University Of Colorado Denver

NIH Research Projects · FY 2024 · 2020-07

DESCRIPTION (provided by applicant): The mountain and plains states of Colorado, New Mexico, Arizona, Wyoming, Montana, North Dakota and South Dakota share a common set of problems that outstrip existing resources: rising population; large groups of underserved and minority workers; region-specific work-related health issues such as mining, energy, and agriculture; and geographic distance from educational centers of excellence in occupational health and safety. The Mountain and Plains Education and Research Center (MAP ERG) was founded in 2007 to incorporate faculty and students from two institutions of higher learning into an integrated, multidisciplinary Center, to improve worker health. The objectives of the MAP ERC are to promote interdisciplinary graduate education in occupational health and safety; to provide needs-based continuing education and outreach in an underserved region; to support pilot research projects that advance the National Occupational Research Agenda; and to improve minority recruitment and retention in the allied fields of occupational health and safety. The MAP ERC incorporates five training programs of the University of Colorado Denver and Colorado State University. Core programs include Industrial Hygiene and Occupational and Environmental Medicine Residency. Three allied programs offer graduate training in Health Physics, Occupational Ergonomics, and Occupational Health Psychology. All provide either graduate or post-doctoral/residency level training. All programs are committed to providing a highly interdisciplinary educational experience through shared courses, field experiences, research collaboration, and conferences. The Pilot Projects Program rigorously reviews and supports R2P community and academic projects that address regional priorities, serving as a stimulus for attracting junior investigators and advancing their careers in the field. Continuing Education places particular emphasis on state-of-the-art online courses for a geographically dispersed occupational safety and health workforce. The Outreach Program is integrated into all programs, bringing together many regional professional organizations and creating collaborative opportunities for addressing workplace challenges. Working in concert with university and community organizations, the MAP ERC will continue to address the need for greater diversity and inclusion of minorities in occupational health and safety professions. The MAP ERC enjoys partnerships with the High Plains Intermountain Center for Agricultural Health and Safety and with the Affiliate Total Worker Health Center for Worker Health and Environment, and with neighboring ERCs and other Training Programs.

NIH Research Projects · FY 2025 · 2020-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The goal of the Molecular Biology Program at the University of Colorado Anschutz Medical Campus is to train outstanding research scientists who will become future leaders in their chosen fields and disciplines. To accomplish this goal, we carefully select high-quality students with commitment, and passion for research. Our training program is flexible and student-oriented to meet individual needs, with a rigorous curriculum and high standards. Our Ph.D. in Molecular Biology is designed as a 5-6 year program with the first year devoted to challenging course work and laboratory rotations; the second to development of a research thesis; and the remaining to completion of the thesis research under the guidance of a training faculty mentor and thesis committee. To further develop their skills, students at all stages of the Program participate in weekly seminar series, an annual Symposium, and our Program retreat. Our curriculum emphasizes the development of critical and creative independent thinking, strong quantitative and statistical analytical skills with a focus on rigor and reproducibility and strong scientific communication skills. Our program’s close ties with the RNA Bioscience Initiative provide students with increased training in RNA biology, high-throughput DNA sequencing, and domain-specific bioinformatics training. A strength of the program is its outstanding and collegial faculty from 12 different departments and divisions who are deeply committed to graduate education and the MOLB Program. As the only NIH-supported training program on our campus focused on research into fundamental molecular and cellular mechanisms, faculty laboratories offer research opportunities for our students in multiple disciplines (biochemistry, genetics, immunology, cell, and structural biology), disease models (cancer, autoimmunity, infectious disease, and developmental disorders), and organismal models (viruses, bacteria, yeast, ciliates, flies, worms, and mice). The success of our training program is evinced by student publications and high-quality postdoctoral, faculty, and industry positions obtained by our students. Given the depth and quality of our student pool and our demonstrated ability to train high quality students, we request 10 students be supported each year.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Brain tumors have become the leading cause of cancer-related death in children. Ependymoma (EPN) accounts for a substantial number of these deaths. Over 70% of children presenting with ependymoma will relapse and almost all children who relapse will eventually die. Unlike medulloblastoma, no effective chemotherapy has been developed in ependymoma to complement the standard, and eventually for most ineffectual, surgery and radiation. In particular, children with the most common type of ependymoma, posterior fossa Group A (PFA1), relapse more frequently and experience more invasive, metastatic disease at relapse. Thus, there is a critical need for more effective therapies to combat high-risk PFA1 tumors. Single-cell RNAsequencing data suggest that the epigenetic silencing of LDOC1 within a specific subpopulation of tumor cells has a profound, direct impact on the tumor biology of PFA1 tumors. The driving hypothesis is that epigenetic silencing of LDOC1 with in the MEC subpopulation, as a result of chromatin remodeling, is the molecular driver in PFA1 EPN, through upregulation of non-canoncial NF-B activation. To address this hypothesis, the studies in aim one will determine the role of LDOC1 expression in EPN by examining 1) the mechanism of gene silencing, 2) the functional role of loss of LDOC1 in vitro and in vivo, and 3) the genomic transcriptional targets of LDOC1. Aim two is designed to 1) determine how LDOC1 regulates non-canoncial NF-B signaling and 2) identify the functional consequences of the NF-B signaling pathway. The collective proposed studies will define the effect of LDOC1 loss, which we hypothesize to be the molecular driver of tumor biology of PFA1 EPN. These studies will significantly add to our understanding of childhood EPN and have the potential to identify rational therapeutic targets for children with this high-risk, poor-outcome pediatric brain tumor.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Taste receptor cells (TRCs) are continually replaced from adult stem/progenitor cells, and the fidelity of this process underlies the relative constancy of our sense of taste. However, a host of cancer therapies perturb taste and we posit this is due to perturbation of taste cell renewal. The Wnt/ß-catenin and Hedgehog pathways are implicated in scores of cancers, and many drugs have been and continue to be developed to target these pathways in tumors; these drugs invariably cause taste dysfunction for patients. Subsets of taste stem cells express the Wnt target gene Lgr5 and the Hedgehog target gene Gli1, and both Wnt and Hedgehog pathways have been shown to regulate taste cell renewal in vivo. Thus, in the long term, understanding the functional relationship of Wnt- and Hedgehog-sensitive stem cells in taste homeostasis, as proposed here, will shed light on how these progenitors are disrupted by chemotherapeutics that cause taste dysfunction, and allow development of strategies to mitigate dysgeusia. In our application, we propose to test explicit hypotheses of the functional relationship of LGR5+ and GLI1+ stem cells in the circumvallate taste papillae of mice. Hypothesis 1: Progenitors expressing high levels of LGR5 are slow cycling, multipotent stem cells that produce rapidly proliferating GLI1+/LGR5low/neg progenitors that give rise directly to TRCs. Hypothesis 2: Upon LGR5+ cell ablation, GLI1+ progenitors expand their potential to reconstitute circumvallate epithelium and give rise to new LGR5+ stem cells. To test these ideas, we combine in vivo molecular genetics, in vitro production of lingual organoids, and single cell transcriptome profiling – all approaches with which we have become skilled. In Aim 1, we test the competency of LGR5 vs GLI1 progenitors to produce taste cell-replete organoids, and further assess the degree to which lineage production by each progenitor type is dependent upon Wnt signaling. In Aim 2, we explore the capacity of GLI1+ progenitors to regenerate both taste cells and LGR5+ stem cells following genetic ablation of LGR5+ cells. In Aim 3, we combine temporally fine-grained lineage tracing with single cell RNA sequencing to transcriptomically define the cell lineages that continually produce each of the functional taste cell types, i.e., glial-like cells and sweet, bitter, umami, salt and sour TRCs. In sum, our proposed studies will lead to significant advances in our understanding of the cellular and molecular mechanisms that maintain our sense of taste.

