University Of Colorado Denver

NIH Research Projects · FY 2025 · 2019-07

The University of Colorado Anschutz Medical Campus T32 training program in Molecular and Systems Toxicology (MST) provides a multi-disciplinary research mentorship program centered around training in systems toxicology including genomics/epigenetics, proteomics, and metabolomics as well as molecular approaches to understand underlying mechanisms of toxicity. The T32 training program is housed under the multi-institutional Toxicology and Pharmaceutical Sciences graduate programs which are based within the Department of Pharmaceutical Sciences, Skaggs School of Pharmacy, but which includes faculty from the Schools of Medicine and Public Health on the Anschutz Medical Campus and also includes faculty from National Jewish Health. The MST training program successfully trained 9 PhD students from 2019-2023. For renewal of the MST T32 training program, we are requesting 3 predoctoral positions/year for 2 years of support (in years 3 & 4 for a total of 15 predoctoral trainees over 5 years). The University of Colorado Anschutz Medical Campus MST training program is be supported by 18 faculty which are focused on systems toxicology research and are listed as mentors on this T32 application. This training program has the primary goal and responsibility of developing predoctoral students into independent and successful environmental health scientists with expertise in systems toxicology. This goal will be achieved by utilizing: 1) internationally recognized, well-funded research mentors in systems toxicology; 2) outstanding institutional support from the UC Anschutz campus; 3) targeted didactic learning approaches; 4) training in responsible conduct in research, grant writing and career development; 5) continued excellence in recruitment and enhancement of diversity; 6) continued student engagement in research, education and career development through various programs; 7) continual development of systems toxicology related coursework and experiences; 8) evaluation metrics to continually improve the MST training program. Ultimately, we expect our trainees to develop into independent scientists with the knowledge and skills to address challenges in toxicology and environmental health using cutting edge ‘omics’ and ‘big data’ approaches.

NIH Research Projects · FY 2025 · 2019-07

ABSTRACT Cancer is a leading cause of morbidity and mortality in the US. However, a significant advancement to treating cancer is the discovery of immune-based therapy. This is a rapidly changing field that requires future clinician researchers to draw upon a unique skillset that is quite distinct from current investigation and practice, as host features play a dominant role over traditional, anatomical-driven parameters. The goal of this proposed T32 program is to train future oncology clinical providers and researchers in the most advanced and promising forms of immune-directed and cell therapy, by integrating those newly required skills into a training plan that addresses those unique needs. The program is designated as the Cell Immunotherapy and Experimental Therapeutics Program (CIETP and is uniquely tailored to adapt to the specific training requirements of clinical fellows, now including medical specialties like Nephrology, Neurology, and Rheumatology that are incorporating cell therapy trials in their clinical research portfolios. CIETP addresses new challenges including a multitude of new agents in development, new mechanisms of action, distinct toxicity profiles, and a lack of patient selection strategies. The opportunity is to invent developmental therapeutics that exploit the power of immune-based therapy, and we need to train our clinical researchers in novel ways. The CIETP has created an ecosystem that teaches advanced research and clinical skills to clinical trainees while giving them the opportunity to contribute to the development and testing of next generation of immunotherapies. We draw from the University of Colorado School of Medicine (UCSOM) basic research and clinical strengths and history of basic immune research (discovery of IL-1 and STING), allergy and autoimmunity research with world- class asthma (National Jewish), and diabetes (Barbara Davis) centers. The UCSOM has supported the creation of in-house facilities to produce clinical-grade cell therapy products, like CAR T and NK-cells and TILs, and to bridge the bench-to-bedside gap focused on creating advanced, humanized mouse models. The UCSOM is a leader in oncology clinical translation with a prestigious Developmental Therapeutics (DT) program, and has created a Personalized Medicine Center designed to facilitate patient-specific therapies, and to link the scientific potential with the delivery of patient care. CIETP draws upon our experience in successfully running this program over the last 4 years. In the first funding period, we successfully attracted candidates from multiple clinical training areas (medical, hematology, pediatric and surgical oncology), with a majority of women scientists, that included individuals from under represented background, and that were productive (based on the number of publications, clinical trials, and grants awarded), developing projects that were evenly distributed between basic and clinical track and covered the immune and cell therapy spectrum. As a measure of our success, all our trainees have continued their careers in academic positions. The CIETP has established the foundation for creating a unique training program, focused on preparing clinical investigators and physician scientists to discover and implement the next generation of immunotherapies. ,

NIH Research Projects · FY 2026 · 2019-06

Project Summary: 8-Oxo-7,8-dihydroguanosine (8-oxoG) is arguably the most important oxidative lesion that is generated within RNA under oxidative stress. As a consequence it is used as a common biomarker of neurodegeneration and disease progression/development, also suggested to be involved in signaling, and in regular RNA metabolism. Over the past decades much research has been devoted to its biological impact on DNA, however, information about its presence within RNA has been overlooked. The aims of this proposal are designed to understand how 8-oxoG affects the structure and function of oxidized RNA. Aim 1 will establish the impact of this oxidative lesion on ubiquitous structural motifs of RNA, known to have many important biological functions, including their role in recognition of/by other biomolecules. Aim2 will focus on the completion of a platform that contains constructs of RNA or DNA for their use in detecting metabolites of interest, denominated as oxidized RNA conjugate aptamers (ORCAs). Aim 3 focuses on exploring the reactivity/interactions between oxidized RNA and RNase R, a ribonuclease that is conserved across all kingdoms of life, thought to be involved in the handling of damaged RNA. Understanding how 8-oxoG impacts the structure and function of RNA is necessary to assess its role in oxidative stress and disease progression/development, as well as intracellular handling. The Resendiz lab has established methodology that ensures the success of each of the proposed aims and has publications that suggest for the proposed hypotheses to hold true. The work within Aim 1 has the potential to establish trends that might be generalized, thus contributing to our understanding of how this oxidative lesion affects RNA structure; furthermore, the computational component is the first step towards using a reliable theoretical approach to determine rules with regards to the impact of 8-oxoG on RNA structure. The proposed aim 2 will use oxidized and canonical RNA to discover metabolites with affinity towards RNA structural motifs of interest, thus leading to molecules with high affinity towards RNA constructs, potentially deriving in the discovery of therapeutic targets. The work proposed in aim 3 represents a step towards understanding the mechanism, or necessary factors, for the intracellular handling of oxidized RNA. The proposed research is highly interdisciplinary and has a high translational impact. This will also serve as a platform to excite and encourage students to pursue careers in the biomedical/biochemical fields, while providing meaningful high-quality training in relevant fields. The PI has a strong track record with supporting individuals to achieve successful transitions to their next academic/professional step. A consequence of the proposed activities will reflect on the strengthening of CU Denver as an institution, with the building of a strong research program that attracts and provide opportunities to students across the state, the nation, and beyond.

