University Of Colorado Denver

NIH Research Projects · FY 2025 · 2017-03

Project Summary This is a competitive renewal application for a Jointly Sponsored Institutional Predoctoral Training Program in the Neurosciences (JSPTPN) for pre-thesis Ph.D. students in the Neuroscience Training Program (NSP) at the University of Colorado, Anschutz Medical Campus (CU Anschutz). The NSP is an interdisciplinary Ph.D. granting degree started in 1986 that has been funded by this training grant since 2001. Presently, NSP has more than 70 faculty members of whom 55 are Training Faculty on this application. NSP aims to train stellar graduate students who develop into independent, thoughtful, and critical thinking neuroscientists who can succeed in the 21st century. This is achieved through broad-based training in neuroscience that includes instruction in the fundamental concepts of neuroscience, diseases of the nervous system, experimental design and statistical methodologies, literacy in quantitative methods, and the responsible conduct of research. Further, our plan trains graduates who are not only high caliber academic scientists but also well-informed about various career options and professionally well-equipped. These specific objectives are achieved through coursework, mainly in the first two years of graduate school, and research projects in training labs. In addition, concepts that are introduced to students in coursework are reinforced and integrated throughout the student’s time in graduate school through various enrichment activities such as seminars and journal clubs and close mentoring by faculty during benchmark exams and Ph.D. thesis committee meetings. Our training plan has been continually enhanced over the years, especially in the area of quantitative methods, reflecting the Program’s identification of these skills as critical for performing high caliber research and for competitive placement of our graduates. Our mission is also to grow and sustain a diverse and inclusive student body. Our training plan includes multiple evaluative components to help ensure effectiveness. These include an annual evaluation by a Neuroscience T32 Advisory Committee of senior faculty at CU Anschutz experienced at leading training grants. The appointments for the Jointly Sponsored Predoctoral Training Program are for the first two years of predoctoral training. As in the prior funding period, we seek to have six student appointees every year, split evenly between first- and second-year students. We are requesting five years of support. The funded student slots will help us meet the research needs of a dynamic and growing neuroscience community at CU Anschutz and is, moreover, justified based on the demonstrated success of our prior trainees.

NIH Research Projects · FY 2026 · 2017-03

The overall goal of this proposal is to elucidate the thiol redox mechanisms that alter neurodevelopment, which can exacerbate cognitive dysfunction in Down syndrome (DS). Down syndrome (DS) is the most common genetic cause of intellectual disability. Importantly, the extent of intellectual disability is highly variable, and parents of affected individuals are often aware of DS during pregnancy. This creates a window of opportunity to improve outcomes through identification of environmental factors and associated mechanisms impacting early development in DS. Preliminary data and reports from our laboratory and others demonstrate that cells from DS individuals exhibit distorted proteostasis, enhanced oxidative stress, and altered metabolism. We hypothesize that these are linked through atypical thiol redox systems in DS cells. This increases vulnerability to thiol-reactive xenobiotics through dysregulation of central carbon metabolism, modifying stem cell fate decisions via altered regulation of Wnt/β-catenin signaling as a mechanism contributing to cognitive dysfunction. Our prior results show that the environmental toxicant, maneb (MB), a neurotoxic dithiocarbamate fungicide, impairs proteostasis, increases oxidative stress and displays greater toxicity in DS cells compared to euploid controls. MB also modifies mitochondrial function, central carbon metabolism and our new preliminary data show that DS cells display significant baseline alterations in the Wnt signaling pathway. Wnt signaling is a vital cell signaling conduit critical for both stem cell maintenance and neurodevelopment. Therefore, elucidating the environmental mechanisms impacting DS development, e.g. Wnt signaling and oxidative stress, will provide a foundation to prioritize environmental chemical surveillance in DS neurodevelopment. Disruption of cellular thiol redox systems, e.g. thiol redox proteome, is a key feature of oxidative stress. This mechanism is also critical for embryonic development, where mitochondrially-derived reactive oxygen species (ROS) trigger stem cells to differentiate. Thus, the approach detailed below will include thiol-reactive toxicants (TRT) as an innovative means to study Gene-Environment interactions affecting neurodevelopment in a special population, DS. This proposal involves three Specific Aims and makes use of a powerful library of euploid and trisomy 21 induced pluripotent stem cells (iPSC) and directed differentiation protocols to investigate the role of thiol redox signaling in the effects of TRT on stem cells derived from DS individuals, and how these exposures alter specific pathways (Wnt and central carbon metabolism) during neurodevelopment. In Specific Aim 1 we will determine if trisomy 21-mediated Wnt dysfunction is exacerbated by TRT exposure, resulting in aberrant iPSC differentiation. Western blotting, qRT-PCR, chemical tools and single cell transcriptomics will be used to interrogate this aim. Specific Aim 2 is designed to study the impact of TRT exposure on the thiol redox proteome and identify mechanistic targets involved in aberrant differentiation. In Specific Aim 3 we will utilize metabolic approaches like metabolomics and extracellular flux analyses to correlate Wnt dysfunction and redox proteomic alterations to metabolic alterations and developmental outcomes. Together, the novel research proposed here will fill a critical gap in knowledge with regard to neurodevelopmental impacts of thiol reactive toxicants in DS and how these exposures can alter neurogenesis, potentially contributing to cognitive variability. Finally, the studies described here are an innovative and data-driven extension of my funded research exploring further into new mechanisms, e.g. Wnt and thiol redox signaling, by which exposures can alter critical neurodevelopmental processes in DS.

NIH Research Projects · FY 2026 · 2017-02

Summary Neurons lack energy reserves and thus their survival depends on an uninterrupted, dynamically regulated supply of blood-borne nutrients, which are delivered through a dense capillary network. Precise control of the blood flow through the brain microcirculation is therefore essential for neuronal health. However, the mechanisms through which blood is distributed within the capillary network remains poorly understood. Furthering our understanding of this process is critical, as it is increasingly appreciated that disruption of brain hemodynamics is one of the earliest pathological events in cerebral small vessel diseases. Pericytes are mural cells that wrap around the endothelial cells forming the capillaries. Our extensive preliminary data show for the first time that pericytes located on the first to fourth order capillary branches constrict and relax in response to luminal pressure changes. This observation implies that the resistance created by the capillary network is not constant and homogeneous, but rather variable and dynamic, casting a new light on blood flow regulation. Specifically, we have found that pressure-induced constriction in pericytes engages the autocrine activation of the epidermal growth factor receptor (EGFR), subsequent inositol trisphosphate (IP3) signaling, and transient receptor potential canonical 3 (TRPC3) activation. Using a well-established genetic mouse model of CADASIL, a hereditary form of small vessel disease, we further propose that pathogenic mechanisms depress the EGFR activation in pericytes, resulting in impaired capillary blood flow autoregulation. To test these ideas, we engage a wide variety of novel, state-of-the-art experimental approaches using intact animals, native tissue, and freshly isolated cells, complemented by sophisticated computational modeling. Taking advantage of our newly developed pressurized arteriole- capillary ex vivo preparation, Aim 1 will explore how EGFR activation by intraluminal pressure and agonist-induced vasoconstriction contributes to γ1 phospholipase C (PLCγ1) activation and IP3- dependent Ca2+ signals. Aim 2 will determine the mechanism linking EGFR and PLCγ1 activation to TRPC3 channel opening to cause membrane depolarization and constriction. Finally, using extracellular matrix disruptions characteristic of CADASIL as a framework, Aim 3 will provide the first insights into the mechanisms by which pericyte contractility is regulated by EGFR and its upstream regulators TIMP3, a matrix metalloproteinase inhibitor, and ADAM17, a metalloproteinase that mediates shedding of the EGFR ligand, HB-EGF. The proposed work has the potential to provide a paradigm-shifting view on how pericytes control capillary blood flow distribution, and as such, should provide the foundation for understanding small vessel diseases of the brain.