NIH Research Projects · FY 2025 · 2020-07

Project Summary Human ASH1L (absent, small, or homeotic discs like 1) mediates proliferation and survival of hematopoietic stem cells and is often upregulated in leukemias. It is required for hematopoietic development and expression of developmental genes, including the HOX gene family. Upregulated activity of ASH1L, found in mixed lineage leukemia (MLL)-rearranged acute lymphoblastic leukemia (ALL), is generally associated with a poor prognosis. ASH1L is a major methyltransferase that methylates histone H3, generating the epigenetic mark H3K36me2 associated with transcriptional activation and elongation. ASH1L contains a unique combination of the catalytic methyltransferase SET domain and adjacent bromodomain (BD), a PHD finger, and a BAH domain with unclear biological roles. Our recent studies reveal that the BD, PHD and BAH domains of ASH1L are epigenetic readers capable of recognizing distinctive states of histone H3. The molecular mechanisms underlying these novel functions of ASH1L are unknown and will be elucidated in the proposed studies. We hypothesize that the concomitant recognition of distinct histone states by the PHD, BD and BAH domains recruits or stabilizes ASH1L at promoters of ASH1L target genes and is necessary for the catalytic activity of ASH1L and methylation of H3K36 at these genes. We seek to understand a crosstalk between the BD, PHD and BAH domains of ASH1L and determine the molecular mechanism and functional significance of the multivalent engagement of ASH1L with chromatin. We will employ complementary in vitro and in vivo approaches to establish the molecular and structural basis and define the biological importance of histone binding by ASH1L readers. This research will provide atomic-resolution insights into ASH1L signaling pathways that may constitute new targets for therapeutic interventions and enhance our knowledge of fundamental principles underlying the epigenetic-driven gene transcription. It will also lead to a better understanding of human cancers associated with aberrant activity of ASH1L, including acute leukemias.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract This proposal for a five-year mentored research career development project focuses on elucidating the role of the type 1 diabetes (T1D) PTPN2 risk allele in loss of B cell anergy. Previously B cells bearing antigen receptors with high affinity for insulin were found only in the anergic B cell compartment of healthy individuals. Importantly, these cells leave this compartment in a proportion of first-degree relatives (FDRs), and in all autoantibody positive pre-diabetics and recent onset diabetics. Departure of these autoreactive anergic B cells in FDRs was shown to be associated with high risk T1D HLA alleles, and three high risk non-HLA alleles, including INS (rs689), PTPN2 (rs1893217), and IKZF3 (rs2872507). Of the three non-HLA risk alleles, only PTPN2 has been previously shown to be a negative regulator of signaling. However these previous studies were completed in mice, not humans, and their exact mechanism by which they contribute to development and signaling has yet to be determined. This application proposes to determine the effect of the T1D risk variant of PTPN2 in maintenance of B cell anergy. Aim 1 will explore the effect of the risk variant on the phenotype of the B cell compartment in FDRs of T1D patients and their response to stimulation. Aim 2 will explore the relationship of loss of anergic B cells with the high risk T1D genotype allele, Ptpn2, using a reductionist mouse model. The potential impact of these studies will lie in understanding how risk alleles conspire to undermine maintenance of immune tolerance to autoantigens in T1D. The candidate is an Instructor at the Barbara Davis Center for Diabetes and has brought together a diverse team of experts to serve on her advisory committee. The outlined proposal builds upon the candidate's previous research but will enable advancement of technical and analytical skills utilizing state-of-the-art technologies and will allow pursuit of new avenues of B cell research. In addition, the training and development plan is comprehensive and tailored to her needs, which will enable her to transition to independence as a highly productive veterinary scientist in the field of autoimmunity.

NIH Research Projects · FY 2024 · 2020-06

ABSTRACT microRNAs (miRNAs) have been proven to promote cardiac regeneration after myocardial infarction. However, current miRNA delivery methods, such as viral vectors or lipid formulations, present safety concerns for widespread use. We have developed an injectable thermo-responsive hydrogel functionalized with carbon nanotubes (RTG-CNT) for the delivery of miRNAs. The RTG-CNT hydrogel transitions from a liquid-solution to a gel-based matrix shortly after reaching body temperature allowing for a liquid-based delivery rapidly followed by a stable-gel miRNA localization. Moreover, this hydrogel has improved short-term (8-week) biocompatibility compared to viral and lipid approaches and it promotes two-fold more miRNA expression than lipid formulations. In this investigation, we propose to test the hypothesis that our novel RTG-CNT hydrogel is far superior delivery model of miRNAs to the heart, through increased biocompatibility, targeted delivery and higher miRNA expression when compared to viral and lipid approaches. We will address our hypothesis with a combination of cell biology and bioengineering by 1) Quantify the biocompatibility and the magnitude of improved localization of our RTG-CNT-miRNA delivery system over liposomal and viral vectors approaches, 2) Measure the improved efficiency of the RTG-CNT hydrogel as pro-regenerative miRNA delivery system vs. liposomal and viral vector deliveries in a mouse MI model and 3) Determine the potential of the RTG-CNT hydrogel to deliver anti-fibrotic miRNAs to further improve myocardial structure and rescue function in a mouse MI model. We believe that the RTG-CNT hydrogel will offer a more biocompatible and far more efficient miRNA delivery system than traditional approaches, that can be realistically translated into clinical applications.