NIH Research Projects · FY 2024 · 2019-06

PROJECT SUMMARY/ABSTRACT Cancer comprises a diverse group of diseases with significant morbidity and mortality. Nearly all common cancers exhibit some form of sexual dimorphism, for example in incidence, prognosis, or response to therapy. This dimorphism has been hypothesized to derive from differences between males and females in hormones, sex chromosomes, and environmental exposures; however the molecular basis of these disparities remains largely unknown. An understanding of this dimorphism is fundamental to precision medicine in cancer, and may lead to discovery of novel biomarkers, therapeutic targets, and improved outcomes. We propose to discover the molecular basis of sexual dimorphism in cancer though the following Aims: Aim 1 will characterize sexual dimorphism in gene expression and its regulation within and between tumor types of NCI's The Cancer Genome Atlas (TCGA). Leveraging TCGA, we will characterize sexual dimorphism at the transcriptome level and its potential genomic and epigenetic causes within and across cancers. Aim 2 will characterize sexual dimorphism in the heritable genetic component of cancer susceptibility across common cancers. Utilizing data from the largest genome-wide association studies of cancer we will search for differences in the genetic architecture between males and females. We determine what proportion of the heritable component of each cancer is shared across sexes, and also estimate how much sex-specific sharing across cancers. Using multiple genetic models, we will perform sex-aware association studies to identify genetic variants that affect males and females differently. We will test a model for genetic risk, whereby the same alleles affect both sexes, but the lower-risk sex would require more or stronger genetic risk factors to develop disease. We will test for specific risk or protective factors encoded on the sex chromosomes that affect the sexes differentially. We will test for gene-sex interactions whereby autosomal loci act in a sex-dependent manner, with or without hormonally-mediated or other sexual dimorphism acting at the gene expression level. Aim 3 will characterize sexual dimorphism in response to therapeutics and define the molecular features and mechanisms contributing to that dimorphism. Utilizing molecular and phenotypic drug response data from over 1000 cancer cell lines from the Cancer Genome Project (CGP), we will build sex- specific predictive models of drug response that we will then apply to gene expression data from the full set of TCGA tumor samples to impute a sex-specific drug response for each TCGA sample. We will correlate imputed drug responses to TCGA tumor molecular features, with focus on those identified in Aims 1 and 2 and candidates suggested by our preliminary data and the literature. We will functionally validate predicted sexually dimorphic response to therapy using cell models, including panels of lymphoblastoid cell lines, human hepatocytes, and cancer cell lines. The results of this study will define genetic and genomic features that underlie sexual dimorphism in cancer biology, susceptibility, and response to therapeutics.

NIH Research Projects · FY 2025 · 2019-04

ABSTRACT Despite the observation of microbial dysbiosis in the intestines of individuals at risk for and with rheumatoid arthritis (RA) and spondyloarthritis (SpA), the mechanisms by which dysbiosis promotes inflammatory arthritis remain unknown. Our group has made the following key discoveries: (1) In patients with axial SpA, paired bacterial metagenomics with intestinal tissue metabolomics screening connected bacterial dysbiosis to tryptophan catabolism into indoles. (2) We identified a novel indole-producing bacterium Subdoligranulum didolesgii present in and targeted by autoantibodies and T cells from individuals at risk for and with RA that can stimulate joint swelling when transferred into mice, whereas a non-indole producing isogenic strain does not. (3) Using the CIA model, we demonstrated that a tryptophan low diet or microbiome depletion with antibiotics results in protection from arthritis, and supplementation with indole is sufficient for disease by modulating both Th17 cell differentiation and autoantibody pathogenicity. The goal of this proposal is to connect these observations and demonstrate the immunologic pathway of indole from its generation by dysbiosis in RA to pathogenic immune functions. Based on ongoing studies, we hypothesize that bacterial dysbiosis associated with RA results in increased tryptophan catabolism into indole, which signals in intestinal dendritic cells to produce cytokines leading to Th17 cell differentiation required for pathogenic autoantibody generation. In Aim 1, we will use paired stool and plasma samples from an established cohort of controls, individuals with RA, and individuals at risk for RA with some of whom transition to disease, to perform bacterial metagenomics of the stool and targeted metabolomics of the plasma for the tryptophan pathway. We hypothesize that bacterial metagenomics will reveal a pattern of tryptophan catabolism to indoles with increased plasma indole metabolites that correspond with the development of RA. Aim 2 will then identify the cellular target of indole in vivo using dual single cell ATAC and RNA sequencing of mesenteric lymph nodes harvested from mice fed a tryptophan low diet ± indole and induced for CIA. Traditional immunologic and molecular biology techniques will then be performed to confirm the signaling pathway and immune effects imparted by indole in human cells. Finally, Aim 3 will examine the requirement of indole-stimulated Th17 differentiation for pathogenic autoantibody formation. We hypothesize that indole- stimulated Th17 differentiation results in IL-21 that is necessary for B cell isotype switching and expression of key glycosylation enzymes. We will block IL-23 and IL-21 in our indole-CIA model and human primary B cells to dissect B cell functions associated with Th17 cell differentiation. Successful completion of these studies will reveal a targetable pathway and disease population for potential therapeutic development by blocking indole in treating RA and SpA.

NIH Research Projects · FY 2026 · 2019-04

It is imperative that we continue to innovate ways to address inequities that challenge the health and well- being of American Indian (AI) youth, families, and communities. The COVID-19 pandemic disproportionately impacted Indigenous communities in the U.S. and made inequities broadly visible, but these inequities are not new. The history of oppression of Indigenous people and attempts at cultural eradication through federal policies and broken treaties has disrupted cultures and communities, and research suggests that this disruption is at the root of elevated substance use problems in these communities. AI youth initiate substance use earlier than most other youth in the U.S. and the links between early initiation and both concurrent developmental disruption and progression to problematic patterns of use are strong. The need for early prevention among AI youth is clear, and evidence suggests that leveraging the cultural strengths may be a particularly effective path toward prevention. We have been working with partners on a Northern Plains Reservation since 2009 to understand risk factors for early initiation of substance use and to develop a culturally grounded prevention program for young adolescents. We have developed and are testing the Thiwáhe Gluwáš’akapi (TG) program for young AI adolescents that reinforces family, cultural and community connections. Through a multiphase optimization trial (TG1 study), we gathered preliminary evidence of the effectiveness of TG; in a randomized controlled trial (TG2 study), we tested the effectiveness of the optimized TG program. This project (TG3 Study) moves this work forward through a hybrid implementation-effectiveness study. First, it will maximize what can be learned from data collected in the TG2 study by lengthening the follow-up period for participating youth and families to four years post-intervention and utilizing Bayesian small sample quantitative analytic methods. Second, this study will build on what was learned in the TG2 study about program delivery to foster the transition from research-based to community-based implementation, engaging existing community organizations to deliver TG to families across the reservation and gathering systematic data on implementation processes and outcomes. This study will expand our understanding of TG program effects, result in a better understanding of the optimal fit for the sustained delivery of the TG program on the Pine Ridge Reservation and begin developing infrastructure for long term program implementation. RELEVANCE (See instructions): Disparities in substance use and high rates of suicide pose significant risk to American Indian (AI) youth and persist despite efforts to create culturally tailored prevention approaches. This study will add to the evidence base regarding the effectiveness of a substance use prevention program tailored for AI youth and families and simultaneously examine community-based implementation of the program to identify strategies to support sustained delivery.

