Colorado State University

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY ABSTRACT Children with Down syndrome (DS) are disproportionately affected by the obesity epidemic, presenting a prevention challenge that is currently unmet. Despite improved life expectancies, people with DS experience poor health.10 Children and adults with DS have significantly higher rates of overweight/obesity (OW/OB) than those with intellectual disability from other causes11 or the general population.12 These risks are rooted in childhood, with rapid increases in OW/OB observed between ages 2 and 6 years in children with DS2–4 and childhood OW/OB conferring a higher risk for OW/OB in adulthood.2,3 However, research examining young children with DS+OW/OB has primarily focused on prevalence rates using retrospective clinical data, with minimal attention to modifiable prevention targets.2,11,13 Consequently, critical questions regarding how DS phenotypic factors such as co-occurring conditions, motor delays, and feeding challenges contribute to OW/OB risk remain unanswered. Despite being a specific, high-risk population for obesity, no prospective, longitudinal DS research has comprehensively characterized OW/OB predictors to guide effective prevention in early childhood when such efforts may enhance downstream health outcomes. Bridging this knowledge gap recently highlighted by prominent DS researchers,18 our team representing expertise in early development in DS, nutrition, and obesity in DS, physical activity (PA), and sleep, will use a longitudinal design to identify early OW/OB prevention targets for young children with DS. Using a staggered wave approach, we will enroll and assess 1/3 of the sample in Wave 1/Y1, with annual follow-up visits (+ 1 month) in Y2&3, using the same approach for Wave 2 (Y2-4) and Wave 3 (Y3-5), thus reducing recruitment demands over time and sites (total project n=160). At T1-3, we will collect data on (a) child factors—objectively measured PA, motor abilities (gross, fine, motor planning), sleep, and weight/height, plus caregiver-reported child dietary intake (energy, macronutrients, and dietary quality), child developmental and medical history, and feeding abilities; and (b) caregiving factors—caregiver feeding and PA practices, family dynamics, caregiver weight/height, and SES. At T1, we will recruit caregivers and their child with DS chronological ages 18-40 months, who walk 15+ feet independently. The age range ensures participant ages align with: (1) the full range of developmental variability expected in children with DS given that 95% of children with DS reach this walking milestone between 18-40 months (median = 24 months),5 and (2) observed weight gain increases between 2 to 6 years of age in DS,2–4 with 4-6 year-olds 61% more likely to have OB than those aged 2-3 years.2 The overarching project goal is to understand variability in risks for OW/OB in DS and identify prevention targets in young children with DS. Our long-term goal is to develop and deliver obesity prevention in early childhood to reduce risks for OW/OB and associated medical sequelae across the lifespan for individuals with DS.