NIH Research Projects · FY 2026 · 2017-01

PROJECT SUMMARY Individuals born with fetal growth restriction (FGR) have dysregulated hepatic glucose production (HGP) as neonates, and higher rates of hepatic insulin resistance and steatotic liver disease with higher hepatic fibrosis scores as children and adults. We discovered, using the same sheep models as in the current proposal, that the FGR fetus has an early activation of HGP and hepatic insulin resistance9, hallmark features of type 2 diabetes (T2D). We also discovered that fetal hypoxemia (HOX model) recapitulates hepatic insulin resistance. Our compelling new data demonstrates hepatic collagen deposition, a sign of fibrosis, in FGR and HOX fetal sheep. Thus, in utero hypoxemia during FGR injures the fetal liver initiating both hepatic insulin resistance and fibrosis. How hypoxemia produces these effects, and the cell-specific mechanisms remain poorly understood, thus limiting our ability to prevent fibrotic liver disease and T2D risk in the former FGR individual. The goal of this renewal is to demonstrate that lactate is more than a metabolic substrate and byproduct of hypoxemia and exerts regulatory control initiating hepatic insulin resistance and fibrosis in the FGR fetal liver. Lactate is abundant in normal mammalian fetuses with higher concentrations during FGR and hypoxemia. Our FGR and HOX fetal sheep also have increased systemic and hepatic lactate (i.e., lactate load). In addition to lactate's role as a precursor for HGP and oxidative substrate in adults and fetuses, emerging studies in adults and cancer cells demonstrate that lactate is a metabolite signal and driver of epigenomic regulation. Given these and our recent data, we propose a mechanism whereby increased lactate production and decreased lactate oxidation increase “lactate load” and enable lactate to be used for HGP; however, this redirection in lactate flux produces energetic and redox stress. In support, FGR and HOX fetal livers have JNK-FOXO1 stress kinase activation and oxidative stress, a major cause of insulin resistance and fibrosis in adults. Moreover, in FGR and HOX fetal sheep, collagen deposition co-localizes with higher lactate entering hepatic periportal regions. Thus, lactate may activate periportal hepatic stellate cells into collagen-producing cells. These findings form the premise for our central hypothesis that increased hepatic lactate load via its metabolic, signaling, and epigenomic effects initiates insulin resistance in hepatocytes and fibrogenic activation in stellate cells with intrinsic programming. Studies in Aim 1 will use our FGR fetal sheep model to demonstrate how lactate is metabolized in the normal and FGR fetal liver. We also will identify novel spatial and cell-specific intrinsic mechanisms to understand how fibrosis and IR develop in the FGR fetus. We propose that lactate-associated metabolism is largely responsible for these effects in the FGR liver. In Aims 2 and 3, we will directly test the role of lactate on insulin resistance and fibrosis by experimentally increasing or decreasing lactate load in fetal sheep. We will also perform in vitro studies using isolated primary fetal hepatocytes and stellate cells to test mechanisms and discern the direct versus indirect effects of lactate. Overall, our studies will delineate the physiologic and hepatocellular effects of lactate as a metabolite and regulatory signal in the fetal liver.

NIH Research Projects · FY 2026 · 2017-01

Project Summary The major genetic determinant in susceptibility to or protection from many autoimmune diseases reside in the human major histocompatibility complex (MHC) that contains the human leukocyte antigen (HLA) region. In type 1 diabetes (T1D), particular HLA class II alleles (e.g. DR4/DQ8) increase the risk for developing disease, whereas others (e.g., DQ6, DQB1*06:02) lead to dominant protection. MHC class II molecules function to present processed antigens to T cells, and the MHC class II−peptide−T cell receptor (TCR) forms a trimolecular complex involving the presentation of self-peptides that shape autoreactive T cell responses in autoimmunity. The goal of our studies is to bridge the gap of knowledge in diabetes-protective MHC class II molecules and insulin-specific T cell responses. Data from the last funding period indicates that T1D protective MHC class II molecules (murine IAb ≈ human DQ6) present insulin, and specifically insulin B chain amino acids 9-23 (B:9-23) to activate CD4 T cells with a regulatory phenotype (Tregs). In the NOD mouse model of spontaneous autoimmune diabetes, insulin-specific FoxP3+ Tregs are present but fail to prevent diabetes onset. The presence of IAb in addition to the NOD diabetes conferring IAg7 MHC class II abrogates all diabetes development. With fluorescent B:9-23 tetramers on each class II molecule, we are able to detect B:9-23/IAb type 1 regulatory T cells (Tr1 cells) in the pancreatic lymph nodes in NOD mice heterozygous for IAb/IAg7, while insulin-IAg7 T cells are less frequent and not activated. Similarly in non-diabetic humans with DQ6, we are able to proliferate Tregs from the peripheral blood that respond to insulin B:9-21/DQ6. Single cell RNA sequencing of these proliferated insulin-Tregs reveals a distinct Tr1 cluster that secrete the anti-inflammatory cytokine, IL-10. However, the necessity for these insulin-Tregs and mechanisms by which these cells confer diabetes resistance remains to be determined. In specific aim 1, we will determine the molecular phenotype and necessity for insulin-Tregs to protect against diabetes development in murine models with a diabetes-resistant MHC class II molecule. Specific aim 2 focuses on parallel studies in humans to identify the protective features of insulin-Tregs restricted to DQ6 and those to DQ8 in individuals with and without T1D. The successful completion of this proposal will provide insights into the function and molecular phenotype of insulin-Tregs restricted to diabetes-protective MHC class II molecules and determine their direct role in conferring diabetes protection, thus aiding the design of therapies that may improve Treg function to treat the underlying autoimmunity in T1D.