NIH Research Projects · FY 2026 · 2019-03

Project Summary This grant will provide infrastructure support to the University of Colorado Consortium Cancer Center and its clinical care partner UCHealth to continue to support the specific aims of the grant: 1. To provide leadership to the NCTN program and participate in scientifically important and clinically relevant NCTN clinical trials. 2. To make these trials available to the greatest proportion of the cancer population of Colorado and Wyoming. 3. To rapidly activate, accrue, caringly provide patient coordination, and accurately report research related data in a timely way to the NCTN components responsible for conducting these trials. 4. To foster and develop young investigators in the NCTN process. Specific support is requested for the administrative, regulatory, clinical coordination, and data management needs to conduct clinical trials of the NCTN safely and efficiently on behalf of the cancer patients in Colorado and our wider catchment area. In addition, we will support enhanced monitoring and auditing to ensure patient safety and proper conduct of the trials at all our sites of clinical care. Quality assessment and improvement are a central feature of our NCTN program. We take seriously the mandate to participate in the development of new concepts, the contribution of our laboratory scientists in these translational projects, and the fostering of new junior investigators to further the clinical impact of therapeutic trials in the NCTN. Dialog and communication are key. Within our institution, we have created a superb forum for our investigators across the entire system (the LAPS Executive Committee) that represents all modalities and disease-oriented groups to prioritize and commit to new trials, to monitor accrual and any other issues related to the conduct of these trials across our system. We support travel for investigators, staff, and scientists to participate in the NCTN meetings and committees. We hope to parlay the Cancer Center's interest in survivorship and prevention to enhance our outreach to the underserved and rural populations and their caregivers in order to enhance interventional accruals for this population that ordinarily would not come to the main facilities.

NIH Research Projects · FY 2026 · 2019-03

Project Summary People with immunocompromising conditions, including end stage renal disease (ESRD) and renal transplantation, are at increased risk of developing severe infections. Vaccination is the preferred mode of protection against infections but people with the highest risk of infectious morbidity tend to have poor responses to vaccines. The recombinant zoster vaccine (RZV) showed excellent efficacy in healthy adults ≥50 years of age and lower immunogenicity and efficacy in transplant recipients. The goal of this study is to define the immunologic and metabolomic profiles associated with decreased magnitude and persistence of immune responses to RZV in people with ESRD before and after transplantation. We will use plasma and PBMC collected in a study of RZV in ESRD sponsored by GlaxoSmithKline, in which participants receive the standard 2-dose RZV immunization regimen while awaiting renal transplantation and are then followed for 31 months. Transplanted participants on stable immunosuppression are randomized to receive a 3rd dose of RZV or not and are followed for 12 additional months. Responses of these immunocompromised hosts are compared with responses of healthy adults ≥50 years of age, who received RZV in previous studies. Research questions will be addressed in the following Aims: Aim 1. Identify immunologic and metabolomic correlates of reduced persistence of immune responses to RZV in people with ESRD. We will compare adaptive and innate memory responses to RZV in 50 ESRD and 50 healthy older adults using flow cytometric and single cell immunogenomic approaches and identify factors associated with persistence. Using systems and computational immunology approaches, we will identify pre- vaccination phenotypic, functional, and genomic immune cell profiles, and inflammatory and metabolomic characteristics associated with decreased responses to RZV in people with ESRD. Associations of inflammatory factors and/or metabolites with characteristics that hamper the immune responses to RZV will be verified in vitro. Aim 2. Define the effect of renal transplantation and of a 3rd dose of vaccine on immune responses to RZV. We will compare adaptive and innate memory responses to a 3rd dose of RZV administered to 30 transplant recipients with responses to the standard RZV regimen in 50 healthy and 50 ESRD older adults using flow cytometry and single cell immunogenomics. We will analyze the T-cell repertoire to define the effect of induction regimens and of the 3rd dose of RZV on the persistence of RZV-expanded T cell clones. We will determine pre- vaccination immunologic and metabolomic profiles associated with responses to the 3rd dose of RZV in transplant recipients. Impact. This study will identify actionable inflammatory and/or metabolomic factors and immunogenomic characteristics associated with decreased responses to RZV in people with ESRD with and without transplantation and will determine if a 3rd dose of RZV can mitigate the negative effect of transplantation on the persistence of immune responses to RZV. We will leverage prior NIH- and GlaxoSmithKline-funded studies.

NIH Research Projects · FY 2025 · 2019-01

ABSTRACT Excessive perivascular fibrosis results from the highly dynamic and dysregulated process of normal tissue repair and is defined by the excessive accumulation of extracellular matrix (ECM) material resulting in decreased vascular compliance and increased vessel stiffness. In contrast, a fibrotic response is critical to the formation and stability of a fibrous cap in the setting of atherosclerosis. While advances have been made in our understanding of fibrosis pathobiology, significant gaps remain. In particular, while mesenchymal cells with the potential to transition into activated myofibroblasts are believed to be key sources of excessive ECM deposition, their origin remain debated. Using cell-specific lineage tracing and RNA sequencing, our group made the paradigm-shifting discovery that a subpopulation of resident vascular stem cells residing within the vascular adventitia originate from mature vascular smooth muscle cells (SMC)(termed AdvSca1-SM cells). SMC reprogramming and AdvSca1-SM cell maintenance is dependent on induction and activity of the transcription factor, Klf4. In contrast, vascular injury-induced or AdvSca1-SM cell-specific genetic Klf4 depletion promote the transition to profibrotic myofibroblasts, which consequently serve as major contributors to perivascular fibrosis. However, the molecular mechanism whereby Klf4 dictates the maintenance and differentiation of AdvSca1-SM cells remains unclear. Leveraging a highly specific AdvSca1-SM cell reporter system and single-cell RNA- sequencing (scRNA-seq), we demonstrate the profibrotic differentiation trajectory of AdvSca1-SM cells. Differentiation is characterized by loss of Klf4 and its downstream effector PI16, a negative regulator of the epigenetic protein, histone deacetylase 1 (HDAC1), and the lncRNA, Meg3, but gain of expression of a profibrotic phenotype. Importantly, these changes were recapitulated in human fibrotic tissue, underlying the translational significance. Statin treatment was previously shown to reduce tissue fibrosis; however, the molecular mechanism is unknown. Querying Connectivity Map database with gene signatures of AdvSca1-SM cells and their profibrotic derivatives indicate that statins are potential candidates to antagonize the profibrotic transition of AdvSca1-SM cells. Finally, while inflammation and fibrosis drive the wound healing process in most adult mammals, scarless regenerative wound healing in adult rodents of the species Acomys has been reported. Our recent findings show a complete absence of fibrosis with rapid regeneration in two models of kidney injury in Acomys. Here, we propose a novel concept that a Klf4-Meg3 axis plays a central role in the maintenance of the stem cell phenotype; loss of Klf4 and/or Meg3 activates a signaling cascade facilitating the phenotypic transition of AdvSca1-SM (Aim One). Further, we propose that Acomys is resistant to pathological vascular remodeling and fibrosis in part due to activation of an AdvSca1-SM-specific regenerative gene expression signature (Aim Two). Finally, statin treatment will inhibit or reverse the profibrotic transition of AdvSca1-SM cells (Aim Three).