NIH Research Projects · FY 2026 · 2022-09

Approximately one-fifth of the world’s couples encounter fertility problems posing physical and emotional health issues. While half of the infertility problems are attributed to the male, insufficient knowledge of sperm physiology prohibits proper diagnosis in roughly 50% of these cases. A key aspect of fertilization involves capacitation, a sperm maturation step within the female reproductive tract. In this step, the sperm acquires the capacity to fertilize an egg, but many aspects of capacitation are still largely unknown. Our long-term goals are to identify and characterize the mechanisms that regulate sperm capacitation. A key player in the capacitation process is the soluble adenylyl cyclase (sAC), an enzyme that efficiently synthesizes cAMP when exposed to bicarbonate and intracellular calcium. When sAC is activated, cAMP levels increase causing the sperm-specific CatSper channel to open, allowing calcium to flow from the extracellular medium into the flagellum. So, how does calcium initially reach sAC for its activation when CatSper is not yet active? Previous reports show that in CatSper-null mice, cAMP levels can increase similarly to wild-type mice, suggesting that the initial calcium influx does not occur as result of CatSper activation. Our preliminary data show that the initial calcium flow indeed takes place in a CatSper-independent manner. Once sAC is activated, it remains active during the whole capacitation process through an unknown regulation. Our preliminary data place Cdc42, a protein known to control multiple cellular functions at the heart of this sAC regulation. While Cdc42 is expressed in mammalian sperm, its role in this cell is unclear. Our central hypothesis is that sAC is differentially regulated to allow both the initial activation that triggers capacitation and maintenance of sufficient cAMP levels throughout the process. Aim 1 will identify a novel mechanism for the initial activation of sAC. Our hypothesis is that the calcium required for the activation of sAC is delivered by ion channels present in the sperm head. Aim2 will determine the role of Cdc42 on the activation of sAC during capacitation. Our hypothesis is that Cdc42 is essential for cAMP production by sAC in the vicinity of the CatSper complex. These two aims will be achieved by using super-resolution imaging to probe the localization of molecules within the flagellum, flow cytometry to assess the activity of signaling molecules at the single-cell level, and patch clamp electrophysiology to measure calcium currents. In addition, transgenic lines will enable us to abrogate CatSper channels and Cdc42 activity. The outcomes from this work will shed light on the initial activation of sAC, will identify the calcium channel responsible for the first calcium influx during capacitation, and will elucidate the mechanism by which sAC remains active in the same region of the CatSper signaling domain. We expect these fundamental capacitation studies will open new avenues to find treatments of male infertility and aid in the development of non-hormonal contraceptives.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Chromosomes are copied by a complex holoenzyme called the replisome. Obstacles are routinely negotiated by the replisome with auxiliary mechanisms, collectively called DNA damage tolerance pathways, that ensure genomic integrity via on-the-fly remodeling. The aberrance of these pathways can lead to chromosome instability and a broad range of diseases including cancer. The Schauer Lab’s long-term goal is to thus understand the molecular basis for genetic and epigenetic fidelity, with the goal of improving the treatment and/or prevention of diseases. In this proposal, the Schauer Lab will use a fully functional replisome reconstituted from over 30 pure polypeptides to study how replisomes bypass obstacles that regularly occur in the genome while enforcing genetic and epigenetic integrity across generations. They also propose to develop whole cell lysate systems to establish active replication forks on double-stranded DNA at natural origins of replication without the need for replication initiation on synthetic forks. DNA damage tolerance mechanisms will be studied using biochemistry, single-molecule biophysics, and structural biology. The structural dynamics of the S-phase damage response will be characterized, with a focus on the mediator kinase Mrc1 and the multiple ways it regulates the elongating replisome. The Schauer Lab also proposes to study the spatiotemporal mechanisms of rescue of lesion-stalled replisomes by translesion