NIH Research Projects · FY 2024 · 2020-06

Although altered metabolism is rapidly emerging as a key feature of epilepsies, it has not been systematically investigated in any genetic form of pediatric epilepsy. Dravet syndrome (DS), a catastrophic childhood epilepsy associated with de novo mutations in a voltage-activated sodium channel, Nav1.1 is one of the most common genetic epilepsies. DS patients suffer with intractable early-life seizures, and debilitating comorbidities. Energy metabolism in comorbidities associated with DS remain virtually unexplored. To address this unmet need, recent collaborative research in our two laboratories revealed decreased glycolytic and oxygen consumption rates in a validated zebrafish model of DS i.e., scn1Lab mutants. This was accompanied by downregulation of key enzymes, pck1 and pck2, in the gluconeogenesis pathway. Here, we hypothesize that energy disruption occurs in DS due to glucose dysregulation resulting in seizures and/or comorbidities. The following aims are proposed to test this hypothesis. Aim 1 will determine if pharmacological inhibition of pck1 and/or pck2 phenocopies metabolic and behavioral deficits in wildtype zebrafish. Aim 2 will determine if pharmacological manipulation of pck1 and/or pck2 is therapeutic in scn1Lab mutant zebrafish. These studies promise to provide a mechanistic explanation of the metabolic defects observed in DS and could suggest novel avenues for therapeutic intervention.

NIH Research Projects · FY 2025 · 2020-05

Lung cancer is the leading cause of all cancer deaths in the U.S. and worldwide. Lung cancer risk and survival are heterogeneously distributed among U.S. populations. African-American men have a higher incidence of and poorer survival from lung cancer than European-American men, even after adjusting for smoking and socioeconomic factors. The tumor-specific biological factors responsible for the racial differences are not yet understood. The goal of this project is to define the mechanisms by which the JAK/STAT3 pathway operates as a key biological contributor of racial health disparities in non-small cell lung cancer (NSCLC), particularly lung adenocarcinoma (LUAD), the most common histological subtype of lung cancer. Our preliminary data suggest that LUADs from African Americans are more likely than LUADs from European Americans to have JAK/STAT3 pathway mutations that directly induce persistent activation of Signal Transducer and Activator of Transcription-3 (STAT3). STAT3 is an oncogenic transcription factor that is hyperactivated in many cancers. It drives expression of genes that regulate anti-apoptotic responses, angiogenesis, cell proliferation, tumor progression, and therapeutic resistance. The premise of this application is that the JAK/STAT3 signaling axis is inappropriately activated by mutations that are more common in LUAD from African Americans than European Americans, and that therapeutic intervention will be of clinical benefit to a molecular subset of patients with LUAD. Given that the molecular subset is more common in African Americans, research on this topic could help narrow the gap in health disparities. Aim 1 will characterize the molecular profiles in LUAD from African Americans and European Americans focusing on JAK/STAT3 and impact on racial differences. In Aim 2, we will utilize CRISPR-mediated genome editing on patient-derived models of cancer from LUAD tumors from African Americans, and other models, to test the hypothesis that aberrant STAT3 activation results from specific mutations in the JAK/STAT3 pathway, and that the mutations drive LUAD development and tumor progression. In Aim 3, utilizing patient-derived LUAD xenografts primarily from African-American patients, we will test the hypothesis that the JAK/STAT3 pathway mutations we identified can serve as predictive biomarkers for effective antitumor response to STAT3 blockade in LUAD, and we will further clarify novel biomarkers of effective tumor response. At the conclusion of this project, we will have uncovered a novel set of biological determinants of NSCLC health disparities. If the results of the study support our hypothesis, they will provide a path to future clinical trials that may improve the clinical outcome of LUAD patients and help reduce lung cancer health disparities.

NIH Research Projects · FY 2026 · 2020-05

Synapses throughout the central nervous system are sculpted by neural activity through changes in their size, shape and molecular composition, which either strengthen or weaken communication between neurons. This “plasticity” in synapse function is widely viewed as the central mechanism for information storage in the brain. While many forms of synaptic plasticity have been discovered and their molecular mechanisms intensely investigated, in many cases there is surprisingly little direct evidence linking them to the cognitive functions they are proposed to control. This has remained a challenge due to a lack of tools for rapidly and locally switching on or off the requisite biochemistry and cell biology underlying different plasticity mechanisms in real time, in vivo. We are developing new tools that fill this void with the long- term goal of addressing fundamental gaps in our knowledge concerning how synapses are modified at the molecular level through development and plasticity, how these modifications influence synapse/circuit function and ultimately the relevance of these mechanisms for important cognitive functions like learning and memory.

NIH Research Projects · FY 2024 · 2020-04

Project Summary: Destabilization of the genome is known to cause and perpetuate many diseases, particularly cancer. Normally, genome stability is maintained by numerous cellular processes devoted to preserving and repairing DNA. As a result, cells rarely acquire new mutations. However, recent findings from our group and others have demonstrated that cells can undergo transient episodes of genome destabilization and acquire numerous genomic mutations simultaneously. These events, termed saltational bursts of genomic instability, drive rapid genome evolution and are posited to contribute to the initiation and progression of multiple types of cancer. Currently, we understand very little about the properties and causes of saltational bursts, largely because a model system with which to rigorously study these events has been lacking. We have recently characterized bursts of genomic instability in the budding yeast Saccharomyces cerevisiae. Based on compelling preliminary data, we hypothesize that stochastic failures in genome maintenance can cause saltational bursts of genomic instability and lead to transient mutagenic episodes. In this application, I propose to use innovative genomic and cellular approaches to comprehensively explore key attributes of saltational bursts of mutagenesis. Specifically, I will 1) investigate the temporal properties of bursts in order to define the duration of these destabilizing episodes, 2) determine whether bursts occur through defects in specific genome integrity pathways, and 3) determine how variable activity of these pathways modulates the frequency and mutational load of saltational bursts. By conducting the above studies, I will contribute much needed insight into the mutational mechanisms that drive rapid evolution. Moreover, these studies will strengthen our understanding of the mutagenic events that give rise to diseases.

