among young women in high HIV prevalence low- and middle-income country settings.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Facioscapulohumeral muscular dystrophy (FSHD) is a devastating muscle disease caused by the inappropriate expression of an early embryonic transcription factor, DUX4, in the skeletal muscle. DUX4 activates hundreds of genes in muscle cells and eventually induces toxicity. Which of the DUX4-driven gene expression changes underlie its pathogenicity is currently unknown. Our previous research has shown that DUX4 significantly affects RNA metabolism in muscle cells. Specifically, DUX4 induces altered splicing and proteasome-mediated loss of RNA quality control, with a resultant increase in aberrant transcripts that encode potentially toxic, truncated proteins. Hence, we hypothesize that DUX4-induced misregulation of RNA metabolism is a key driver of its pathogenicity in FSHD. To test this hypothesis, we seek to determine the mechanistic underpinnings of how DUX4 increases aberrant RNAs in the cell. We will do so by determining the mechanism of DUX4-induced aberrant splicing (Aim 1) and identifying factors that induce loss of RNA quality control via the ubiquitin-proteasome system upon DUX4 expression (Aim 2). We will use a combination of biochemistry, genome engineering, proteomics, and genomics to address these questions in a temporally controlled, inducible DUX4 expression system. Successful completion of these studies will allow us to determine how DUX4, a transcription factor, rewires the post-transcriptional gene regulatory network of muscle cells. Given that perturbed RNA metabolism is a common mechanism underlying many muscle diseases, a better understanding of its role in FSHD could also aid in novel therapeutic discovery.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY The goal of this proposal is to identify the mechanistic link between fetal glucagon and placental nutrient delivery as a novel, adaptive mechanism for matching nutrient supply to fetal metabolic demand under adverse uterine conditions. Pregnancies complicated by intrauterine acidosis, hypoxia, and growth restriction are often characterized by elevated fetal glucagon. While fetal metabolic hormones have historically been considered for their function in regulating fetal growth, recent evidence from the Rozance laboratory has suggested glucagon may be regulating maternal nutrient delivery via placental signaling. In nine-day fetal glucagon infusions in sheep, we observed dramatically reduced uterine blood flow which resulted in decreased placental transfer of amino acids, glucose, and oxygen to the fetus. This decrease was accompanied by a robust reduction in maternal placental lactogen (CSH) concentrations, which we have previously demonstrated results in lower uterine blood flow and placental nutrient delivery in an in vivo model of placental CSH RNAi in sheep. Thus, we hypothesize that fetal glucagon regulates placental nutrient delivery by inhibiting placental trophoblast CSH secretion and uterine blood flow. This proposal will demonstrate that fetal glucagon acts by matching placental nutrient delivery to fetal growth and metabolic demands by regulating maternal CSH secretion and NO-mediated vasodilation to alter blood flow. Aim 1 will establish the mechanism by which fetal glucagon inhibits CSH secretion by the trophoblast. Aim 2 will identify the link between fetal glucagon, CSH, and uterine blood flow, and how this relationship regulates vasoactivity and placental nutrient delivery. Expected outcomes: This proposal will be the first mechanistic physiological investigation into fetal glucagon as an inhibitor of trophoblast CSH production, regulating uterine blood flow and placental nutrient transfer. Furthermore, it will also demonstrate the novel vasodilatory role of CSH in uterine circulation. Lastly, this proposal will provide me critical training in fetal physiology, improve the translatability of my research by working with human samples, augment my grantsmanship and scientific writing skills, and cultivate key professional relationships to establish successful future collaborations as a tenure-track, assistant professor specializing in fetal-placental-maternal communication. Impact: The research outlined by this proposal will identify a previously unknown mechanism for matching nutrient delivery to fetal metabolic demand to increase survival during pregnancy complications.