synthesis polymerases, and how both Mrc1 and ubiquitination of DNA sliding clamps regulates this response. When replicating chromatin, nucleosomes present a strong block to replication fork progression in the absence of histone chaperones. The Schauer Lab will study histone dynamics at the replication fork in reconstituted chromatin, with a focus on regulation of histone deposition symmetry by histone chaperones and in the molecular mechanisms of various replication- coupled histone chaperones themselves. Tools will be developed to track histone fate and dynamics at the single-molecule level. The goal is to get a better understanding of the processes that control epigenetic inheritance, important for maintaining cellularity during cell division. Finally, the Schauer Lab proposes to study collisions between the replication machinery and an actively elongating transcription complex, since these conflicts can be highly mutagenic. Transcriptional regulation by the rpb4/7 heterodimer will also be studied. Transcription will be reconstituted from either purified proteins, or whole-cell extracts, or a combination of the two. The Schauer Lab is developing biochemical and single-molecule tools for these projects that will afford an unprecedented glimpse into the molecular mechanisms behind these critical processes. Single-molecule fluorescence resonance energy transfer (smFRET) will be employed to track intermolecular interactions, allowing a characterization of the structural dynamics of these systems. Further, technology will be developed to track dynamic motions of fluorescently labeled proteins on double-tethered DNA at the single-molecule level. The mechanistic insight afforded by these studies will be beneficial for the medical research community.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY DNA repair by homologous recombination (HR) in tumor cells accelerates the development of resistance to chemo- and radiotherapy and leads to the recurrence of disease. Hence, inducing HR deficiency in HR-proficient tumors is a promising strategy to increase the efficacy of DNA-targeted therapies. Yet, we still do not know which stage of the HR reaction is the most sensitive to inhibition and consequently the most promising to target. However, inhibition of HR pathway intermediates during synapsis and strand invasion may be particularly effective. The long-term goal of our study is to lay the groundwork for the development of novel HR-directed anti- cancer therapeutics. The central hypothesis of our project is that human cells have evolved multiple pathways of strand invasion. The rationale for this project is that a detailed understanding of the molecular mechanisms of the multiple pathways of strand invasion is likely to offer a strong scientific framework whereby new strategies to cancer therapy can be developed. The overall objectives in this application are to (i) elucidate the molecular mechanisms of the multiple pathways of strand invasion in HR in human cells, and (ii) determine the steps in these pathways in which the HR functions of the RAD51 activators RAD51AP1, RAD54L, and RAD54B intersect. The central hypothesis will be tested by pursuing two specific aims: 1) Dissect the non-epistatic and epistatic relationships between RAD51AP1, RAD54L, and RAD54B; and 2) Determine the functional roles of the RAD51AP1-RAD54L and RAD51AP1-RAD54B protein complexes. Under the first aim, isogenic human cancer cell lines will be used to determine the phenotypic consequences of RAD51AP1, RAD54L and/or RAD54B deletion. Proven knockout strategies and assays to evaluate the effect that loss-of-function has on cytotoxicity, genome stability, replication and recombination will be employed. For the second aim, biochemical assays of strand invasion utilizing nucleosome-free and nucleosome-containing DNA substrates will be carried out, and mutants defective in protein complex formation will be tested for complementation in cell survival assays. The research proposed in this application is innovative in the applicant’s opinion, because it focuses on unraveling the poorly understood interplay between the multiple pathways of strand invasion that exist in human cells, the intra-pathway synthetic interaction between RAD51AP1 and RAD54L, and the role of human RAD54B in HR. The proposed research is significant because it is expected to provide strong scientific justification for the continued development of inhibitors that target HR stimulators of strand invasion. The knowledge gained herein also has the potential of offering new opportunities for the development of novel cancer therapies.