NIH Research Projects · FY 2026 · 2020-04

While traditional genetic tools will always have their place in the molecular and cellular biologist’s armamentarium, increasingly scientists require more nuanced and precise tools for probing biological processes. Over the past 15 years, our group has been developing methods that allow researchers to acutely perturb biochemical processes, in real time, with fast, reversible, and tunable control. A focus of our research is the development of tools that utilize induced protein interactions that can be activated by light or chemicals to control downstream cellular processes and a variety of protein assemblies, including dimerization, oligomerization, clustering, and biomolecular condensates. In our ongoing work, we will continue to develop, validate, and explore the use of novel optogenetic and chemical genetic tools to control protein function and assembly, extending these tools into new areas. The long term goals of the project are to enable a set of modular precision tools for probing and manipulating dynamic biochemical processes, and begin to utilize these tools to uncover new biology with relevance to health and disease states.

NIH Research Projects · FY 2026 · 2020-04

CENTER OVERVIEW PROJECT SUMMARY/ABSTRACT The University of Colorado Anschutz Medical Campus (CU-AMC) is world renowned for basic, translational, and clinical diabetes research and treatment of both Type 1 and Type 2 diabetes (T1D and T2D), and their related complications. During its first funding cycle, the CU-AMC DRC has already strengthened and enhanced diabetes research in Colorado by providing: 1) state-of-the-art research technologies and specialized resources to maximize effective and innovative research; 2) access to patient samples and data; 3) enrichment programs, education and seminar series to the diabetes community; and 4) by attracting new scientists into the diabetes field through the Pilot and Feasibility program and active recruitment activities. These objectives have been realized by providing new and improved infrastructure and access to specialized reagents and resources that are critical for diabetes-related research, and an environment that promotes scientific interactions, research discoveries, and progress towards diabetes treatment and cures. The CU-AMC DRC is primarily located on the Anschutz Medical Campus (AMC) in Aurora, Colorado where the majority of CU-AMC DRC clinical and research departments are located. Now that the DRC is established, we have also welcomed members from the University of Colorado Denver and Boulder campuses, Colorado State University and the School of Mines to diversify our research base. Our broad diabetes research base currently includes 103 basic, translational, and clinical scientific investigators across three AMC schools (Medicine, Nursing and Pharmaceutical Sciences) and includes 27 departments/divisions. The membership currently brings diabetes/diabetes-related direct cost grant support totaling > $65 million in individual grants and >$10 million in center and training grants. Drs. Lori Sussel, PhD. and Jane Reusch, MD bring together complementary areas of scientific backgrounds and expertise to oversee the organization and scientific focus of the CU-AMC DRC. Despite the challenges brought on by the COVID-19 pandemic, we have successfully established four Biomedical Research Cores to provide cutting-edge technologies in Clinical Resources, Tissue Procurement and Processing, Diabetes Modeling and Cell and Tissue Analysis. The CU-AMC DRC Pilot and Feasibility Program has also successfully recruited promising young faculty into diabetes-related research and encouraged established investigators from other fields to enter the diabetes field. The Dean of the CU-AMC DRC School of Medicine has assisted with recruitment packages for bringing new diabetes expertise to campus, supported the purchase of equipment for our DRC core facilities and pledged to supplement the P&F budget by $150,000 annually. Finally, the CU-AMC DRC Enrichment Program has promoted the interaction between diabetes researchers at CU-AMC by providing seminar forums for exchange of research findings, providing opportunities to form collaborative relationships, and encouraging cross-pollination between the diverse diabetes communities.

NIH Research Projects · FY 2026 · 2020-03

The overall goal of the Colorado StARR Program (CSP) is to recruit, train, and retain outstanding resident- investigators focused on translational research in heart, lung, and blood disorders. The initial cycle of the CSP (3/1/2020-2/29/2024) was funded to support 3 scholars per year, and we are requesting support for 4 scholars per year in the renewal application. In the initial cycle of the CSP, we have: 1) successfully established our Program on campus, 2) developed strong interest and a positive reputation among several housestaff programs, 3) extended our Program to include Surgery housestaff (in addition to Medicine and Pediatric housestaff), 4) partnered with the University of Pittsburgh for a research survey course, 5) established an effective approach to ongoing oversight of the mentor-mentee relationship, and 6) fostered a community of physician-scientist trainees at CU including StARR scholars, MSTP students, PSTP trainees, and early faculty. To date, we have committed to support 11 scholars for a total of 12 years of resident-investigator support (2 scholars have completed the program but have not yet transitioned to fellowship, 6 are currently supported by the program, and 3 will initiate their mentored research in the latter half of 2023), and we plan two more recruitment cycles (spring and fall, 2023). The CSP will continue to provide a career defining research experience by leveraging a successful clinical and research enterprise that includes outstanding Medicine, Pediatric, and Surgical housestaff, a diverse spectrum of accomplished, experienced, and committed mentors, state-of-the-art clinical and research facilities, and successful fellowship and collaborative research training programs in heart, lung, and blood disorders. The CSP is designed to provide our resident-investigators with a comprehensive, integrated, and formal career development experience with the goal of positioning them to become the next generation of academic leaders in heart, lung, and blood disorders. This research training and career development will be achieved with planned periods of research and clinical responsibilities that have been approved to lead to board-eligibility in Medicine, Pediatrics, or Surgery, a structured milestone-driven training program that includes individualized career development plans, pairing our resident-investigators with outstanding, dedicated mentors with extensive disease- specific research expertise and a wide array of cutting-edge approaches to research, and providing appropriate oversight of the mentor-mentee relationship. Our vision is to: x Train the next generation of highly skilled and engaged academic leaders x Provide cutting-edge research training opportunities in broad areas of heart, lung, and blood disorders x Ensure access to a wide range of cutting-edge methodology/technology to enrich the experience x Achieve the highest standard of excellence for mentorship and research training x Foster values that sustain and enrich research careers in academic medicine, including team science