NIH Research Projects · FY 2025 · 2024-08

In the natural world, animals rely heavily on their sense of smell to safely navigate their environment, and experimentally mice can be taught complex decision-making tasks which rely on the animal's ability to integrate experience and olfactory stimulus. Association of context and olfactory stimuli is thought to be achieved through the encoding and retrieval of memories through the hippocampus. The hippocampus, a curved structure nestled deep within the temporal lobe of the brain, serves three primary functions: the regulation of emotions, formation of new memories, and memory retrieval. It plays a vital role in the creation, organization, and retention of fresh memories, as well as in linking certain sensations and emotions (valence) to these memories. The dorsal and ventral hippocampus regions have been implicated in acquisition, consolidation, and retrieval of temporal information in optogenetic stimulation of trace fear conditioning by a significant number of studies and importantly, lesions to dorsal CA1 (dCA1) result in impaired memory retrieval. Calbindin 2 pyramidal cells in dCA1 have additionally been implicated in learning during the go-no go associative discrimination task, as inhibition slows learning. Interestingly, within dCA1 exists a population of pyramidal cells that during the go-no go associative learning task demonstrate divergent stimulus responses, which take place at discrete times during the go-no go task, thereby exhibiting ‘time tiling’. This can be thought of as temporally discrete divergence in activity related to stimulus valence and are called ‘decision-predicting time cells’. With the recent discovery of decision-predicting time cells, there is currently no literature investigating their molecular composition, their influence on behavior, and their interactions with interneurons. Of note, it has been demonstrated that PV interneurons play crucial roles not only in regulating the excitability of cell populations, but also in providing precisely timed inhibitory input. Currently, no studies have characterized the roles of parvalbumin-positive interneurons in dCA1 hippocampus during complex olfactory discrimination tasks, particularly related to decision-predicting time cells. Aim 1 will probe if decision-predicting time cells and calbindin 2 positive and utilize advanced optical techniques such as two-photon holographic stimulation to selectively impair divergent time tiled responses. Aim 2 will characterize the role of PV interneurons related to decision-predicting time cell activity and determine the effect on licking behavior and accuracy during the go-no go olfactory task if PV interneurons are inhibited during the task.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Restoring the integrity of the gastrointestinal tract could be key to counteracting the spectrum of inflammation- associated debilitating illnesses and increased mortality among persons living with HIV-1 (PWH) on antiretroviral therapy. This may be accomplished by harnessing pathways that maintain gut homeostasis, of which IgA- microbiome interactions are emerging as fundamental mechanisms. In fact, using gut biopsies from an age/sex- matched cohort of PWH and HIV-1-uninfected individuals, we demonstrated alterations in the IgA repertoire among PWH that associated with specific bacterial taxa. Testing these IgA-microbiome correlations experimentally is a major challenge, as the human antibody repertoire is extremely diverse, with over a trillion distinct antibodies possible. Interestingly, recent high-throughput studies revealed the existence of 'public' antibodies: clonotypes that were shared among unlinked individuals that encode the same V and J genes and highly related CDR3 antigen-binding regions. The biology of public antibodies remains mysterious; statistically, these antibodies should not exist unless there is a shared antigenic history and/or evolutionarily-conserved function. Notably, in inbred, specific-pathogen-free mice, a subset of public IgA interacted with the gut microbiome, suggesting a potential role in gut homeostasis. To date, whether these findings extend to humans remains unclear. Using our next-generation sequencing dataset, we uncovered over a dozen public IgA clonotypes specific to PWH versus HIV-1-uninfected controls, and vice versa. As a critical first step in understanding their biology, our main goal in this exploratory study is to prepare recombinant public IgA clonotypes specific to HIV-1-uninfected individuals or PWH, and interrogate their reactivity against fecal bacterial communities and microbial glycans. We will then test if bacteria-reactive public IgA recovered from PWH gut biopsies influence HIV-1-mediated CD4+ T cell death and inflammation in a robust ex vivo Lamina Propria Aggregate Culture model that we developed previously. These studies promise to provide the field with a first look at the nature and immune properties of public IgA clonotypes in the human gastrointestinal tract, that may provide promising leads for therapeutic modulation of mucosal inflammation in chronic HIV-1 infection and a wide variety of medical conditions associated with a 'leaky gut' syndrome.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract Type 1 Diabetes (T1D) is an autoimmune disease thought to be mediated by autoreactive CD4 and CD8 T cell that orchestrate the death of insulin-producing beta cells within pancreatic islets. This proposal aims to investigate how antigen-specific immunotherapy (ASI) with a CD4-specific neoepitope can selectively suppress autoreactive T cells by tolerogenic delivery of antigens in the NOD mouse model of autoimmune diabetes. Previously, our lab demonstrated that induction of tolerance to a dominant CD4 neoepitope, the 2.5 Hybrid Insulin Peptide (2.5HIP), can prolong islet graft survival in diabetic NOD mice. In the outlined studies, we will investigate the mechanistic impact of ASI on the differentiation and function of effector CD4 and CD8 T cells in islet grafts. The role of IL-10-producing regulatory T cells, expanded by 2.5HIP tolerance induction, will also be examined. We hypothesize that induction of tolerance to the 2.5HIP expands antigen-specific IL10-producing regulatory T cells that, directly or indirectly, arrest the effector differentiation and function of autoreactive CD4 and CD8 T cells within islet grafts. The specific aims of the study are to (1) determine the differentiation and functional states of antigen-specific CD4 and CD8 T cells following induction of tolerance to the hybrid insulin peptide, and (2) determine the contribution of IL10-producing regulatory T cells to prolonged graft survival and the suppression of autoreactive T cells. The completion of the proposed studies is expected to generate high-impact information on the mechanisms underlying autoreactive T cell suppression and differentiation following induction of antigen- specific tolerance. Findings from this research will also critically inform antigen-specific therapeutic approaches for human T1D and other autoimmune diseases.