NIH Research Projects · FY 2025 · 2022-09

Project Summary The long-term goal of the proposed research is to determine the central mechanisms by which undernutrition and realimentation impact reproduction through regulation of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, using male and female sheep as an animal model. Puberty onset integrates various internal and external cues resulting in an increased release of GnRH from the hypothalamus that imparts the capacity for sexual maturation and reproductive success. Inadequate energy intake (undernutrition) has a significant negative impact on GnRH, and subsequently LH secretion, thereby delaying puberty onset. However, the central mechanisms responsible for the suppression of GnRH/LH secretion during undernutrition or the increase of GnRH/LH secretion following re-feeding (realimentation) remain largely unknown. Thus, the objectives of this proposal are 1) to determine the role that AgRP signaling plays in regulating GnRH and kisspeptin neurons during undernutrition and realimentation, and 2) to determine the role that microglia play in regulating GnRH and kisspeptin neurons during undernutrition and realimentation. In Aim 1, we will characterize changes in AgRP signaling in GnRH and kisspeptin neurons in feed-restricted (FR) sheep, examine the in vivo effect of AgRP immunoneutralization in the arcuate nucleus (ARC) of the hypothalamus on LH secretion in FR sheep, and characterize changes in AgRP signaling in GnRH and kisspeptin neurons in refed sheep. In Aim 2, we will characterize changes central immune signaling in GnRH and kisspeptin neurons in FR sheep, examine the in vivo effect of central infusion of an interleukin-1 receptor antagonist on LH secretion in FR sheep, characterize changes in central immune signaling in GnRH and kisspeptin neurons in refed sheep, and examine the in vitro effect of low glucose and insulin on microglia phenotype and function. Herein, with our expertise in whole-animal physiology, in vivo drug delivery, immunohistochemistry, and in vitro cell culture we have designed experiments to apply the highly innovative technique of a fluorescent in situ hybridization assay, RNAscope, for detection of signaling components for AgRP (Aim 1) and interleukin-1β (Aim 2). The proposed experiments will not only address the neuronal networks by which changes in metabolic state impact reproduction, but also provide important insight into the role the immune system likely plays. Thus, this work will provide a greater understanding of how undernutrition impacts central networks that regulate GnRH/LH secretion and yield novel and critical insight that may allow for better control of the timing of puberty, and ultimately lead to improved human health through prevention of disorders (e.g. cardiovascular disease and depression) associated with delayed puberty.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY Over 100,000 newborns receive mechanical ventilation through an endotracheal tube (ETT) each year in the United States. Intubating newborns is challenging due to their size and delicate nature, and unfortunately, nearly 40% of the initial intubation attempts are incorrect, and the tube is inadvertently placed in the esophagus instead of the trachea, or too deep in the main stem bronchus, leading to ventilation of only one lung, or with the tip of the tube too high in the trachea. It is critical to detect malpositioning of the tube promptly. The goal of this research is to develop Simultaneous Multi-Source Electrical Impedance Tomography (SMS-EIT) technology for the bedside to correctly and instantly identify ETT position or malposition. In this application we will combine (1) deep learning EIT-based confirmation of ETT placement with (2) EIT images of lungs being ventilated. Together, this would provide clinicians and bedside staff with a real- time, closed-loop system for determining if (1) the ETT was inserted in the correct lumen (trachea, not esophagus) and (2) if the lungs are being ventilated appropriately to detect left or right mainstem bronchial malplacement. The same system with no change in electrode placement could be used to monitor for inadvertent extubation and for the onset of emergency conditions such as pneumothorax. EIT is a noninvasive, non-ionizing functional imaging technique in which images are formed from voltages measured on electrodes on the body arising from imperceptible applied currents. Since EIT is a safe and portable technology with no damaging side effects, it can be used both for continuous monitoring and as needed. Our interdisciplinary team from GE Research, Colorado State University, and Stanford University will develop and validate the specialized SMS-EIT system through three specific aims. The first aim is to develop and implement an electrode configuration, reconstruction algorithms, and hardware modifications of the GE SMS-EIT system for the special needs of neonates and this project. In the second aim, training data and a deep learning classification algorithm to classify intubation as correct, esophageal, too high, or mainstem bronchial misplacement will be developed. The efficacy and clinical feasibility of the SMS-EIT system and algorithms for the real-time detection and classification of ETT malplacement will be evaluated in a study of 30 infants in the Level IV NICU at Stanford University Medical Center.