NIH Research Projects · FY 2025 · 2017-01

PROJECT SUMMARY In type 1 diabetes (T1D), autoimmunity is established and self-perpetuating within the islets at diagnosis. Autoreactive T cells in T1D patients destroy endogenous and grafted beta cells, yet there is a gap in knowledge about the mechanisms by which T cell pathogenesis is modulated in the islets. Evidence from mouse models of T1D and human samples suggest that myeloid cells play an essential role in this process. The myeloid compartment represents the largest immune population in non-diabetic and many T1D human islets. Islet myeloid cells can contribute to islet destruction or protection, yet a significant gap in knowledge remains about how the islet myeloid compartment plays such divergent roles in islet autoimmunity during T1D progression. We have shown that Mertk signaling in islet myeloid cells suppresses autoreactive T cell responsiveness to antigen and prevents rapid progression of T1D. Mertk mediates apoptotic cell uptake (efferocytosis), and its signaling is immunoregulatory. However, we do not yet understand the mechanisms by which Mertk signaling in islet myeloid cells suppresses the ability of autoreactive T cells to respond to locally presented antigen. Our scRNA-seq analyses of myeloid cells from the islets of non-diabetic and T1D organ donors support that, similar to our mouse data, efferocytosis is enhanced and antigen processing and presentation are suppressed during the active period of disease following T1D onset. Thus, our overarching hypothesis is that in the islets during T1D, stimulatory myeloid cell subsets promote pathogenic T cell functions through antigenic stimulation and inhibitory islet myeloid cell subsets suppress the pathogenic T cells in a manner that is dependent upon Mertk mediated efferocytosis within the pancreatic islet. To begin to address the gaps in knowledge about islet myeloid cell function, we propose the following aims: Aim 1: Elucidate the mechanisms by which Mertk expressing myeloid cells modulate effector and regulatory T cell responses in the islets. Aim 2: Characterize the myeloid cell subsets that actively present antigen to effector and regulatory T cells in mouse islets and how this is altered by Mertk signaling. Aim 3: Determine the human islet myeloid subsets that perform Mertk-mediated efferocytosis and drive T cell signaling in human islets. The successful completion of this proposal will result in: (1) an improved understanding of the mechanisms used by Mertk- expressing islet myeloid cells to suppress the islet T cell response, (2) an enhanced understanding of pancreatic islet myeloid cell subsets and pathways that drive pathogenic T cell activation versus those that regulate islet autoimmunity and (3) identification of novel therapeutic targets to potentially skew the autoim- mune T cell response toward protective immunity in type 1 diabetes.

NIH Research Projects · FY 2024 · 2016-12

MoTrPAC Project Summary The Molecular Transducers of Physical Activity Consortium (MoTrPAC) will discover and characterize the range of potential molecular transducers that underlie the health benefits of exercise in humans. MoTrPAC was launched in 2016 with six adult Clinical Centers and a pediatric Clinical Center that have collaborated to generate extensive Manuals of Operations to guide research protocols involving all aspects of clinical operations (Phase I). Phase II began in the fall of 2019 with all Clinical Centers demonstrating excellent progress toward initial recruitment goals and implementation of the protocol. The initial goal set forth by the Consortium was to recruit 270 children (10-17 years of age) and 1,980 sedentary adults (aged 18+ years of age) who are randomized to endurance exercise training (170 youth, 840 adults), resistance exercise training (840 adults), or no-exercise control (50 youth, 300 adults) interventions. Additional groups of highly active endurance trained (50 youth, 150 adults) or resistance trained (150 adults) individuals serve as comparator groups and do not undergo the MoTrPAC exercise interventions. The recruitment and enrollment approaches are sex balanced, with participants across a wide age range (10-17, 18-39, 40-59, and 60+ age groups) and of different races and ethnicity. Due to the COVID-19 pandemic, some MoTrPAC activities were suspended for more than a year, beginning in March 2020, with continued constraints through 2022. Despite the numerous challenges encountered as a result of the pandemic, recruitment activities at the adult and pediatric Clinical Centers have accelerated to a rate that is projected to successfully achieve the target enrollment numbers by the end of the new award period. This led the NIH Common Fund to release the current NOFO (RFA-RM-23-010) to provide MoTrPAC with funding to complete recruitment and follow-up for the clinical studies, including finishing mechanistic randomized controlled trials of sedentary adults and children and observational studies of highly active adults and children. This will enrich the participant cohorts that are critical to understand the heterogeneity of exercise adaptations across age, gender, and minority groups. This extension will enable MoTrPAC to complete the intended goals as originally envisioned and will provide a more complete public database of the health benefits of exercise and provide insight into how physical activity mitigates disease.

NIH Research Projects · FY 2025 · 2016-09

PROJECT SUMMARY/ABSTRACT Chronic conditions, such as obesity, asthma, depression, and developmental disabilities, are increasing among children worldwide. Further, the age at diagnosis for many such conditions is decreasing, pointing to early-life origins. The developmental origins of health and disease (DOHaD) paradigm posits that exposures in utero and during early life influence phenotype and physiology as well as behaviors that shape lifelong health. Epidemiologic studies of DOHaD phenomena have historically focused on single exposure-health outcome associations, despite calls for more holistic approaches to capture the totality of exposures and their causal pathways. Other important limitations of prior work include study samples that diminish generalizability and limit opportunities to identify differences in health outcomes across subgroups and infrequent or lack of granularity in assessment of exposures and health outcomes across ontogeny. Together, these limitations hamper identification of when and how single and combinations of risk factors across early life culminate in chronic disease. This proposal addresses the above gaps by leveraging the Environmental influences on Child Health Outcomes (ECHO) Program to investigate a broad range of early-life exposures, from society to biology, in relation to pediatric health outcomes among ~50,000 caregiver-offspring pairs. The Colorado team will implement ECHO’s Core and Specialized Protocols in the Healthy Start Study, a Colorado pre-birth cohort that recruited and followed 1,250 mother-child dyads. In this cohort, exposures, and outcomes relevant to ECHO were collected during pregnancy, at birth, during infancy and the toddler years, and in early childhood (4-5 years). In the first ECHO Cycle, the Colorado team followed 765 children, to date, through middle childhood (8-12 years) and transferred extant data from pregnancy onward to the ECHO platform. In response to RFA-OD-22-019 and in collaboration with other ECHO components, our team will implement the ECHO Cohort Data and Biospecimen Collection Protocol with high fidelity and use community-engaged retention strategies to follow 750 participants aged 11-19 years to achieve two scientific objectives: (1) characterize patterns of contextual, chemical, and physical exposures during the in utero period and early childhood that predict co-occurring clusters of major ECHO outcomes through adolescence; and (2) examine joint effects of exposure to in utero overnutrition and childhood psychosocial adversity on the Specialized Outcome of pediatric obesity. The work proposed will facilitate a holistic understanding of how the totality of early-life exposures shape chronic disease risk in children; examine the involvement of a potentially modifiable biological mechanism (DNA methylation); and provide insights into multi-level targets for preventive action, including family- and individual-level factors that build resiliency in the face of unfavorable early exposures. The Colorado team brings to ECHO a group of investigators with complementary and synergistic expertise who are uniquely poised to lead innovative team science to steer solution-oriented research.