NIH Research Projects · FY 2026 · 2019-01

PROJECT ABSTRACT This is a renewal application from University of Colorado Anschutz Medical campus that expands the original focus of our existing “PRIDE Academy: Impact of Ancestry and Gender on omics of lung diseases (AGOLD)”. The new descriptive title is “Health AI and Data Science in Cardiovascular and Pulmonary Disease-Application and Bioethics (HARP-BIO)”. In this renewal, we propose to continue program focus on impact of gender and ancestry on interpretation of omics data but will now include data sciences in general, artificial intelligence and machine learning and how biases inherent in coding or programming of computers and/or instruments results in biased outputs and ultimately health disparities in pulmonary and cardiovascular diseases. This topic is well aligned with NHLBI interest areas of “Emerging technology and informatics, such as artificial intelligence/machine learning and Integration of wearable technology in research and clinical care” and Genomics, “populomics”, and precision health to advance the science of tailored treatment approaches for HLBS conditions. The overall objective remains the same: to introduce scholars from under-represented backgrounds to various omics platforms and how data sciences should be used ethically, interpreted and applied when working with under-represented populations. This academy will be housed at the Pulmonary and Critical Care, Cardiology Divisions of Dept of Medicine and the Department of Bioinformatics using resources available through the Centers for Personalized Medicine and Health Artificial Intelligence as well as the Center for Bioethics and Humanities. Over the last 4 years, AGOLD has trained 18 scholars, all underrepresented in Science or Medicine; all scholars received small research project funds leveraged to obtain additional extramural funds as well as peer-reviewed publications. The Pulmonary Division has a distinguished record of training leaders in pulmonary medicine and the new Bioinformatics Department has been instrumental in elucidating to what extent genetics can explain health disparities in complex diseases, particularly asthma. We propose a PRIDE summer academy that will include didactic and hands-on workshops in genomics and proteomics of lung and cardiovascular diseases, ethical use of data sciences and machine learning and introduction of the concept of academic “coaches”. A coach is not intended to supplant the mentor, but rather guide team members through the process of successfully navigating the academic world using well-tested social science approaches. Through 2 consecutive Summer Academies, the scholars identify a mentor and mentoring team, receive instruction on “omics” and ethical use of data sciences and artificial intelligence, are paired with coaches who ensure that milestones are achieved; participate in grant-writing workshops and mock study sections; implement an evaluation plan that measures how the program is achieving its objectives. By using the combination of coaching and social science practices such as cultural capital and communities of practice that allow team members to feel connected with each other, we will ensure their academic persistence and ultimate success.

NIH Research Projects · FY 2025 · 2018-09

Project Summary/Abstract Postsynaptic kinase/phosphatase networks in amyloid b-induced synaptic dysfunction Impaired synaptic function and synapse loss are early hallmarks of Alzheimer’s Disease (AD). There is strong evidence that amyloid beta (Ab) leads to AD-related synapse dysfunction and cognitive impairment. For example, long-term potentiation (LTP), a key form of plasticity for learning and memory, is disrupted in mouse models harboring familial AD-linked mutations that lead to Ab accumulation. Even acute applications of soluble Ab oligomers block LTP within minutes and lead to synapse loss within days. It is increasingly appreciated that Ab hijacks normal synaptic signaling pathways involved in plasticity, biasing them toward long-term depression (LTD), or synapse weakening/elimination. LTP and LTD in the hippocampus are ultimately driven by insertion and removal of AMPA-type glutamate receptors (AMPAR) from synapses. Central to the signaling pathways regulating AMPAR trafficking during LTP and LTD is a postsynaptic Ser/Thr kinase/phosphatases signaling network that is coordinated by the scaffold protein AKAP79/150 and includes CaMKIIa, PKA, and PP2B/calcineurin (CaN). During the last funding period we made several important discoveries regarding how Ab engages local, postsynaptic LTD-associated signaling pathways to impair LTP. Central to the current proposal, we observed that either acute Ab application or prolonged Ab exposure in the 5xFAD mouse model of AD leads to accumulation of Ca2+-permeable AMPARs (CP-AMPARs) at hippocampal synapses. CP-AMPARs are normally excluded from synapses but can be trafficked to synapses downstream of AKAP-anchored PKA signaling to modify synaptic strength during plasticity in the healthy brain. However, in many cases, the incorporation of CP-AMPARs biases subsequent synaptic signaling toward LTD, consistent with Ab-triggered synapse depression and elimination. Thus, a new central premise established by the research previously funded by this multi-PI R01 is that Ca2+ influx through CP-AMPARs plays a previously overlooked, but key role in mediating Aβ synaptotoxicity. Additional data indicate that Ab may engage AKAP-PKA signaling via G-protein- coupled b2-adrenergic (b2AR) and/or group I mGluR receptors. During the next funding period we will test the overall hypothesis that b2AR/mGluR-AKAP-PKA signaling regulates synaptic recruitment of CP-AMPARs to mediate not only acute impacts of Ab associated with LTP inhibition but also chronic impacts leading to dendritic spine/excitatory synapse loss, long-term synaptic dysfunction, and cognitive impairments in vivo in 5xFAD mice.

NIH Research Projects · FY 2025 · 2018-09

Project Summary/Abstract In Type 1 Diabetes (T1D) autoreactive CD4 T cells mediate the destruction of insulin producing beta-cells. The reasons for this misguided attack are poorly understood. Post-translational protein modifications could provide plausible explanations for the existence of autoreactive T cells in T1D. Hybrid insulin peptides (HIPs) are a form of post translationally modified antigens that form in beta-cells through the covalent ligation between proinsulin fragments and other beta-cell peptides. The resulting HIPs contain non-genomic amino acid sequences making them plausible targets for pathogenic T cells in T1D. Various HIP-reactive CD4 T cell clones were shown to trigger diabetes in non-obese diabetic (NOD) mice, a major animal model for the study of T1D. In addition, HIP-reactive CD4 T cells were identified in residual pancreatic islets of T1D organ donors. Significantly elevated levels of HIP-reactive T cells were also be detected in the peripheral blood of recent-onset T1D patients, but not in non-diabetic control subjects. Importantly, we applied mass spectrometric analyses on islets and confidently validated the presence of several HIPs that participate in T1D pathogenies. Autophagy is a cellular mechanism that removes unnecessary or dysfunctional components of the cell through lysosomal degradation. Our data indicate that autophagy plays a critical role on HIP-content in islets. Here we propose to modulate autophagy in human islets through various mechanisms, including cytokine treatment, with the objective to increase cellular HIP content. This will facilitate our identification efforts of novel HIP through mass spectrometric analyses on islets. We will also study the role of metabolic stress that may lead to the accumulation of a new sub-group of HIPs in beta-cells that could provide a trigger for T1D. Furthermore, we will study the mechanism of HIP-formation in human islets. Understanding this mechanism may provide us with new therapeutic targets that could be used to block HIP-formation in beta-cells and remove epitopes for disease- driving T cells in T1D. Additionally, we will use ELISPOT analyses to establish the role of identified HIPs as T cell epitopes in T1D pathogenesis. Such HIP-reactive T cells could serve as T1D biomarkers and allow us to improve current disease-prediction models. Lastly, we will advance and benchmark an innovative computer algorithm for the mass spectrometric identification of hybrid and native peptides. This algorithm may allow us to identify other types of hybrid peptides that cannot be discovered through conventional methodologies. In summary, success in this project will deliver new technologies, biomarkers, and therapeutic targets for the study and prevention of T1D. Identification of novel HIPs will also deliver targets for the induction of antigen specific tolerance induction in T1D, which may be required for the reversal of T1D.