NIH Research Projects · FY 2025 · 2020-02

Project Summary/Abstract United States military Veterans from recent conflicts are coping with symptoms related to posttraumatic stress disorder (PTSD). Many Veterans are resistant to conventional health and mental health interventions (e.g., medication, psychotherapy), and often symptoms are not significantly improved by traditional treatments. Alternative treatment methods are needed. An underlying feature of PTSD is exaggerated inflammation, both peripherally and in the central nervous system, which is thought to play an important role in the vulnerability to, aggravation of, and perpetuation of adverse consequences of this condition. Therefore, an innovative intervention strategy would be the use of anti-inflammatory/immunoregulatory probiotics to reduce inflammation. In this study, we will investigate the effects of an 8-week oral administration of Lactobacillus rhamnosus GG (LGG; ATCC53103), a probiotic shown to have anti-inflammatory and immunoregulatory effects on both biological signatures of systemic inflammatory processes and proximal signatures of probiotic administration. LGG is a commensal organism that colonizes the human gut mucosa and suppresses mucosal inflammation via inhibition of the production of proinflammatory cytokines. The specific aim of the study is to identify the effect of probiotics on systemic inflammation, as well as PTSD symptoms, microbiota composition, gut permeability, stress response, and decision-making. Outcomes will be assessed using a longitudinal, double blind, randomized placebo-controlled design. After initial evaluation procedures to confirm PTSD and Functional Bowel Disorder diagnoses, 59 participants will be randomized to probiotic supplementation and 59 will be randomized to placebo supplementation. The proposed line of research addresses the NIH funding opportunity purpose, “to accelerate translational and clinical Phase IIa” trials regarding “probiotic[s]” to increase “understanding regarding underlying mechanisms of their action(s), and variability in responses to these interventions”. Long-term, this study may lead to a paradigm shift in the manner by which we target clinical symptoms associated with PTSD by beginning the process of supporting a multitargeted, neuroprotective approach.

NIH Research Projects · FY 2026 · 2020-02

Project Summary Nearly 1 million people in the United States alone are affected by Multiple Sclerosis (MS). MS is an inflammatory, demyelinating disease of the central nervous system (CNS). While MS is classically regarded as a disease of the white matter, the number of white matter lesions does not correlate with physical disability or cognitive impairment. Recent evidence indicates that gray matter areas have significant myelin loss and increased cortical lesion load is associated with increased cortical atrophy and cognitive decline. Functional imaging of patients with MS reveals increased hyperexcitability within primary motor cortex and throughout the motor network. Moreover, functional recovery in MS patients is associated with normalization of aberrant cortical activity, suggesting a relationship between motor network hyperexcitability and impaired motor behavior. However, our understanding of how myelin loss influences the activity of single neurons, or neural circuits, within grey matter is extremely limited. Our overarching goal is to understand how demyelination results in motor dysfunction. Previously, we used longitudinal characterization of single neuron firing patterns in motor cortex following demyelination to demonstrate early neuronal hyperexcitability that recovers during remyelination. Moreover, we found that enhancing neuronal activity through skilled motor learning after demyelination enhances oligodendrocyte regeneration. However, additional work is needed to determine the dynamic nature of electrophysiological changes across myelin loss and repair to elucidate the neural foundations for behavioral dysfunction. This proposal aims to dive deeper into the cell and circuit mechanisms involved in the relationship between myelin loss and behavioral deficits. By combining three-photon in vivo imaging, high-density electrophysiology, and computational approaches, we will probe the impact of myelin loss across spatial and temporal scales in the motor system, and gain a foundational understanding of the impact of myelin loss on behavior.

NIH Research Projects · FY 2025 · 2020-01

PROJECT SUMMARY Arboviruses cause serious human disease. Viremia level following arbovirus infection of vertebrates is a critical determinant of viral transmission cycles, global viral spread, and disease severity in individuals. Surprisingly, the factors that dictate viremia following arbovirus infection are poorly defined. We found that multiple arboviruses, including chikungunya (CHIKV), Ross River (RRV), o’nyong nyong (ONNV) and Zika viruses, are cleared from the circulation by phagocytic cells. Experiments in splenectomized mice showed that the spleen is dispensable for arboviral clearance. Instead, virus accumulates in the liver and clearance is independent of natural antibodies and complement factors, suggesting a non-opsonic mechanism. Consistent with this idea, clearance of circulating alphaviruses was blocked by competitive inhibitors of scavenger receptors (SRs) that mediate non- opsonic uptake of non-self and modified-self ligands. Remarkably, we found that single lysine (K) to arginine (R) mutations in the E2 glycoproteins of CHIKV and ONNV (E2 K200R), as well as RRV (E2 K251R), abrogated clearance of circulating alphavirus particles by phagocytic cells, and promoted rapid viral dissemination to distal tissues. Moreover, substitution of CHIKV E2 K200 with a variety of other amino acids also allows for clearance evasion, suggesting a specific interaction between key K residues and a host factor. Ks are targets for post- translational modification (PTM), and mass spectrometry analysis of E2 in virions revealed that CHIKV E2 K200 is ubiquitinated. These experiments have revealed a previously unrecognized pathway that controls arbovirus viremia and dissemination in vertebrates. We hypothesize that PTM of key Ks in viral glycoproteins licenses the capture of circulating arboviruses via SRs expressed on liver Kupffer cells (KCs). In Specific Aim 1, the cell types that capture circulating arboviruses will be defined. We also will determine the role of KCs in viral clearance and dissemination, and the development of anti-viral immunity. Finally, we will evaluate the role of phagocytic cells, and KCs specifically, in the clearance of a genera-spanning panel of arboviruses. In Specific Aim 2, we will define the spectrum of blood-borne arboviruses susceptible to SR-mediated clearance. We will use ELISA and cellular binding assays to determine the murine and human SR(s) that bind virus particles. Using SR knockout mice, we will determine the role of specific SRs in clearance of circulating arboviruses. In Specific Aim 3, we will define the role of E2 ubiquitination in the clearance of circulating CHIKV and RRV. We will use mass spectrometry-based proteomics to determine K residues in arboviral particles that are modified with ubiquitin or other PTMs. Finally, we will use a collection of reverse genetics systems to define the role of specific modified Ks in arboviral clearance from the circulation. This work will provide new mechanistic understanding of arbovirus clearance from the circulation. Elucidating these mechanisms could provide new insight into viral transmission, dissemination, and pathogenesis, identify new risk factors of severe disease, and reveal new therapeutic targets for the treatment of arboviral disease.