NIH Research Projects · FY 2025 · 2016-08

ABSTRACT Distinctive 2′-hydroxyl (OH) groups on every ribose make RNA an easy target of some endonucleases, which damage RNA, creating products with 5′-OH and 2′,3′-cyclic phosphate termini. Like DNA repair systems that surveil and repair lesions in the genome to preserve the genetic code, the products of RNA damage are substrates for coupled end modification and processing steps including ligation, stabilization, and degradation. However, unlike the DNA damage response, we are only beginning to understand how RNA cleavage, end modification, and processing are integrated, and how together the “RNA damage response” promotes RNA processing and orchestrates regulatory control. In this MIRA, we continue to explore the role of RNA damage and repair, focusing on 4 specific questions: 1. What are the RNA targets of human kinase- mediated decay?; 2. Do coupled 3′-end modification and 3′⟶5′ RNA decay control RNA fate?; 3. What are the targets and physiological roles of bacterial Rtc RNA repair enzymes? 4. How do 2′-phosphate modifications inhibit exonuclease degradation?

NIH Research Projects · FY 2025 · 2016-08

PROJECT SUMMARY Mucus and macrophages protect the lungs in health, but they can also contribute to disease following lung injury. How their protective vs. pathological functions are calibrated is poorly understood. We seek to determine mechanisms that control their interactions during responses to lung injury. For mucus-mediated defense, the mucin glycoprotein MUC5B is essential. In mice, absence of Muc5b causes particles and bacteria to accumulate in the lungs, ultimately resulting in early mortality. Despite its requirement for health, MUC5B is an important risk factor in human pulmonary fibrosis, where it is misexpressed in bronchiolar club cells and type 2 alveolar epithelia. Overexpression of Muc5b in these cell types in mice potentiates fibrosis following bleomycin-induced lung injury. These data suggest that the levels and locations of MUC5B/Muc5b- expression are significant factors in the pathogenesis of pulmonary fibrosis. Nonetheless, we do not yet fully understand cellular and molecular mechanisms that explain how MUC5B/Muc5b promotes lung fibrosis. Our recent work suggests that defensive and pathologic effects of airway mucus are regulated by interactions between Muc5b and airspace macrophages (AMs). Resident AMs are present constitutively and are required for non-inflammatory defense. In response to injury, AM pools increase through recruitment of blood monocytes that mature into macrophages. These recruited AMs are more inflammatory than resident AMs, but they are also short-lived, resulting in transient expansion and then contraction of the AM pools. Mechanisms of acute and resolving inflammation that distinguish resident and recruited AM types also impinge on fibrotic repair. The presence of both MUC5B/Muc5b and AMs in distal airspaces, along with our prior observation of AM dysfunction in Muc5b knockout mice, implicate a connection between MUC5B/Muc5b and AM functions. We identified a potential mechanism mediated by mucin glycans and AM glycan receptors. MUC5B/Muc5b is heavily coated with sialic acid (SA) that is attached to galactose via an α2,3-linkage. It is also a ligand for sialic acid binding immunoglobulin like lectin-F (Siglec-F), an inhibitory signaling molecule found almost exclusively on AMs in healthy lungs. We found that a Muc5b-SA-Siglec-F axis is critical for resolving inflammation, as shown by prolonged recruited AM accumulation in mice lacking each component. We now also show that bleomycin-induced lung fibrosis is suppressed in Muc5b-SA-Siglec-F axis disrupted mice. Thus, while protective in response to bacterial inflammation, this mechanism appears to be detrimental in a pro-fibrotic injury setting. We hypothesize that fibrotic repair of lung tissues is mediated by a Muc5b-SA-Siglec-F dependent AM programming mechanism. This will be tested in three aims that test whether 1) promotion of lung fibrosis by Muc5b requires α2,3-sialylation; 2) ligation of Siglec-F by sialylated Muc5b mediates fibrosis; and 3) the Muc5b-SA-Siglec-F axis regulates fibrotic programming of resident vs. recruited AMs.

NIH Research Projects · FY 2025 · 2016-08

PROJECT SUMMARY The hypoxia of high-altitude (HA, >2500 m) increases the frequency of fetal growth restriction (FGR) three-fold. The normal pregnancy rise in blood flow to the uteroplacental circulation (termed “uterine” here) is also reduced in FGR at HA or at low altitude, but lower uterine blood flow is not solely responsible for FGR because O2 supply still exceeds fetal O2 consumption, even at HA. Thus, the mechanisms by which lower uterine blood flow reduces fetal growth and their temporal relationship remain unclear. Our prior work implicates AMPK in the regulation of uterine vascular function, blood flow, and fetal growth, and our preliminary data show that FGR vs. appropriate for gestational age (AGA) pregnancies in La Paz, Bolivia (3850 m) have lower third-trimester uterine blood flow; greater placental AMPK activation, suppressed mitochondrial oxidative metabolism, and metabolite profiles supporting impaired fatty acid and amino acid metabolism. We propose human and sheep studies to be conducted under chronic maternal hypoxia in order to determine whether placental AMPK signaling serves as a nexus between uteroplacental perfusion and placental metabolism to regulate fetal growth through its dual role as a potent vasodilator and metabolic sensor. In HA residents with AGA or FGR pregnancies women at unlabored C-section, we will measure blood flows, perform four-vessel sampling on both sides of the placenta, collect placental and human uteroplacental and fetoplacental arteries regulating blood flow for vasoreactivity studies, and conduct biochemical assays. Because vasodilation is impaired in FGR, we will test whether pharmacologic mediated modulators of mitochondrial oxidative metabolism and redox status restore impaired AMPK- vasorelaxation.Since access to human blood vessels and placenta are only available at delivery, we will perform parallel studies in a sheep model of hypoxia-associated FGR in order to measure these same variables but also with metabolic tracers both before and after FGR (i.e. at mid- and late-gestation respectively) in order to identify when uterine O2 supply decreases, and test the temporal relationship between O2 supply, O2 consumption, nutrient uptake, and fetoplacental metabolism relative to the initiation of FGR. As in the human studies, we will also assess the effects of AMPK activation on uterine vasoreactivity and placental nutrient metabolism, and test whether restoring mitochondrial oxidative metabolism improves vasodilation in key uterine resistance vessels. The proposed studies will enable our understanding to move beyond the conventional idea that insufficient fetal oxygenation triggers FGR to one in which we know when and how the hypoxia-associated FGR develops. Such information is essential for refining therapeutic strategies for restoring fetal growth under conditions of hypoxia, a goal that has, to date, proven elusive.