NIH Research Projects · FY 2025 · 2024-08

Abstract Advancing patient care in otolaryngology is critically dependent on training future leaders in the field to become successful independent researchers and drive advancements in patient care. Most otolaryngology residency programs offer limited research training and there is a dire need for well-trained independent physician investigators in the specialty. New approaches are required to recruit budding physician investigators into academic research-focused careers in Otolaryngology. Therefore the goal of this new R25 application is to provide exceptional research training in otolaryngology-related research to carefully selected medical students and Otolaryngology residents. Trainees will receive research training and career development to set them up with the skills required to become outstanding clinician scientists able to perform high impact research. Resident trainees will undergo 18 months of research integrated into their 6 year residency training program. Research training for medical students will occur in the third year of medical school with a dedicated 9 month research block. One resident and one medical student with exceptional research promise and dedication to an academic career in Otolaryngology will be selected from diverse applicant pools and admitted each year into the research training program. The Department of Otolaryngology has a balanced, well-funded and expanding research portfolio and an excellent track record of producing physician scientists through our existing NIDCD T32 training grant. Funded faculty with active research programs will serve as research mentors and clinically-focused faculty will serve as career mentors. The wider community at The University of Colorado Anschutz Medical Campus has major strengths in all areas related to Otolaryngology including hearing, balance, smell, taste, speech, language and disorders of communication and head and neck cancer. The research training faculty has existing extensive research collaborations which will continue to grow and foster an outstanding team-based training environment and unique training experience for each of our trainees. The intensive research and mentoring program for each trainee will involve individualized development plans, training in innovative research methods, structured didactics and rigorous ethics education.

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT The Mountain West region experiences a higher burden of injury mortality than many other parts of the nation and the region includes significant populations including rural and frontier populations, Latinos, American Indians, Veterans, and uninsured individuals. The Injury and Violence Prevention Center (IVPC) at the University of Colorado Anschutz Medical Campus was established with the vision of contributing to the significant reduction in injury and its outcomes in Colorado, the Mountain West region, the U.S., as a function of high-quality research, education, and effective practice. We seek to accomplish this with three specific aims (1) Nurture cutting-edge prevention-focused research on injury and violence that responds to and guides practice in regional and national contexts; (2) Enhance skills of the current and future injury prevention workforce, engage related professionals and trainees across disciplines, and increase awareness of prevention strategies, and (3) Provide leadership in addressing injury problems by working with community members, practitioners, and policy makers. We will achieve these aims through objectives and activities organized into 3 cores: Administrative Core, Outreach Core, and Training and Education Core. As we work toward these aims, and our mission of driving evidence-based practice through research, education, training, and practice, we affirm these core values: (1) Advancing Health Equity; (2) Ethical Research and Practice; (3) Translation of Research to Practice, and (4) Collective Impact. The four proposed research projects address research priorities for the National Center for Injury Prevention and Control. As part of an established partnership working to address substance use and adverse childhood experiences (ACE’s), Project 1 will use a community- based approach to identify the social determinants of health- and substance use disorder-related needs of a rural, largely Latino community in Colorado. Project 2 will use a randomized control trial design to test messages to prevent firearm suicide among Veterans. Project 3 will identify social and structural level factors of suicidal ideation for Black girls and young women to inform and develop effective suicide prevention efforts. Project 4 will use an innovative methodological approach, and a nationally representative sample, to understand parents’ decisions regarding their children’s contact and/or collision sports participation. We are uniquely well-positioned to provide leadership in the region and nationally. We will build on our solid foundation of a team of nationally and internationally recognized injury prevention leaders and researchers and strong collaborative relationships with practice partners.