NIH Research Projects · FY 2025 · 2018-09

PROJECT SUMMARY Bacteriophages (phages) are gaining traction as antibacterial therapeutics largely due to several high profile uses as emergency-approved experimental biologics. A perceived problem with phage therapy is that the development of bacterial phage resistance will curtail the use of phages as clinically relevant therapies. This conclusion overlooks a fundamental flaw in the development of phage resistance, that is, bacteria often incur reduced fitness as a result of acquired phage resistance. These fitness tradeoffs include enhanced antibiotic susceptibility, reduced virulence, and the inability to stably colonize their host. The work described in this proposal will capitalize on the emergence of phage resistance as a means to successfully treat recalcitrant opportunistic pathogens that reside in the intestine. We will use the Gram-positive intestinal commensals and opportunistic pathogens Enterococcus faecalis and Enterococcus faecium as model organisms to determine how phage resistance phenotypes influence their fitness in the intestine and whether these outcomes can be leveraged as novel therapeutic approaches. Specifically, we will define the mechanisms that drive phage resistance fitness defects of E. faecalis and E. faecium within the intestine by executing three specific aims: 1) to define the mechanism(s) driving the intestinal colonization deficiency of phage resistant strains harboring cell surface exopolysaccharide mutations; 2) to examine how phage-mediated mutations in the peptidoglycan hydrolase gene sagA result in antibiotic sensitivity; and 3) to determine if phage-antibiotic combinations reduce enterococcal intestinal colonization.

NIH Research Projects · FY 2025 · 2018-08

SUMMARY Different regulatory pathways work together to coordinate gene expression, and our guiding philosophy has been that exploring the interface of different types of regulation is thus a good way to discover new and important biology. Building on discoveries from our previous MIRA award, we propose to focus on understanding how different strategies for post-transcriptional regulation—mRNA decay, translation, and protein decay—act together to control gene expression. In the first of our two themes, we will explore the idea that protein decay acts as a meta-regulator of the post-transcriptional landscape of the early embryo. Using Drosophila melanogaster as a model system, we will investigate one of the earliest steps of development, the maternal-to-zygotic transition. During this process, maternal gene products are replaced with zygotic ones, and we have discovered that removal of three maternal RNA binding proteins is critical and determined how their destruction is developmentally controlled. We propose to extend this line of investigation to other RNA binding proteins that are destroyed by distinct and unknown mechanisms in the early embryo. We want to answer three critical questions: what are the mechanisms by which these other RNA binding proteins are destroyed? How is their degradation developmentally controlled? How does their degradation in turn change post- transcription regulation? Our research is significant because our results will reveal how destruction of maternal proteins shapes the regulatory landscape of the early embryo, and they will likely provide a conceptual framework applicable to other types of developmental transitions. In the second theme, we will explore how the open reading regulates mRNA decay and translation. Poor (or “nonoptimal”) codons lead to reduced protein output, and understanding the underlying mechanisms remains an open area of research for the field. We have excitingly found that nonoptimal codon usage represses translation initiation in human cells. This pathway is at least as potent as previously described pathways like mRNA decay. Our proposed program will build upon these results at a mechanistic level and will answer the following three important questions: What is the role of the poly(A) in translational repression due to nonoptimal codons? What factors mediate translational repression? How does codon-mediated regulation change during early development? This last question represents a new direction for our lab and leverages our unique combination of skills in the Drosophila MZT and codon-mediated regulation. Our research will reveal the molecular basis for a major repressive pathway mediated by nonoptimal codons. The systems we establish have the potential to reveal other types of developmentally-coordinated translational control, and we anticipate that our research will provide a launching point to explore the how gene regulation changes in biological space and time.

NIH Research Projects · FY 2025 · 2018-08

PROJECT SUMMARY/ABSTRACT The goal of my laboratory is to understand the role of extrachromosomal DNAs (ecDNA) in development and stress response and how these processes are dysregulated during disease pathogenesis. EcDNA are linear or circular fragments of DNA separate from the cells normal complement of chromosomes and have been identified in multiple species and healthy tissues. ecDNA are elevated in cancer where they frequently harbor oncogenes and can contribute to drug resistance and therefore may be important potential therapeutic targets. However, we currently lack a fundamental understanding of the biogenesis, regulation, maintenance and selection of ecDNA. My laboratory seeks to answer these fundamental questions surrounding ecDNA to facilitate understanding the role of ecDNA in normal biological processes and their dysregulation in disease. We have uncovered that multiple environmental stress responses cause cells to use targeted DNA rereplication events to produce ecDNA. This MIRA proposal aims to understand which stress responses utilize ecDNA production, identify the epigenetic regulators of these loci and develop methods to assess the importance of the extra gene copies independent from the chromosomal locus. This work will provide fundamental insights into a relatively unexplored pathway in environmental stress response that is quite commonly dysregulated in cancer. The work in this MIRA represents roughly 50% of the work in the laboratory. Our other research projects synergize with this work as they investigate the role of ecDNA in two disease contexts, breast cancer and Down syndrome (DS). The work in breast cancer leverages newly discovered ecDNAs produced during epithelial to mesenchymal transition as a potential novel therapeutic target to help reduce metastatic breast cancer. Our work in DS investigates how cells with trisomy of chromosome 21 (T21; the genetic root of DS) fail to produce ecDNA from stresses associated with comorbidities for individuals with DS. In the next 5 years, the combined work from these projects will synergize to provide greater understanding on the generation of ecDNA and their importance both in normal stress response and dysregulation in disease. We will uncover novel methods to prevent ecDNA formation allowing us to assess the importance of the extra gene copies independent of the endogenous chromosomal locus. We will establish a better fundamental understanding of how epigenetics establish permissive chromatin environments for ecDNA production and have identified specific enzymes that could be leveraged in the future as potential therapeutic targets. We will identify signaling and regulatory pathways that control ecDNA connecting stress sensing to ecDNA production.

NIH Research Projects · FY 2025 · 2018-08

Project Summary The human genome exists in the cell nucleus as chromatin, a complex of the DNA with histone proteins. Though genetic information is encoded in the DNA sequence, another layer of information, is encoded in the histone proteins, specifically in the form of post-translational modifications (PTMs). This layer of information is often referred to as epigenomics, and provides instructions on how the genome is to be regulated. Chromatin and the epigenomic content, is highly dynamic, constantly restructuring in response to developmental and environmental cues. One of the most important questions in biology is how this information is interpreted by transcriptional and other regulatory complexes, leading to gene regulation and cell fate. Histone modifications are “read” through small subdomains within the regulatory complexes called reader domains, and specificity for a unique epigenomic pattern is thought to be achieved through the integrated activity of multiples of these reader domains. However, though much is known about the association of reader domains with fragments of histones, the molecular mechanims underlying how they associate with histones in a chromatin relevent context, or how they function together to readout a specific epigenomic state, are not well understood. This research program addresses this fundamental question in chromatin regulation. We are pioneering the use of NMR spectroscopy to study the association of reader domains with the basic unit of chromatin, the nucleosome. We are combining this with fluorescence microscopy, cryo-electron microscopy, and basic biochemistry for an overall multidisciplinary approach to building models of these complexes. Over the next five years we will focus on how the conformation of the nucleosome regulates readout of epigenomic signatures. We will continue our investigation of the nucleosome conformation itself and how known cancer mutations dysregulate this. In addition, we will determine the kinetic and structural basis of association of reader domains from the PBAF chromatin remodeling and PRC1 histone modifying complexes with nucleosomes. Through collaborative studies we will investigate the functional consequence of these interactions. We will continue to build towards our long-term goal of understand of how multiple reader domains integrate to allow regulatory complexes to navigate and respond to a dynamic chromatin substrate. Results from this research program will reveal fundamental mechanisms of chromatin regulation, provide insight into the etiology of a number of human diseases, and lay the groundwork for the development of targeted therapeutics.