NIH Research Projects · FY 2025 · 2016-07

ABSTRACT Despite major breakthroughs over the past few years in our basic understanding of the cellular and molecular changes that lead to cancer, many key steps in carcinogenesis and changes in early cancers that promote invasion and metastasis, still remain poorly defined. Rigorous training of future young investigators in cancer biology will be essential in our quest for a deeper understanding of carcinogenesis and for the development of better methods of early cancer detection, improved diagnosis, and effective new cancer treatments. The Training Program in Cancer Biology (TPCB) is a multidisciplinary program across all cancer subtypes that capitalizes on the unique strengths and training opportunities at CU Anschutz, including world renowned research in functional genomics, experimental therapeutics, steroid receptor signaling, stem cells and organ specific cancers (in particular lung, breast and blood). Our goal is to provide interdisciplinary training at the cutting edge of cancer research to best prepare our trainees to compete in a biomedical research environment increasingly focused on translational applications of basic research. Our training plan consists of laboratory training, didactic activities, attendance at scientific conferences, professional development and career mentoring, development of communication skills, and exposure to the clinical perspective. Based on feedback from trainees and faculty, for this renewal we have added new training components, including mandatory mentor training and training in rigor and reproducibility. The renewal also includes expanded opportunities for training in quantitative biology and bioinformatics, and expanded clinical/translational and career development opportunities. The co-PIs of this training program have been leaders in Cancer Biology training at the University of Colorado Cancer Center and the Graduate School during the past 10 years. Resources from the Cancer Center, the Graduate School, and individual departments are committed to support the training program. The infrastructure of the Cancer Center is particularly important as it provides core resources available to training faculty that are therefore available to the trainees. The proposed program will include pre- doctoral training through the graduate training program in Cancer Biology, which is housed in the Graduate School, and post-doctoral training for PhD and PhD/MD scientists. A total of 38 training faculty were selected based on their scientific expertise and track record of mentorship. The first cycle of this T32 grant was highly successful, with all slots competitively filled. For the second cycle of this grant, we propose to support 4 postdoc and 4 predoctoral trainees each year. We will select trainees from external and internal pools based on their research and academic records and commitment to cancer research. We are committed to providing comprehensive training at all levels so as to best prepare our trainees for successful careers in biomedical science inside or outside academia.

NIH Research Projects · FY 2025 · 2016-06

Cardiovascular diseases such as vascular calcification are a leading cause of death in patients with chronic kidney disease (CKD). However, there is no effective therapy for vascular calcification available. In addition to uremic toxins such as indoles and phosphorus, inflammatory cytokines such as TNF play a major causative role in the regulation of CKD-dependent vascular calcification. Our long-term goal is to identify new pharmacological strategies for the prevention of vascular calcification. Our studies have demonstrated that simultaneous activation of the endoplasmic reticulum (ER) stress and IKK-NFB-inflammation pathways in vascular smooth muscles cells (VSMCs) are major events in the induction of vascular calcification in CKD. We have also revealed that ER stress-mediated integrated stress signal (ISR) in VSMCs plays a causative role in the pathogenesis of vascular calcification. Unexpectedly, however, the inhibition of IKK-mediated inflammation drastically exacerbated vascular calcification in CKD mice. In addition, both ER stress-ISR (ATF4-CHOP) activation- and IKK inhibition-mediated vascular calcification are highly associated with vascular cell death. There results led us to hypothesize that one of the regulated cell death (RCD) pathways is a major player in the initiation of vascular calcification. To find clues about the mechanism, we recently screened a library of chemicals that inhibit RCD. Based on the RCD chemical library screening, we identified an RCD pathway that selectively contributes to IKK inhibition-induced and CHOP-induced vascular calcification. We therefore propose two specific aims to elucidate. Aim 1 will examine whether the RCD pathway affects vascular calcification by altering the secretion of calcifying macrovesicles in cultured cells. Aim 2 will examine whether modulation of the RCD pathway affects CKD-dependent vascular calcification in vivo. Completion of this project will provide novel therapeutic targets for CKD-mediated vascular calcification.

NIH Research Projects · FY 2025 · 2016-06

Project Summary The central nervous system (CNS) is protected by two barrier systems, the blood brain-barrier (BBB) and the blood-cerebrospinal fluid barrier (B-CSFB). These barrier systems have unique cellular properties that regulate the molecules and cells that can enter or exit the CNS and the CSF. CNS barriers are essential for development and health but breakdown in a variety of diseases, causing or exacerbating CNS pathology. A detailed under- standing of CNS barriers is also essential for efficient drug delivery to the brain and spinal cord. The development and function of the B-CSFB at the level of the meninges, a trilayered structure that surrounds the CNS, is poorly understood. This is despite evidence implicating meninges-located barriers in perinatal and adult diseases as an early site of immune cell activation and entry in neuroinflammation. One of two barrier structures in the me- ninges is the arachnoid barrier layer, which segregates the outer meningeal dura and its non-barrier vasculature, from the CSF and cell types in the subarachnoid space. Unlike the BBB and other parts of the B-CSFB, nothing is known about mechanisms of arachnoid barrier cell specification, timing of layer maturation or acquisition of functional properties. Further, only a few studies have looked at arachnoid barrier dysfunction in CNS diseases and so far, no studies have tested if an immature arachnoid barrier has enhanced vulnerability to breakdown. We have combined our knowledge of CNS vascular and BBB development with our unique expertise in the meninges to develop new tools to study the arachnoid barrier. Experiments proposed here build upon our initial discoveries to identify mechanisms that underlie arachnoid barrier layer development, investigate arachnoid bar- rier maturation and function, and measure its response in insult. To do this we will: 1) utilize in vivo and culture models to uncover the molecular mechanisms of arachnoid barrier cell specification, 2) use our new model where we perturb arachnoid barrier formation prenatally to determine its role in establishing separate meninges immune cell and vascular compartments and in protecting the fetal brain in an animal model of maternal infection 3) identify the cellular and molecular mechanisms of arachnoid barrier breakdown in bacterial meningitis. Comple- tion of this work will substantially advance the field of CNS barrier systems. It will provide the first model of arachnoid barrier development including the cellular and molecular mechanisms and the timing of emergence of barrier properties. It will provide important information about the function of the arachnoid barrier. Experiments proposed here focus on the prenatal brain however findings will set the stage for future studies in postnatal and adult function. Third, it will provide the most detailed analysis to date of arachnoid barrier response to CNS insult, paving the way for future studies in other CNS diseases. In the long term, this new knowledge has the potential to be used to design new ways to limit crossing of molecules and cells at the arachnoid barrier to treat disease or increase crossing of drug therapeutics to access the CNS.

NIH Research Projects · FY 2024 · 2016-06

Project Abstract Intravenously injected nanoparticulate drugs are cleared by phagocytes and elicit immune reactions, including cytokine release and pseudoallergy (up to 10% in Doxil® and 30% in Onpattro®). There is substantial preclinical evidence on the involvement of the serum complement system in these responses. Understanding the mechanisms of complement activation in patients and designing preventive strategies can improve the safety and efficiency of nanoparticle-based drugs. We found that cell membrane-derived inhibitors of complement can effectively shut off complement activation and prevent the uptake of nanoparticles by leukocytes in the blood of healthy donors. Furthermore, the inhibitors that are designed to target complement deposits on the nanoparticle surface showed picomolar activity, for all nanoformulations we tested. The available data suggest a novel hypothesis wherein less than 1% of nanoparticles act as initiators of the complement cascade. Our long-term goal is to conduct a clinical trial of combination of nanomedicines with complement inhibitors. Such a trial will provide the nanomedicine field with the ultimate answers on the role of complement in hemocompatibility, clearance, and infusion reactions in humans. The main objectives of this proposal are 1) to further understand mechanisms of complement initiation by nanoparticles; 2) to design a rapid companion test to measure complement activation by nanoparticle-based drugs; 3) to study the efficacy of nanoparticle-binding inhibitors in blood of defined cohorts of patients in vitro and in dogs. This research will shift the existing paradigm of stealth design: it will enable the control and fine- tuning of the biocompatibility of nano-sized drug delivery and imaging systems using specific inhibitors.