NIH Research Projects · FY 2025 · 2024-07

1 Research: Tyrosine kinase inhibitors (TKIs) such as alectinib have transformed survival outcomes for patients 2 newly diagnosed with anaplastic lymphoma kinase (ALK) positive non-small cell lung cancer (NSCLC). For 3 most patients, initial TKI therapy results in tumor response but with variable levels of disease shrinkage and 4 control. Extrinsic factors for this variability, such as the immune system, have not been well studied as these 5 tumors are considered immunologically “cold.” However, our data suggests immune cells within the tumor 6 microenvironment (TME) shape the extent of TKI response. Patients with ALK+ NSCLC experience faster 7 disease progression on TKI with increasing numbers of neutrophils in pre-treatment biopsies. Further, their 8 overall survival is significantly shorter on TKI when cancer cells have increased transcriptional activity of 9 CXCL5 and CXCL8, potent chemoattractants for neutrophils. We believe neutrophils within the ALK+ NSCLC 10 TME impair tumor reactive CD8+ T cells. Murine models of ALK+ NSCLC require CD8+ T cells, but not CD4+ 11 T cells, for a sustained alectinib response and tumor clearance. Analogous to the patients, the ALK+ NSCLC 12 murine model that upregulates potent chemoattractants for neutrophils, contains 1) increased Ly6G+ 13 neutrophils and 2) fewer activated CD8+ T cells in the TME, and 3) reduced responsiveness to alectinib. Our 14 central hypothesis is that the recruitment of neutrophils by tumor mediated cytokines decreases alectinib 15 responsiveness by impairing anti-tumor T cells. In this proposal, using orthotopic mouse models of ALK+ 16 NSCLC, we will determine how neutrophils modulate the TME and CD8+ T cell responses in ALK+ NSCLC. 17 Candidate: Dr. Erin Schenk's career goal is to become a leading laboratory-based physician scientist 18 identifying critical TME immune cells that drive treatment response in ALK+ NSCLC. She will apply innovative 19 technologies and model systems to generate scientific rationale for clinical trials testing novel immunotherapies 20 in patients with NSCLC. To reach this goal, Dr. Schenk will master CRISPR to modify the immune TME 21 content in mouse models of ALK+ NSCLC, test for TKI response, and apply unbiased approaches to explore 22 the TME with scRNAseq, full spectral flow cytometry, and spatial transcriptomics. In parallel, she will acquire 23 the skill sets necessary for clinical trial development and implementation. 24 Environment: The University of Colorado is the only NCI-designated cancer center in Colorado and has an 25 international reputation in clinical and translational work in ALK+ NSCLC. Dr. Schenk has ready access to 26 multiple shared resources (cores) across the cancer center, on line and in person graduate level didactic 27 courses, institutional support from the Thoracic Oncology Program and the Division of Medical Oncology. She 28 has assembled a panel of accomplished mentors including TME expert Dr. Raphael Nemenoff, tumor 29 immunologists Drs. Eduardo Davila and Jill Slansky, neutrophil expert Dr. Sean Colgan, and international 30 ALK+ NSCLC expert and clinical trialist Dr. Ross Camidge. 31