NIH Research Projects · FY 2026 · 2018-06

Project Summary Hair cells are auditory mechanoreceptors that use an apically located hair bundle to convert mechanical vibrations due to sound into electrical activity, a process termed mechanotransduction. The mechanotransduction process is essential to cochlear amplification, which is responsible for our excellent sound level sensitivity, large dynamic range, and amazing frequency discrimination. Loss of mechanotransduction leads to loss of cochlear amplification. Control of the range of sensitivity of the mechanotransduction process is hypothesized to be a key contributor to augmenting the dynamic range. In this proposal, we will investigate two mechanisms that can control the range of stimuli to which the hair cell is responsive. Mechanotransduction adaptation can adjust the range of sensitivity in the presence of an ongoing stimulus. One type of adaptation described decades ago is termed slow adaptation due to its kinetics. Slow adaptation was hypothesized to function via the motor model of adaptation. We recently overturned the motor model of slow adaptation, and we now propose a new model of slow adaptation requiring the phosphoinositide PIP2. In this proposal, we build upon our existing data by studying specific mutations in TMC1, the putative mechanotransduction channel, that we hypothesize to have a role in mediating PIP2 binding to modulate channel function. These experiments will provide mechanistic insight for slow adaptation. The second mechanism of sensitivity control is through cAMP. We recently discovered that cAMP functions to reduce gating spring stiffness, thereby controlling the sensitivity of the mechanotransduction channel. In this proposal, we will study the upstream and downstream signaling pathways using knockout mouse models to identify the molecular contributors to the pathway. These models will allow us to also identify the physiological role of the cAMP pathway in the cochlea. These experiments will lead us one step closer to understanding the role of cAMP in normal hearing function and identifying the molecular component(s) of the gating spring, which is the final downstream target.

NIH Research Projects · FY 2024 · 2018-06

The overall mission of this research program is to determine how the antioxidant enzyme, extracellular superoxide dismutase (EC-SOD or SOD3) regulates redox-sensitive signaling pathways responsible for inflammation and fibrosis in pulmonary vascular diseases across the age span, and harness this knowledge to design new and precise therapies. The different research projects are based on three complementary themes. Theme 1 interrogates the regulation of SOD3 expression, activity and distribution in the healthy and diseased pulmonary circulation in the mature and immature lung. These studies would include in vitro, and in vivo studies using animal models, as well as activity translating the work through new human studies. They will address the multiple levels of SOD3 regulation, including genetic polymorphisms, epigenetic regulation, or other post-translation SOD3 modifications, that can influence gene expression, enzyme activity, half-life and localization. Theme 2 evaluates how changes in SOD3 activity or binding properties impact redox sensitive signaling pathways that are responsible for the development of pulmonary vascular disease, in particular, inflammation and subsequent vascular remodeling and fibrosis. These experiments utilize a unique series of SOD3 mouse strains, including a mouse with knock-in of a known human SOD3 polymorphism, to interrogate how individual changes in SOD3 location or content can influence disease pathogenesis and severity. Based on the unique extracellular localization of SOD3, studies will test the effects of insufficient SOD3 on matrix integrity, matrix-cell interactions, cell-cell interactions and communication between extracellular signals and intracellular cellular responses. Ongoing studies are testing how the loss of vascular SOD3 increases the susceptibility of two key redox-sensitive targets localized to the extracellular matrix (ECM): activation of latent TGF-β, which enhances PASMC and fibroblast growth, inflammation and synthetic function, or oxidative fragmentation of hyaluronan, which binds to macrophage CD44 receptors and activates the NLRP3 inflammasome. Future planned studies will test how altered SOD3 impacts the redox landscape to modulate innate immunity, cellular metabolism and mitochondrial dysfunction responsible for vascular fibrosis in PH. Theme 3 translates the findings into new therapeutic strategies to replenish deficient SOD3 to restore redox homeostasis. This framework is supported by a new initiative, funded by a Dean's Strategic Infrastructure Research Committee Award for the purchase of an electron paramagnetic resonance spectrometer, to develop a collaborative and interdisciplinary UCD Redox Biology Shared Resource Facility to advance the study of Redox Biology. These studies collectively will provide new insight relevant to the mission of the Precision Medicine Initiative, as they will uncover how individual variables that influence SOD3 impact the development of inflammation and fibrosis in pulmonary hypertension.

NIH Research Projects · FY 2026 · 2018-06

PROJECT SUMMARY Cardiac hypertrophy and failure involve a rewiring of gene expression through alternative splicing, but it remains unclear which splicing changes are relevant to disease development, and how splice variants affect protein function. Although many alternative transcript isoforms have been discovered, not all are translated into proteins and instead may be degraded via non-sense mediated decay or co-translational protein quality control. Proteomics methods that can identify and quantitate splice variant proteins empirically and on a large scale provide essential tools to study how alternative splicing regulates cardiac gene expression. We and others showed that tissue-specific splice variant proteins may be identified using a combined RNA sequencing and mass spectrometry approach. Accordingly, our goal here is to examine the mechanisms by which alternative splicing regulates the genetic program in hypertrophic and failing hearts, by identifying the proteins and pathways that are coordinately regulated by splicing, the resulting complement of protein isoform species (`proteoforms') in the heart, and the consequences of proteoform sequences on protein structure and function. Specifically, we plan to: (1) apply a quantitative RNA-guided proteomics framework to identify key isoform switches at the transcript and the protein level, with emphasis on the changes in splice factors and RNA-binding proteins in mouse models of systolic and diastolic dysfunction; (2) combine RNA-guided proteomics and proteome-wide biophysics approaches, we will interrogate the impact of splice variants on protein structure and thermal stability, and discover significant isoforms through alternative splicing, intrinsically disordered, and regulatory post-translational modification modules. We anticipate the successful completion of these aims will generate new conceptual insights into how alternative splicing regulatory networks reprogram the cardiac proteome in pathological remodeling and heart failure, and more generally, contribute to methods and concepts to elucidate the regulation and function of alternative splicing in the heart.