NIH Research Projects · FY 2025 · 2016-02

Project summary: We and others have demonstrated that antigens derived from infectious viral infections persist in the host for extended periods of time, well beyond the time in which the infection is cleared from the host. Our lab has specifically identified that antigens derived from both vaccination and viral infections persist or are archived by the host lymphatic endothelial cells (LEC)s, identifying the source of archived antigens. We have published that this archived antigen maintains a more effector like pool of antigen specific memory cells which enhances the clearance of a secondary infectious challenge. Identification of key mechanisms involved in antigen archiving during vaccination is critical for our understanding of enhanced protective immunity to vaccination. While we have established many important criteria for antigen archiving and protective immunity, in this renewal application we aim to dive deeper into the cell types involved and the processes required. We aim to better appreciate how the expression of subset specific genes, now discovered in both lymphatic endothelial cells and dendritic cells, may be required for antigen handling, the implications of which could affect antigen archiving and protective immunity. We have established a novel methodology leveraging the 10x genomics platform to identify DNA-antigen conjugates for the study of antigen dispersal over long periods of time. With this methodology and technology in hand we now have the capability to accurately and faithfully measure cell types that acquire antigens as well as the exact number of antigens within each cell over time. Using these studies we have identified several novel findings based on antigen amount and transcriptional signature to lead us to the hypothesis that specific LECs and DCs, based on their transcriptional program, contribute to the acquisition, retention and exchange of antigens. Furthermore, this handling of antigens by LECs and DCs can be manipulated by other inflammatory events that cause antigen release and presentation, and as a result, improve immune responses to secondary and heterologous infections.

NIH Research Projects · FY 2026 · 2015-04

Abstract This project proposes to continue our highly successful research and mentorship program for undergraduate students to enhance diversity in the environmental health sciences (EHS) based at the University of Colorado Anschutz Medical Campus. Our program, the Colorado Undergraduate Research in Environmental Health Sciences (CUrehs), will draw students from the University of Colorado downtown Denver campus which has a large underrepresented population of students. Our program intends to expose students to research experiences and mentorship in EHS for one year and will include an intensive summer program that trains them in the responsible conduct of research, and provides them with the confidence and guidance to pursue graduate education. Overall, our CUrehs program provides an integrated 3-pronged approach to developing student interest in environmental health sciences research and careers: (1) Laboratory Experience; (2) Social/Community Development; (3) Educational/Field Trip Experiences. We have trained a total of 43 students during the past funding cycle with success as measured by students applying to professional or graduate school, publications and presentations at local and national conferences. Our goal remains to enhance diversity in the environmental health sciences by meeting the following aims: 1. Advance our successful research-training program for undergraduate students in the environmental health sciences. 2. Strengthen the undergraduate studies in EHS at the University of Colorado while improving the participants' preparedness for graduate or professional school. 3. Enhance the diversity of students progressing to graduate or professional school in environmental health sciences.

NIH Research Projects · FY 2026 · 2015-04

PROJECT SUMMARY Medulloblastoma (MB) is the most prevalent malignant brain tumor in children and demonstrates high level of heterogeneity. Treatment for MB includes chemotherapy and radiation often resulting in long-term morbidity. MYC driven MB in particular are high risk tumors with poor long-term survival. We previously identified WEE1 as a target in MYC driven MB. WEE1 regulates MYC driven replication stress and high throughput chemical screening identified high degree of synergy with gemcitabine. Further data suggest that MB tumors become resistant to WEE 1 inhibition and that this mechanism is mediated by CDK7.. We hypothesize CDK7 re-sensitizes MB cells to WEE1 inhibition by reprograming the enhancer landscape to alter metabolic pathways and suppressing homologous recombination DNA repair networks. To CDK7 that inhibition of inhibition MB and homologous targeting approaches for validate inhibition in combination with WEE1 as a therapeutic strategy we will pursue three key questions. 1. How dose CDK7 alter enhancer landscape to modulate sensitivity to WEE1 inhibition? 2. Is combination of WEE1 CDK7 inhibition therapeutically effective in MB in vivo? 3. Can addition of agents that target recombination mediated DNA repair potentiate CDK7/WEE1 mediated therapeutic of MB? The results of this work are expected to yield important insights into that could be used to inhibit MYC function in high-risk MB, and provide a rationale the treatment of MYC driven MB, which represents the long-term goal of our research.

NIH Research Projects · FY 2025 · 2015-04

PROJECT ABSTRACT The Inflammatory Bowel Diseases (IBD), including Crohn’s disease and ulcerative colitis, remain among the most debilitating inflammatory disorders of the western world. It is estimated that more than 3 million Americans suffer with IBD, with incidence rates on the rise in many populations. The precise etiology of IBD is not known but emerging evidence implicates shifts in the constellation of microbes in the intestine (dysbiosis) as a significant factor. Our interest in this proposal is aimed at capitalizing on the importance of microbiota- derived metabolites in promoting intestinal homeostasis. In particular, we aim to better understand how microbial- derived factors, such as short chain fatty acids (SCFA, esp. butyrate), contribute to mucosal barrier function and wound healing. Our work in progress has focused on developing and testing a panel of butyrate mimetics that may best serve mucosal barrier function and wound healing in models of IBD. Additional ongoing work using unbiased single cell RNA sequencing (scRNAseq), we identified a cohort of butyrate- induced genes with potential importance in barrier function. In this proposal, we will elucidate whether butyrate mimicry can elicit selectively integrated epithelial functional responses that promote barrier function and coordinate wound healing. Three synergistic specific aims are proposed to address this goal. In Aim 1, we will expand on molecular mimicry of butyrate analogs to optimize epithelial function. Aim 2 will elucidate the mechanisms of selective epithelial control butyrate analogs. Specific Aim 3 define disease-relevant influences of butyrate mimicry on signaling and gene expression in acute and chronic mucosal inflammation models in vivo. Results from these experiments will provide new insights into innate regulation of mucosal barrier and an expanded physiological role for SCFA produced by commensal bacteria. It is our hope that extensions of this work will lead to the discovery of new therapeutic templates for mucosal inflammatory disease.