NIH Research Projects · FY 2024 · 2019-09

PROJECT SUMMARY. Trisomy 21 (T21) causes a different disease spectrum in people with Down syndrome (DS), protecting these individuals from some diseases, while strongly predisposing them to others. For example, >50% of adults with T21 are affected by one or more autoimmune conditions, including a wide range of immune skin conditions. Unfortunately, the mechanisms driving this different disease spectrum are poorly understood, which creates a challenge in the clinical management of DS. We recently discovered that T21 causes consistent activation of the interferon (IFN) response across diverse cell types, which is likely due to the fact that four of the six IFN receptors are encoded on chr21. Accordingly, T21 cells are hypersensitive to IFN stimulation, display hyperactivation of JAK/STAT signaling, and overexpress IFN-Stimulated Genes. Furthermore, dozens of inflammatory cytokines are dysregulated in people with DS, and T21 drives production of potent neurotoxic metabolites via the IFN- inducible kynurenine pathway. Therefore, we hypothesize that hyperactivation of IFN signaling drives immune dysregulation and various pathologies in DS, and that pharmacological inhibition of IFN signaling could have multidimensional therapeutic benefits in this population. Accordingly, we propose here to complete a first-in-kind clinical trial for a JAK inhibitor in DS. our Specific Aims are: 1. To define the safety profile of JAK inhibition in people with Down syndrome. We will perform an open- label Phase II clinical trial for Tofacitinib, a JAK1/3 inhibitor, in people with DS and an active immune skin condition, with the main primary endpoint being the assessment of safety. 2. To determine the impact of JAK inhibition on the immune dysregulation caused by trisomy 21. Using blood samples collected during the trial, we will define the impact of JAK inhibition on a) IFN scores derived from the transcriptome of white blood cells, b) circulating levels of inflammatory cytokines elevated in people with DS, c) levels of neurotoxic metabolites in the IFN-inducible kynurenine pathway, and d) levels of key autoantibodies involved in autoimmune thyroid disease and celiac disease, two common co-occurring conditions in DS. 3. To define the impact of JAK inhibition on immune skin conditions in Down syndrome. Using proven metrics currently employed in clinical trials of JAK inhibitors for immune skin conditions, our main secondary endpoint will be to determine whether JAK inhibition reduces skin pathology in DS. 4. To characterize the impact of JAK inhibition on cognition and quality of life in Down syndrome. Using a battery of tests to evaluate cognition in DS, we will explore the impact of JAK inhibition on diverse cognitive functions. Decreased skin pathology may also affect overall perceived health and enjoyment, as well as have social implications, which will be measured by quality of life assessments.

NIH Research Projects · FY 2025 · 2019-09

Project Summary This R35 MIRA renewal application is designed to investigate mechanisms responsible for the exaggerated effects of alcohol intoxication on the gut, lung, and brain after burn injury. Of the million people per year who suffer burn injuries in the United States, nearly half of the adult patients are intoxicated at the time of injury. Intoxicated burn patients have increased morbidity and mortality compared to those who had not been drinking. The lung is the most frequent organ to fail after a remote injury such as cutaneous burn, with 45% of burn patients showing some form of lung damage even in the absence of inhalation injury. Pneumonia and acute respiratory distress syndrome (ARDS) are among the major complications seen in intoxicated burn patients. Additionally, cognitive impairment is common among burn patients in the ICU, and it is associated with a breach of the blood-brain barrier (BBB) and neuroinflammation. Little is known about the mechanism by which alcohol intoxication upregulates the post-burn systemic inflammatory responses that lead to excessive pulmonary inflammation and increased susceptibility to lung infection nor is it known how cognitive dysfunction arises in this patient population. A common feature of these two critical organs is the aberrant activation of local populations of macrophages, alveolar macrophages (AM) in the lung and microglial cells in the brain. These cells are highly sensitive to stimulation by bacteria and bacterial products. Interestingly, both the post- burn pulmonary inflammation and the incidence of delirium in intoxicated burn patients follow a disruption in the integrity of the intestinal epithelial barrier secondary to burn. This, along with dysbiosis of the fecal microbiome, is critically involved in the systemic inflammation seen after alcohol intoxication and burn injury. To date, we know very little about how and when the intestinal microbiome recovers after remote injury, such as a cutaneous scald burn, and even less about how environmental factors, including alcohol intoxication prior to injury, alter that process. In this research program, we will use our clinically-relevant murine model of alcohol intoxication and burn injury along with clinical samples from burn patients, many of whom misuse alcohol, to address the following questions: 1) Can innate immunity be restored in the lung after alcohol and burn injury? 2) Do microglia play a role in the heightened cognitive impairment and neuroinflammation when alcohol proceeds burn injury? 3) Will restoring the gut barrier integrity and microbiome reduce systemic inflammation, improve the pulmonary response to infections, and lessen neuroinflammation and cognitive impairment? Taken together, these studies will expand the knowledge of how alcohol exposure alters the gut in the context of remote injury, such as burns, and may lead to the development of novel therapies to improve outcomes of patients with burns and other forms of trauma.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY/ABSTRACT The precision and accuracy of vertebrate movement is mediated, in part, by the cerebellum. Two primary divisions of the cerebellum – the cortex and nuclei – are clearly delineated modules that contribute to behaviorally-relevant computations. Sensorimotor information is first processed by the cortex and is then relayed to the nuclei by Purkinje cells (PCs). The resultant output of the cerebellar nuclei has profound influence on downstream motor control, motivating questions of how nuclear activity is controlled by PCs. In a model behavior, mouse reaching, reach endpoint dysmetria, a hallmark of cerebellar damage, is attributed to the dysfunction of anticipatory braking signals from the cerebellar interposed nucleus that slow the limb near the intended target location. This proposal builds on observations in the previous cycle that the firing rates between Purkinje neurons and their targets in the interposed nucleus are inverse of one another, consistent with a population rate code mediating information transfer. However, in addition to inverse rate coding, we find, as in other species and behaviors, that Purkinje neurons synchronize simple spike firing selectively during behavior. Our proposed studies investigate the sufficiency of PC rate and temporal coding to generate behaviorally relevant adjustments to reach kinematics in mice.The objective of this proposal is to testthe central hypothesis that cerebellar cortical circuits selectively transmit population activity through a synergistic rate and temporal code, imparted by local cortical inhibition, and that this code is refined by learning. We propose to delineate the behavioral significance of PC population coding, how it is generated by cerebellar circuitry, and how motor learning engages the dual nature of this synergistic rate and temporal code. RELEVANCE TO PUBLIC HEALTH: Future therapies targeted at ameliorating cerebellar disease will increasingly leverage understanding of computational mechanisms of the structure, thus identifying those principles, the goal of this proposal, is of central importance.