NIH Research Projects · FY 2026 · 2018-05

PROJECT SUMMARY A key step in the assembly of the large double-stranded DNA (dsDNA) viruses is packaging of a genome into a pre-assembled procapsid by an ATP-driven motor complex. In the herpesviruses and many bacteriophages, packaging is catalyzed by a terminase enzyme that utilizes a concatemeric genome substrate. To accomplish this, terminase enzymes assemble into distinct initiation, motor and termination complexes to processively excise an individual genome from the concatemer, and concomitantly package it into the capsid. This requires that the enzymes cycle between stable nuclease and dynamic motor intermediates. While our understanding of packaging initiation and motor translocation is extensive, termination of genome packaging remains ill-studied and poorly characterized in all viruses, primarily because defined experimental systems have not been developed. Phage  is an exception wherein rigorous biochemical assays allow molecular dissection of the entire assembly pathway. This multi-PI application proposes to use phage  to interrogate termination, the final and most poorly characterized step in the packaging pathway. Two fundamental questions central to genome packaging are addressed; (i) how does the translocating motor recognize the genome end while also sensing that a sufficient length of DNA has been packaged, transition to a site-specifically bound nuclease complex, and (ii) how do “finishing proteins” promote end maturation and terminase ejection from the nucleocapsid without loss of the tightly packaged DNA. We describe highly integrated and synergistic biochemical, biophysical, single-molecule and structural approaches to characterize this conserved and essential, yet largely unstudied step in virus assembly. Given that this process is strongly conserved in all of the dsDNA viruses, both prokaryotic and eukaryotic, and the commonality of initiation-translocation-termination pathways in biology, the results will have broad implications in virology and cell biology.

NIH Research Projects · FY 2025 · 2018-02

Project Summary One of the most fundamental challenges in cell biology is understanding how cells migrate in three- dimensional extracellular matrices (ECM) at specific times and to specific locations. Cell migration helps shape all tissues and organs during development and the disruption of the normal mechanisms that normally control migration dramatically enhance the lethality of cancers. Our recent studies identified members of Rab40 sub- family as important regulators of cell migration. Rab proteins are the largest family of small monomeric GTPases belonging to the Ras oncogene superfamily. Among them, Rab40 sub-family is unique because it contains a suppressor of cytokine signaling (SOCS) box located at the C-terminal end of the protein. Importantly, we and others have shown that Rab40 sub-family of proteins bind to Cullin5 via the SOCS box to form a ubiquitin E3 ligase complex that regulate various aspects of cell migration in vitro and in vivo. Consequently, Rab40 proteins emerged as important coordinators between membrane trafficking, cytoskeleton dynamics and cell signaling, and understanding the molecular machinery governing functions of Rab40 sub- family during cell migration is a major focus of this proposal. In all vertebrates Rab40 family consists of two members, Rab40b and Rab40c. Our recent work has shown that Rab40b and Rab40c are important for targeted secretion of matrix metalloproteinases, as well as regulation of actin dynamics during breast cancer migration, invasion and metastasis. We also have shown that Rab40b/Cullin5 complex mediate ubiquitylation of several regulators of cell migration, including Rap2, while Rab40c/Cullin5 mediates protein phosphatase 6 (PP6) complex ubiquitylation and inactivation. Based on our published and preliminary data, we hypothesize that Rab40b and Rab40c mediate coordination between signaling, membrane trafficking, and actin dynamics during cell migration. The following aims are designed to test this hypothesis by combining of the unique expertise from Dr. Rytis Prekeris (Rab GTPases and actin dynamics), Dr. Kristin Artinger (zebrafish and neural crest cell migration), and Dr. Traci Lyons (breast cancer). First, we will define the roles of Rab40b/Cullin5 and Rap2 complexes in regulating membrane and actin dynamics during cell migration by mapping Rab40b-dependent Rap2 ubiquitylation sites and dissecting how Rap2 regulates actin and focal adhesion site dynamics at the leading edge and/or invadopodia. Second, we will elucidate the roles of Rab40c/Cullin5-PP6 complex in regulating signaling during cell migration. To that end, we will use protein binding assays in combination with protein mutagenesis and various microscopy approaches to determine biochemical properties of PP6 binding to Rab40c and the consequences of this binding on PP6 complex stability and activity. Third, we will determine the functions of Rab40b/Cullin5-Rap2 and Rab40c/Cullin5-PP6 pathways during neural crest cell migration in vivo using zebrafish since zebrafish model allows direct and real- time in vivo analysis of cell migration.

NIH Research Projects · FY 2026 · 2017-09

ABSTRACT Background: Sex Chromosome Trisomies (SCT) including Klinefelter (XXY), XYY syndrome, and Trisomy X (XXX), occur in 1 out of every 500 births. In childhood there are increased risks for language and learning disabilities, ADHD, autism, and emotional disorders. Medically, SCTs are associated with testicular failure in XXY, ovarian failure in XXX, and all have increased morbidity and mortality due to high risks for insulin resistance, seizures, and other health conditions. Prenatal SCT diagnosis has dras- tically increased over the past decade in the US with more widespread noninvasive prenatal screening (NIPS). The eXtraordi- narY Babies Study was launched in 2017, and has enrolled the largest and most diverse prenatally diagnosed SCT cohort to date, including 271 infants followed prospectively from 2 months to 3-4 years of age with detailed medical, hormonal, and developmental phenotyping coupled with a longitudinal biobank including over 1250 biospecimens. Results have identified medical features not previously described in SCT, detailed acquisition of developmental milestones, and identified differ- ences in early speech and behavior profiles known to be ‘red flags’ of later diagnoses such as autism, dyslexia, and ADHD. Parents shared experiences highlighting the need for improved genetic counseling models. Follow-up of participants into the school-age years is critical as important comorbidities such as reading disabilities, ADHD, autism and endocrine dysfunction being to emerge, phenotypic variability broadens, and developmental and health outcomes become more predictive of later functioning. In this renewal project we aim to: (1) Describe and compare the natural history of neurodevelopment, medical problems and hormonal profiles of SCT through prospective study of the eX- traordinarY Babies cohort into early school age, (2) To identify of poor developmental and health outcomes in SCT, with special attention to modifiable factors of development, health and environment to guide future intervention trials, and (3) To develop an evidence-based, parent-informed best practice model for prenatal genetic counseling unique to the needs of the SCT population. Approach: Current study participants (n=262; XXY=174, XYY=25, XXX=54, XXYY/XXXY=9) and 60 newly recruited children will complete annual assessments up to 7-8 years of age. New recruitment will target those from underrepresented racial and ethnic groups, low socioeconomic status, rural locations, and XYY and XXX. Demographics, health and family history, and education/interventions will be collected, along with assessments of: (1) cognitive, psychological and motor functioning; (2) physical and gonadal measures and (3) quality of life. Statistical models will contrast longitudinal profiles for each SCT group and compare to population norms. Linear models and logistic regression will be used to test the association between poten- tial early risk factors and selected outcomes at age 7. Biological samples will be added to the biorepository. Parent experi- ences with the prenatal SCT diagnosis will be analyzed via a mixed method approach to develop evidence-based genetic counseling resources. Impact: Longitudinal study of the largest cohort of prenatally identified children with SCT provides a novel resource that will inform the natural history of developmental and medical profiles in SCTs, guide genetic counseling, identify targets for intervention trials, inform newborn screening, and provide an invaluable data and biospecimen repository for future research.