NIH Research Projects · FY 2026 · 2024-07

Every year, more than 75,000 children and 750,000 adults in the United States are admitted to an Intensive Care unit with a diagnosis of sepsis. Sepsis-associated acute kidney injury (SA-AKI) has a high morbidity and mortality rate and unfortunately there are no methods to predict, prevent, or treat SA-AKI. Emerging evidence implicate the role of complement activation as a key driver of SA-AKI development. We have shown that urine complement factor Ba is associated with incident AKI in both critically ill children and adults with sepsis. However, whether complement activation is involved in SA-AKI pathogenesis in critically ill children and adults is unknown. We hypothesize that urine Ba elevation temporally precedes development of SA-AKI and associates with urine cellular changes. This proposal will examine the association between urine Ba and AKI in critically ill patients with sepsis. First, we will prospectively enroll critically ill children with sepsis and longitudinally obtain urine Ba levels to determine the association between urine Ba levels and severe AKI outcomes, and then validate these findings in a large multicenter trial of pediatric sepsis (Aim 1). We will validate this association through two biorepositories from clinical trials in critically ill septic adults (Aim 2). This will determine when urine Ba peaks and identify cutoff values and identification of an ideal window to study use of therapeutic trials of factor B inhibition in the future. Finally, we will measure complement deposition on shed tubular epithelial cells in the urine to determine if complement activation is occurring in the kidney (Aim 3). As an Assistant Professor in Pediatric Critical Care with a strong publication record, I am establishing myself in the field of critical care nephrology. A K23 Career development award would support the following research goals: 1) strengthen the evidence implicating the complement cascade’s role in SA-AKI development; 2) identify a potential therapeutic window to stratify patients for treatment with a complement therapeutic in future R01 proposals; 3) obtain mechanistic insight discriminating systemic versus localized kidney complement activation. My career development plan includes dedicated time to 1) train and gain experience in conducting patient-oriented research and clinical trials; 2) work with a multidisciplinary team of experts in Adult and Pediatric Nephrology, Emergency/Critical Care, and Biostatistics; 3) pursue training in biostatistics and biomarker analytics; 4) gain skills in interpretation of complement quantification methods; 5) develop professional skills including manuscript preparation, grant writing, presentation skills, etc.; and 6) establish the foundation for an independent research career aimed at improving outcomes for critically ill patients with SA- AKI. My training environment is outstanding with mentorship from Dr. Kendrick, an adult nephrologist with clinical research expertise and a strong record of successful mentoring, and co-mentors Drs. Thurman and You, as well as a robust team of methods advisors. At the completion of this project, I will be well-equipped to develop an independent research program focused on intervenable mechanisms that relate sepsis with AKI.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY. CANDIDATE: Mathew Wingerson is a doctoral student at the University of Colorado Anschutz School of Medicine pursuing research innovations to support clinical management of concussion. His doctoral research has investigated novel approaches to prescribing aerobic exercise in clinical settings to facilitate concussion recovery. The training plan and research proposed in this F31 application will further equip him for an independent research career investigating post-concussion diagnosis, prognosis, and rehabilitation. DOCTORAL TRAINING PLAN: The training plan aims to (1) develop expertise as a clinical trialist in rehabilitation sciences, (2) expand knowledge in advanced data science methods for clinical research, and (3) gain meaningful experience in concussion fluid biomarkers of recovery and brain pathophysiology. These objectives will be achieved through coursework, research experience, conference attendance, and mentorship from a multidisciplinary team. SPONSOR, CO-SPONSOR, & COLLABORATORS: Dr. David Howell, an expert in concussion research, and Dr. Brianne Bettcher, a specialist in brain biomarkers, will serve as the project sponsor and co-sponsor. Additional contributions from Dr. Jonathan Smirl, a cerebrovascular physiologist, and Dr. Patrick Carry, an applied biostatistician and epidemiologist, will supplement the training plan, providing their expertise to facilitate Mathew’s doctoral education and successful execution of the research project. Dr. Julie Wilson, a physician-researcher and director of concussion clinical care at the Children's Hospital Colorado Sports Medicine Center, offers an expert clinical perspective throughout the execution of the training plan and research, having provided clinical management to children and adolescents with concussion for >13 years. RESEARCH: Active rehabilitation after concussion, including engagement with aerobic exercise, is standard-of-care clinical practice. Existing guidelines for prescribing aerobic exercise are complex, resource- and time-inefficient, and impractical for some clinical settings. Concussion-induced physiological impairments, such as cerebrovascular dysfunction, persist beyond symptom resolution and may not be effectively treated by current rehabilitation guidelines. This proposed research challenges existing clinical practice recommendations by investigating moderate-to-vigorous physical activity (MVPA), an innovative, clinically pragmatic rehabilitation strategy, as an efficacious approach to treating post-concussion physiological disturbances. Leveraging resources and data from an NIH-funded parent study (R01HD108133; enrollment goal: n=288), the project will identify important longitudinal relationships between post-concussion MVPA and cerebrovascular recovery. The study will deliver precise MVPA-based rehabilitative recommendations poised for immediate clinical implementation to facilitate recovery of concussion- induced impairments in physiological systems.