NIH Research Projects · FY 2024 · 2019-09

Summary The University of Colorado Anschutz (CU Anschutz) has a strong history of training undergraduate, pre- and post-doc scientists in the biomedical sciences, and specifically in cancer research. The overarching goal of the Cancer Research Experience for Undergraduates (CREU) program is to provide a mechanism through which undergraduate students can explore cancer research as a future career. In this research- intensive experience, students will spend 10 weeks in a laboratory working with a mentor from the University of Colorado Cancer Center. Students will choose from 52 qualified mentors with expertise in basic and clinical/ translational cancer research. CREU is primarily a research-intensive experience, however students will also attend weekly seminars that explore fundamental questions in cancer biology and professional development, including career mentoring. In addition, students will select from a variety of clinical/translational workshops (CTWs) based on their interests. CTW's will typically be ½ day experiences with faculty and biotechnology companies on the CU Anschutz campus and will be structured so as to capitalize on unique strengths and cancer training opportunities on our campus in personalized medicine and bioinformatics, cancer pathobiology and molecular diagnostics, and experimental therapeutics and biotechnology. A total of 26 students will be selected to participate in CREU from a pool of national applicants. To assure representation of diverse trainees, we have established “links” to two minority serving institutions, Chaminade University of Honolulu and the University of New Mexico. Applicants from these institutions will be given preferential consideration for participation in CREU, provided they meet our admissions standards. We will also actively recruit under- represented students at regional and national minority recruitment events. The CREU program is designed to provide potentially transformative experiences for college students who are interested in careers in biomedical research, but who have had limited immersive experiences in cancer research. Our goal is to increase the number of students, particularly from underrepresented minorities, who choose to pursue cancer research for a career. We will determine how well we meet our goals using short and long-term evaluation tools.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY/ABSTRACT Alzheimer's disease (AD) currently affects more than 5 million Americans and is a significant burden for patients, caregivers, and our healthcare system. Currently, there is no known cure for AD, and approved therapies do not reverse the underlying pathogenesis or inhibit disease progression. Amyloid deposits and their associated amyloid beta oligomers (Aβ) are linked to multiple cellular dysfunctions in the brains of AD patients and in animal and cell culture models of AD. Some of these dysfunctions include microtubule instability, loss of dendritic spine density, and depressed hippocampal long-term potentiation (LTP). Many hypotheses have been put forward to explain the routes by which Aβ contributes to these phenotypes. However, a unified mechanistic understanding has not yet been achieved. This project builds on previous studies that have shown that Aβ inhibits the activity of a select set of microtubule motor proteins. Among them, Kinesin-5/EG5/KIF11 is of particular interest due to its diverse functions, which include regulating microtubule stability, microtubule polymerization, and dendritic architecture, all of which are negatively impacted by Aβ. Based on these findings, the long-term goal of this project is to determine whether Kinesin-5 activity may serve as an effective target for preventing or reversing A β -induced AD phenotypes: 1) by overexpressing Kinesin-5 to maintain its activity, or 2) by using small molecule drugs we identified in a screen that block Aβ-mediated inhibition of Kinesin-5 activity. In order to address these goals we are using a mouse model system in combination with cell culture and biochemical tests that aim to 1) Determine whether Kinesin-5 overexpression improves learning and memory in a wild-type mouse without AD pathology and whether Kinesin-5 overexpression rescues Aβ-induced AD phenotypes in the an AD mouse model; 2) Determine whether Kinesin-5 overexpression improves long-term potentiation (LTP) and whether deficits in LTP caused by Aβ are rescued by Kinesin-5 overexpression; and 3) Determine whether Kinesin-5 overexpression impacts neural process outgrowth, spine density, and morphology in wild-type and 5xFAD mice. Our proposed research will also provide an increased understanding of the role of Kinesin-5 activity in regulating diverse neuronal processes. The clinical significance of this work comes from the identification of underlying mechanisms linked to aberrant cellular functions that may uncover an entirely new therapeutic approach to AD. The innovative aspects of this project are rooted in the identification of Kinesin-5 as a potential therapeutic target for the treatment of AD and include: 1) interrogating of as yet uncharacterized functions of Kinesin-5 in learning, memory, and AD-related phenotypes; and 2) pursuing an entirely new avenue of investigation into desperately needed AD treatments using Kinesin-5 activity as a therapeutic target.