NIH Research Projects · FY 2026 · 2017-09

PROJECT ABSTRACT Recreational cannabis legalization (RCL) is swiftly expanding in the United States. Ideally, such policy shifts are informed by evidence; however, little is known about the effects of RCL. This is particularly true for long- term effects. Data from states that were the first to legalize cannabis can help understand RCL’s long-term effects. With public opinion indicating that further RCL expansion is likely, it is imperative to clearly understand its public health consequences. Our original grant suggested that adults living in RCL states used cannabis 20% more frequently (than those in non-RCL states) but had virtually no other psychosocial consequences. However, increasing evidence suggests that cannabis use is most consequential when it is frequent and persistent. In the context of this literature, it is worth noting that our original grant assessed behavior within approximately five years of legalization. It is unclear whether continued access to cannabis in RCL environments increases persistent use, leading to negative consequences. We propose a renewal grant to examine the long-term impact of RCL in approximately 4,500 participants from two large, prospective twin cohorts in Colorado (CO; full RCL since 2014) and Minnesota (MN; RCL passed in 2023, dispensaries expected to open in 2024). Both CO and MN participants have been assessed at least three times before RCL began in CO (adolescence, emerging adulthood, young adulthood) and twice since RCL began in CO. The proposed project would collect the third post-RCL assessment in CO and the first since RCL began in MN. We will leverage this study design to control important familial confounds related to cannabis use and its potential consequences. Further, we will incorporate the longitudinal nature of these cohorts to control prior behavior (e.g., cannabis use, mental health) while examining the effects of RCL. Thus, we will use these data to address whether years of RCL access affect cannabis use, alcohol use, mental health, and physical health (Aim 1). We hypothesize that having more years of RCL access is associated with greater cannabis use and consequences. Further, we will determine whether pre-RCL mental and physical health problems predict increased post-RCL cannabis use (Aim 2). This aim is based on evidence, including our preliminary data, that suggest health problems are a common motive for cannabis use. Thus, we hypothesize that pre-RCL health problems are associated with greater post-RCL cannabis use. Finally, we will identify individual differences uniquely related to post-RCL cannabis use and consequences (Aim 3). We hypothesize that post-RCL cannabis and its consequences are associated with greater perceived cannabis benefits, greater ease of access, and personality factors that may have impeded cannabis use in non-legal contexts (higher harm avoidance, lower rule-breaking). In summary, this study will equip key stakeholders with well-controlled, rich evidence on the consequences of RCL.

NIH Research Projects · FY 2025 · 2017-09

Pain-sensing sensory neurons of the dorsal root ganglion (DRG) and dorsal horn (DH) can become sensitized (hyperexcitable) in response to surgically induced peripheral tissue injury. Because of insufficient knowledge about the mechanisms for this sensitization, current treatment for postoperative pain has been limited to somewhat non-specific systemic drugs (opioids) having significant side effects or potential for abuse. The important role of voltage-gated calcium channels (VGCCs) in pain processing has been recognized for a while since calcium (Ca2+) is the major trigger for the release of synaptic vesicles from neuronal presynaptic terminals in response to noxious stimulation. An increase of intracellular Ca2+ in pain sensing neurons (nociceptors) can also influence the excitability of these cells. We established that CaV3.2 (T-type) calcium-channels make a previously unrecognized contribution to sensitization of pain responses by enhancing excitability of peripheral nociceptors. We also showed that the blockade of CaV3.2 currents in nociceptive DRG neurons by 5-reduced neuroactive steroids (NASs) underlies their potent peripheral anti-nociceptive effects in a clinically relevant rodent model of perioperative pain model of plantar skin incision. In addition, we have shown that NASs that inhibit CaV3.2 channels are effective in alleviating mechanical hyperalgesia post-surgery when administered preemptively whereas morphine provides dose-dependent pain relief only when administered once the pain had developed. However, limited aqueous solubility and potent hypnotic/sedative effects linked to their direct or indirect (via metabolic pathways) effect on GABAA receptors may hinder future development of NASs for novel pain therapies. Hence, we will follow up on our exciting findings that inhibition of neuronal CaV3.2 in pain pathways underlies effective post-operative analgesia with novel analogues of NASs that have more favorable pharmacokinetic and pharmacodynamic properties, as well as better solubility. The Specific Aims are: Aim #1: To develop NAS CaV3.2 inhibitor analogues with diminished potential for conversion to GABAA positive allosteric modulators and test hypothesis that their analgesia is mediated via inhibition of CaV3.2 channels. Aim #2: To study analgesic potency of novel analogues of NASs and their interactions with morphine using a rodent model of postoperative incisional pain and chronic constrictive injury (CCI) of sciatic nerve. Aim #3: To define the role of novel NASs in modulating synaptic transmission and neuronal excitability of nociceptive dorsal horn (DH) neurons using optogenetics. The proposed work is innovative and medically significant because we anticipate that our studies will identify novel therapies for perioperative pain that may greatly decrease the need for narcotics and potential for drug abuse.

NIH Research Projects · FY 2024 · 2017-07

Project Summary Sarcoidosis is a systemic granulomatous disorder of unknown etiology that affects the lung in greater than 90% of cases. The disease is characterized by the accumulation of activated CD4+ T cells in the lung and other sites of disease activity. Evidence suggests that these T cells are intimately involved in the pathogenesis of sarcoidosis. In the previous version of this proposal, we identified lung CD4+ T cells from HLA-DR3-expressing Löfgren’s syndrome (LS) subjects expressing related T cell receptors (TCRs) and determined that these TCRs recognized peptides derived from NAD-dependent protein deacetylase (NDPD) expressed in a common airborne mold species, Aspergillus nidulans. Using HLA-DR3-NDPD tetramers and IFN-γ ELISPOT, we validated those findings and showed that a significantly greater number of NDPD-responsive CD4+ T cells exists in the lungs of LS subjects; thus,we have identified a potential causative agent in the genesis of LS. This study of Swedish subjects with LS serves as a “proof of concept” for the current renewal whose focus is to identify T cell epitopes for CD4+ T cells derived from the lungs of sarcoidosis subjects expressing HLA-DRB1*11:01, the HLA allele strongly linked to sarcoidosis in Caucasians and African-Americans in the US. Thus, we hypothesize that expanded CD4+ T cells in the lungs of HLA-DRB1*11:01-expressing US sarcoidosis patients are accumulating in response to etiologic sarcoidosis antigen(s) and recognize those antigens in an HLA- DRB1*11:01-restricted fashion. This proposal harnesses the strengths of a multidisciplinary research team and focuses on a sarcoidosis cohort in the US. Using a single cell RT-PCR approach, Aim 1 will characterize αβTCR pairs expressed on CD4+ T cells derived from the lungs of US sarcoidosis patients and generate hybridomas expressing disease-relevant TCRs. The second specific aim will determine the peptides that stimulate the CD4+ T cell hybridomas expressing the TCRs of interest. The final aim will use functional assays and HLA-DR11-peptide tetramers to identify and enumerate antigen-specific CD4+ T cells in the lungs of sarcoidosis patients and determine if the frequency of these T cells can serve as a biomarker for diagnosis and/or prognosis. Thus, using a novel yet proven scientific approach, we will address critical knowledge gaps in the etiologic T cell antigens involved in the pathogenesis of sarcoidosis in US patients, further advancing our understanding of this enigmatic disease.