NIH Research Projects · FY 2026 · 2015-04

PROJECT ABSTRACT Our goal is to improve skeletal muscle growth and body composition in the fetus, neonate, and adult affected by intrauterine growth restriction (IUGR). Fetal skeletal muscle growth is profoundly limited as a result of placental insufficiency and leads to lifelong reductions in muscle mass (sarcopenia) and metabolic disease risk, making restoration of muscle mass during the perinatal period a high priority. During the previous project period, we found multiple defects in fetal skeletal muscle growth in a highly relevant sheep model of IUGR, including lower muscle protein synthesis (MPS) rates, lower muscle mass relative to brain and whole-body weights, smaller myofibers with lower myonuclear number, and fewer total myofibers compared to normally-growing controls. Based on our preliminary data, we have identified adaptations within branched-chain amino acid (BCAA) catabolism that result in lower MPS in the IUGR fetus. Weight-specific BCAA uptake rates by the IUGR hindlimb are reduced despite normal circulating BCAA concentrations, the likely result of lower Na+K+-ATPase activity to drive BCAA into the myocyte. Branched-chain aminotransferase (BCAT) protein expression is higher, which is the first step in BCAA catabolism and results in de novo alanine and glutamine synthesis to support gluconeogenesis, anapleurosis, and energy production. Despite our recent progress, however, critical knowledge gaps remain regarding the mechanisms by which placental insufficiency programs the IUGR myocyte to reduce BCAA uptake and increase BCAA catabolism. Furthermore, it is not known when during an IUGR gestation these mechanisms become intrinsic to the myocyte or how they may be prevented and/or reversed to improve muscle growth. We have assembled a research team of highly qualified experts in metabolism and state-of-the-art metabolomic and proteomic techniques to fill these knowledge gaps. We will test the hypothesis that lower Na+K+-ATPase and higher BCAT activity are intrinsic to the fetal myocyte to increase BCAA catabolism and limit MPS by the end of an IUGR gestation, but that a targeted therapy (L-alanyl-L-glutamine, AG) delivered during a critical developmental window will ameliorate these defects to increase MPS. In Aim 1, we will determine the cellular mechanisms that reduce MPS and test the extent to which these adaptations are intrinsic to the myocyte and/or induced by extrinsic circulating factors the IUGR fetus. In Aim 2, we will address when MPS becomes limited during the course of an IUGR pregnancy. This information is critical to inform the timing of therapies during pregnancy aimed to increase fetal growth. In Aim 3, we will test the capacity of an AG infusion into the IUGR fetus to activate Na+K+-ATPase, stimulate BCAA uptake, and reverse BCAA catabolism to increase intramuscular BCAA for MPS. In summary, this proposal will address gaps in knowledge about how BCAA utilization is regulated in the fetus exposed to placental insufficiency when myofibers are forming, myonuclei are accreting, and hypertrophy rates are some of the highest during the lifespan. These studies are a necessary prerequisite for designing novel approaches to restore muscle growth.

NIH Research Projects · FY 2024 · 2015-03

PROJECT SUMMARY Changes in placental structure and function not only cause serious pregnancy complications but also determine life-long health by programming the fetus for future metabolic and cardiovascular disease. Unfortunately, the mechanisms linking altered placental function to poor short- and long-term outcomes are complex and remain largely unknown. To better understand placental biology and pathophysiology, a multidisciplinary approach utilizing a wide array of cutting edge technologies is required. Progress in this area is hampered by the paucity of scientific meetings focused on placental biology. The primary objective of this R13 grant proposal is to meet the urgent need of a multidisciplinary, interactive forum for dissemination of novel concepts and exchange of ideas in placental research by featuring exceptional speakers working in cutting-edge areas of research. The secondary objective is to provide a low cost, high-quality learning environment, in the area of placental biology, which will encourage attendance and active participation by a diverse group of in training and early investigators. In our conference plan we seek support for an annual one- day conference as a satellite meeting the day before the Annual Scientific Meeting of the Society of Reproductive Investigation (SRI). By this design we will maximize the impact of the meeting and will allow for attendance of a diverse group of basic scientists and clinical investigators ranging from graduate students to well established researchers. The meeting will allow ample time for interaction, informal discussion and networking and is anticipated to attract 130-150 attendees each year. The program will have a balanced mix of “state of the art” lectures presented by the leaders in the field, emerging concept presentations, and shorter talks by trainees. All speakers will be mandated to provide ample time for questions and discussion. This proposal is significant because the meeting is timely and will promote novel scientific inquiry into placental biology, which is expected to pave the way for future innovation to develop approaches to monitor placental function in vivo and to target the placenta for intervention. The proposal includes numerous innovative aspects. For example, we propose to facilitate the utilization and adaptation of emerging concepts from other research fields by inviting one speaker each year that works in a research area other than placental biology. To provide ample opportunities for early career investigators to connect, discuss, and interact with the speakers we propose to organize the conference lunch so that in training investigators will have direct access to speakers in a small group. The proposed meeting is expected to have significant and sustained impact on the field because it is unique in bringing together world-leading investigators representing diverse but complementary expertise in a creative and interactive forum, which is necessary to address the complex scientific questions pertaining to the role of placenta in determining health and disease from fetal life to adulthood.

NIH Research Projects · FY 2025 · 2014-09

The goal of this competitive T32 renewal resubmission application is to continue a research training program that is preparing postdoctoral physician and PhD fellows for careers as scientists addressing priority research topics at the interface of geriatrics and palliative care. Funding is requested for 4 post-doctoral trainees per year, the same number of trainees as in year 5 of the current T32 award. This T32 research training program supports postdoctoral training for both physician-scientists and Ph.D.-scientists. The primary impetus for the current T32 training program, and this requested competitive renewal, is the urgent need for a pipeline of well-trained scientists positioned to advance the field of palliative care, ultimately improving care for older adults with serious illness. This T32-supported research training program addresses a scientific knowledge and research workforce gap in aging and palliative care, namely, the paucity of robust research evidence and a pipeline of investigators. The goals of our T32 program include: 1) recruiting and retaining outstanding post-doctoral trainees; 2) supporting their research training through experiential and didactic instruction, leveraging the program faculty’s expertise in intervention development and testing in pragmatic clinical trials; 3) ensuring the highest level of academic success through conscientious monitoring of thoughtfully constructed individual research career development plans; and 4) enhancing the mentoring skills of early stage mentors through co-mentoring with more experienced senior mentors. These goals remain at the heart of this program and are reflected in the organization and focus of this training grant. To continue to achieve these goals, in response to trainee and external advisory board feedback, we are implementing the following innovations to our curriculum: 1) hybrid delivery with 3 times a year in-person intensives and 2) longitudinal scientific development series. The proposed competitive renewal builds on the demonstrated successes over the first two funding periods (established 2014, renewal 2019). Success in the 2 initial funding periods of this T32 is demonstrated by ability to: 1) fill positions with 15 outstanding multi-disciplinary trainees and foster an ongoing pipeline of trainee candidates; 2) produce academically successful and productive trainees who are publishing peer-reviewed manuscripts, presenting in national venues, obtaining intra- and extra-mural grant funding and attaining research-focused faculty positions; 3) develop the cohort of research mentors by engaging more early stage faculty as co-mentors; and 4) enhance geriatric palliative care research capacity through deliberate and strategic interactions amongst programs and faculty, successfully expanding the cohort of engaged multi-disciplinary experienced mentors. This T32 has, and with ongoing funding will continue to have, immediate local, regional, and national influence by developing a cadre of skilled, productive scientists.