NIH Research Projects · FY 2025 · 2019-08

PROJECT SUMMARY Eukaryotic cells contain within them a myriad of spatially distinct sites that serve a variety of functions. To facilitate this organization, eukaryotic gene expression is routinely spatially regulated through the trafficking and sequestration of thousands of different RNA molecules to distinct cellular locations. Misregulation of this process leads to detrimental phenotypes in a wide range of systems, from developmental defects in Drosophila to neurological disease in humans. Despite this importance, our knowledge of the regulation of RNA localization is quite limited. For other modes of post-transcriptional regulation like splicing, our understanding of how the interactions of RNA binding proteins (RBPs) and RNA motifs lead to specific outcomes is much more mature. This relies on many years of work by many groups that have defined the regulatory language of splicing and allows us to make predictive and combinatorial models about how splicing is regulated across conditions and cellular environments. We lack such an ability with regards to RNA localization, in large part because we lack the analogous “parts list” that defines the language of localization regulation. The overall goal of the Taliaferro lab is to learn this language and be able to build predictive models that explain how RNAs are trafficked to their intracellular destinations. This includes identification of sequences within RNAs that govern their transport as well as RNA-binding proteins that bind these sequences and mediate the process. Historically, these features have been identified for one RNA at a time through a time consuming and laborious process. Although these studies form the foundation of the RNA localization field, the amount of effort they require has limited their application to a large number of RNAs. Further, it is often hard to take findings from these single-transcript experiments and generalize them into larger principles that underlie rules that govern the transport of thousands of RNAs. The Taliaferro lab aims to approach this problem from the opposite direction by isolating subcellular transcriptomes and identifying features shared in common among RNAs localized to a particular destination. This strategy identifies elements associated with the transport of hundreds of RNAs at once, parallelizing the one-transcript-at-a-time approaches used in the past. During the course of the proposed experiments, over the next five years, the Taliaferro lab will use a variety of high-throughput approaches to both discover new sequence elements that regulate RNA localization as well as deeply characterize previously identified elements in order to discern how they work. Additionally, we will develop new technologies that will facilitate the definition of RNA localization mechanisms in organisms and cell types that thus far have been intractable. We expect that results from these studies will be a positive step toward our goal of understanding and eventually applying the molecular language of RNA localization regulation.

NIH Research Projects · FY 2026 · 2019-08

PROJECT SUMMARY Red blood cells (RBCs) are a perfect model to study oxidant stress. RBCs are the most abundant cell in the body (84% of total human cells) and play an essential role in oxygen transport and thus in the regulation of all oxygen-dependent metabolic processes. To facilitate this task, RBCs are loaded with hemoglobin (Hb) and iron. Indeed, 66% of total bodily iron is in RBCs. As a result, the mature RBC faces significant oxidant stress deriving from iron-dependent Fenton and Haber-Weiss redox chemistry. The lack of nuclei and organelles prevents RBCs from synthesizing new proteins to replace oxidatively damaged components during the average lifespan of 120 days in circulation. Every day over 200 billion RBCs are removed from the bloodstream and de novo generated via erythropoiesis, a process that relies on the uptake of circulating iron in the ferric (oxidized) state, and its reduction to the ferrous state. This process is catalyzed by the ferrireductase STEAP3 – a transcriptionally-regulated target of tumor protein p53. Both p53 and STEAP3 are critical to erythropoiesis and polymorphic in humans, whereby mutations in p53 - occurring in >50% of all cancers, 0.2% of all healthy humans - are not just inherited, but commonly accumulate during organismal aging or exposure to carcinogens. In genetic studies on murine models of in vitro aging of RBCs (i.e., under conditions that mimic blood storage in the blood bank) we have documented that hypermorphic STEAP3 is associated with poor blood storage quality, owing to increased oxidant stress and elevated lipid peroxidation. Iron-mediated non-apoptotic cell death via lipid peroxidation is a hallmark of ferroptosis, a novel process of cell death investigated extensively in nucleated cells, but hitherto ignored in iron-loaded RBCs. More than other cells, RBCs rely on antioxidant systems to keep oxidant stress in check. A key antioxidant system is represented by the soluble tripeptide glutathione, glutathione-dependent detoxification systems (e.g., glutathione-peroxidase 4 - GPX4 to counteract lipid peroxidation) and oxidized glutathione recycling via NADPH-dependent enzymes. The main pathway that sustains NADPH synthesis in RBCs is the pentose phosphate pathway. Glucose 6-phoshate dehydrogenase (G6PD) is the rate-limiting enzyme of this pathway, an X-linked gene that is mutated in ~500 million people. Oxidant damage to protein triggers formation of isoaspartyl damage, a process counteracted by the enzyme PIMT. Relevant to this proposal, in other cell types p53 promotes ferroptosis by up-regulation of STEAP3 and down-regulation of G6PD, while being itself negatively regulated by PIMT. Even though genetic and pharmacological tools are available to regulate ferroptosis, these approaches are untested in mature RBCs, which is the focus of this project. Relevance to public health: RBC responses to hypoxia and oxidant stress regulate hemolysis, an etiological contributor to physio/pathological adaptations to pro-oxidant challenges in vivo (e.g., high altitude, exercise) or ex vivo, such as blood bank storage of RBCs for transfusion purposes, ranked 1st in the list of breakthroughs in the history of mankind that are credited with saving the most lives.

NIH Research Projects · FY 2026 · 2019-08

PROJECT SUMMARY Predicting functional consequences of genomic variation remains the biggest barrier to precision medicine. While nonsense variants are considered the best understood of all genetic variants, our work has revealed significant gaps in our knowledge base for their functional interpretation. My group studies how variability in mRNA surveillance impacts the functional consequence of human genetic variants. Protein-truncating genetic variants often cause loss of gene function because their encoding transcripts are subject to degradation by an RNA quality control process called nonsense-mediated RNA decay (NMD). NMD senses transcripts with a premature stop codon and triggers their degradation to prevent the production of truncated proteins. However, the efficiency with which NMD acts on a target transcript is variable across genes, tissues, and even individuals. We have shown that non-canonical translation events such as stop codon readthrough and translation reinitiation can cause NMD evasion by a nonsense-containing transcript, though the rules governing such events are unknown. My research program builds on these discoveries to systematically investigate factors that cause variable NMD efficiency. Specifically, we combine massively parallel reporter assays with targeted gene editing to identify and validate sequence features that permit NMD escape. Through this work, we aspire to build new rules for the functional classification of variants and use such rubrics to build more accurate and predictive models for phenotypic consequence of nonsense variants. Such a model will allow us to identify variants that are likely to cause disease and to develop novel therapeutic approaches to counter genetic disorders.