NIH Research Projects · FY 2026 · 2014-04

PROJECT SUMMARY: Age-related macular degeneration and several other diseases can cause rod and cone photoreceptors to die. This results in permanent visual impairment because the human retina does not regenerate itself. One potential way to restore vision is by replacing the lost photoreceptors. Audacious strategies to restore vision include reprogramming the diseased retina to regenerate its own photoreceptors and directly replacing the lost photoreceptors from stem cell sources. Implementing these strategies requires specific and efficient methods of programming cells to become rods and cones. However, we face a major barrier to achieving these therapeutic strategies due to our limited understanding of the mechanisms that govern how photoreceptors normally develop. Most of the undifferentiated progenitor cells of the retina will activate the transcription factor Otx2 and acquire the potential (or competence) to become photoreceptors and bipolar cell interneurons. These competent Otx2+ cells then choose between rod, cone, and bipolar cell types. This fate decision is controlled in part by the transcription factors Prdm1 and Vsx2. Loss of Prdm1 profoundly increases bipolar cells at the expense of photoreceptors. Conversely, Vsx2 mutants lack bipolar cells. Neither photoreceptors nor bipolar cells are generated when Otx2 is mutated. Therefore, the gene regulatory networks responsible for Otx2, Prdm1, and Vsx2 expression determine the cell type composition of the retina and its ability to function normally. Our goal is to decipher how this gene regulatory network functions to control cell fate decisions during retinal development. Our first objective is to determine how Otx2 expression, which is necessary for photoreceptor and bipolar cell competence, is regulated during mouse retinal development. We have identified three non-coding DNA regulatory elements (enhancers) that are essential for Otx2 expression at different times in development. Perturbing enhancer function also suggests that they dynamically interact in a tight three-dimensional structure that allows them to substitute for one another. We will test how these enhancers cooperate and substitute for each other in normal and perturbed conditions using high-resolution chromosome conformation capture and other genetic approaches. Our second objective is to understand how Otx2+ cells choose between photoreceptor and bipolar cell types. We found that deleting the bipolar-specific enhancer of Vsx2 prevented bipolar cell formation. Our results from mutating this enhancer along with Prdm1 suggested that transient Vsx2 expression permanently drives bipolar formation. Using a combination of mouse mutants, mutagenesis, misexpression, and CRISPR inhibition tools, we will determine how Vsx2 and the Vsx2 bipolar enhancer control bipolar fate choice. Our experiments will unravel the gene regulatory network that controls photoreceptor and bipolar cell competence and fate choice. Gaining this knowledge is important because it will help us create regenerative strategies to replace lost photoreceptors and restore vision in millions of people suffering from blinding diseases.

NIH Research Projects · FY 2025 · 2013-08

Project Summary Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the destruction of insulin-producing beta cells in the pancreas. CD8 T cells play a crucial role in this process, but the specific antigens they target and the molecular mechanisms driving their pathogenicity remain poorly understood. This knowledge gap hinders the development of targeted therapies to prevent or reverse T1D progression. Our proposed research aims to address this critical need by comprehensively investigating CD8 T cell antigen specificity and molecular signatures across different stages of T1D development. This project will employ innovative high-throughput screening methods, single-cell RNA sequencing, and advanced computational analyses to achieve three main objectives: (1) Identify novel epitopes recognized by islet-infiltrating CD8 T cells from T1D organ donors, focusing on both native proteins and potential neoepitopes, such as hybrid insulin peptides and RNA splice variants, preferentially expressed by ‘stressed’ islets. (2) Determine the molecular phenotypes specific to islet-reactive CD8 T cells within pancreatic lymph nodes of individuals at various stages of T1D development, comparing them to virus-specific T cells to elucidate unique features associated with autoimmunity. (3) Characterize the molecular signatures of preproinsulin-specific CD8 T cells in peripheral blood across different stages of T1D development, from prediabetic (islet autoantibody-positive) individuals to those with new-onset T1D. By integrating data from these complementary approaches, we expect to gain unprecedented insights into the antigen specificity and functional states of autoreactive CD8 T cells throughout T1D progression. This research has the potential to revolutionize our understanding of T1D pathogenesis and enable the development of novel biomarkers for early detection and monitoring of disease progression. Moreover, the identification of key molecular pathways involved in T cell activation and beta cell destruction may guide the design of more effective, targeted immunotherapies for T1D prevention and treatment. Ultimately, this project aims to improve the lives of individuals at risk for or living with T1D by identifying novel T cell epitopes and molecular signatures specific to disease progression. These findings will pave the way for developing more accurate predictive biomarkers and antigen-specific immunotherapies, bringing us closer to the goals of early intervention, disease prevention, and potential reversal of beta cell destruction in T1D.

NIH Research Projects · FY 2025 · 2013-07

Despite recent progress, accelerated implementation of highly effective and sustainable public health policies will be required to meet the Sustainable Development Goals’ (SDG) targets for maternal and child health by 2030. The broad goal of this application is to continue to participate in all collaborative activities relevant to the mission of the Global Network (GN), which aims to develop and test potential sustainable interventions through collaborative, large-scale, high impact multi-site common research protocols. The primary objective of our research unit (RU), which represents a partnership between the University of Colorado and Guatemala, is to develop and test interventions to mitigate the priority threats to maternal-child health posed by malnutrition and infection. The specific research expertise of our team is nutrition, infectious diseases, and biological effects of external stressors. This objective will be met through the following specific aims: 1) continue and enhance a strong research record and productivity as GN members; 2) continue to develop common GN protocols through high-impact clinical trials and observational studies that are transformative for maternal-child health; and 3) continue to foster a progressively stronger research partnership with our colleagues and collaborators in Guatemala and other GN sites. During the past 5-yr cycle, this RU has built a strong research record, evidenced by more than 60 GN publications (approximately 40% led by our team); by completion of longitudinal growth and neurodevelopment follow-up through 2 years in the four sites of the preconception maternal nutrition intervention trial; actively consulting on child neurodevelopmental follow-up for two other major GN trials; strengthened research capacity in Guatemala; successful implementation of all GN-initiated common protocols, including COVID-19 surveillance, with contribution of a high percentage of participant numbers and retention to each study; and securing external funding to support common protocol implementation. Major contributions to the GN by this RU include service on 2 subcommittees and co-chairing the oversight committee for the ongoing Maternal Newborn Health Registry. The positive impact of our continued participation in the GN will be accomplished by leveraging our combined expertise in nutrition, infectious diseases, and other external stressors to design interventions to improve maternal, infant, and child health and resilience.