NIH Research Projects · FY 2024 · 2022-07

Project Summary Inflammatory and infectious diseases are among the leading causes of death worldwide. Coordination and regulation of immune defenses are essential to combating infection and preventing aberrant inflammatory responses. This is especially important within the gastrointestinal tract where potential pathogens must be differentiated from commensal microbes. Deleterious alterations in our gut microbial community (dysbiosis) are associated with numerous diseases. At the forefront of host-microbe interactions are intestinal cells which act as a critical barrier and interface with our luminal environment. Intestinal cells possess a repertoire of innate immune receptors and defense mechanisms, allowing them to directly respond to and modulate gut bacteria. Thus, they are thought to play a critical role in establishing and maintaining the beneficial symbiotic relationships we develop with our gut microbiota. Remarkably, several fundamental processes underlying these host-microbe interactions remain poorly understood, including how intestinal cells differentiate between commensals and pathogens or which intestinal cell responses promote commensal selection in the gut. The long-term objective of this proposal is to determine how intestinal cells directly contribute to the selection and maintenance of commensal gut bacteria. The proposed research approaches this objective by leveraging the tractability and simplicity of the model organism C. elegans to provide the fine-scale spatial resolution needed to dissect the intricacies underlying host-microbe interactions. C. elegans is a model of intestinal development and innate immunity that also harbors a diverse gut microbiome in natura, making C. elegans an excellent system for studying microbial- induced innate immune responses. Aim 1 will determine how bacteria- and community-specific immune responses are organized in the intestine by evaluating the transcriptional response of intestinal cells to commensal and pathogenic gut bacteria using single-cell RNA-sequencing (scRNA-seq). This will reveal unique innate immune signatures among intestinal cells throughout the gut based on bacterial exposure. Preliminary evidence from our embryo scRNA-seq dataset highlight several spatially distinct immune genes including the C- type lectins (CLECs), which have a well-defined role in bacterial recognition in mammals. Aim 2 will determine how intestinal cells regulate commensal colonization in the gut by testing the role of innate immune receptors, specifically CLECs, in selecting for and maintaining commensal bacteria. Using a combination of molecular biology and microscopy techniques, aim 2 will elucidate the cohorts of CLECs required to recognize and respond to commensal gut bacteria. Results from this proposed investigation will clarify how innate immunity is regulated on a transcriptional level throughout the intestine and how it contributes to the selection and maintenance of host-microbe relationships in the gut. By combining the tractability of C. elegans with innovative mechanistic analysis we will move the C. elegans research community forward and, importantly, reveal conserved pathways enabling the creation of generalizable concepts not limited solely to C. elegans.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY/ABSTRACT Mechanical circulatory support (MCS) is a critical tool to treat heart or lung failure, in the form of extracorporeal circulation through membrane oxygenation or through a ventricular assist device. Thrombosis and bleeding remain major complications with MCS. As a result, patients receive systemic anticoagulation to prevent thrombosis. However, this can increase the risk for bleeding, which is the most common complication in MCS. To counter this issue, there has been a large effort to eliminate or minimize the need for anticoagulation. Surprisingly, even if anticoagulation is eliminated, studies demonstrate that bleeding remains highly prevalent, while thrombosis remains relatively unaffected. Therefore, there is a need to focus on alternative pathways to bleeding. Almost all patients on MCS experience the bleeding disorder acquired von Willebrand syndrome. Furthermore, patients, especially pediatric patients, experience platelet dysfunction and can exhibit low platelet counts. We attribute these events to the flow environment in MCS. While many groups have focused on the effect of shear stress on blood, our group discovered an unprecedented role for turbulence in driving loss of high and even intermediate molecular weight von Willebrand factor (VWF) multimers, reducing the ability for VWF to bind to platelets and to collagen. Furthermore, there is strong evidence that flow in MCS is causing signals for platelet activation, but also clearance and cell death, with an unknown effect of turbulence. The combination of signals in response to flow can lead to both thrombosis and hemorrhage, depending on the balance of events. Our goal is identify what specific conditions lead to VWF or platelet functional loss in response to flow by pursuing three aims. 1) We will quantify changes in thrombus growth in response to turbulence relative to laminar shear conditions for various anticoagulants. 2) We will quantify the increased cleavage occurring in turbulence relative to laminar flow for similar shear stress conditions and how VWF function varies after flow exposure with and without flow-induced extension. 3) We will assess platelet state after exposure to different flow regimes and how this changes with the presence of VWF or with potential new therapeutic targets. Altogether, this work will distinguish the impact of turbulence relative to shear stress on blood, which could lead to improved design criteria for blood-contacting medical devices and potential therapeutics if we identify specific pathways leading to dysfunctional hemostasis.

NIH Research Projects · FY 2025 · 2022-06

Abstract The prevalence of pulmonary nontuberculous mycobacterial (NTM) infections caused by Mycobacterium abscessus complex (MABSC) and Mycobacterium avium complex (MAC) species is increasing worldwide and poses a particular threat to susceptible individuals with structural or functional lung conditions such as cystic fibrosis, chronic obstructive pulmonary disease and bronchiectasis. The intrinsic recalcitrance of these pathogens to chemotherapeutic treatments and alarming treatment failure rates place a high priority on the development of more effective treatment approaches. The ability of MABSC and MAC to persist intracellularly and extracellularly within granulomatous lesions in a non-replicating state is likely to contribute to the drug tolerance of these microorganisms and to treatment failure in chronically-infected individuals. Further compounding this problem is the ability of MABSC and MAC to form what appears to be genetically programmed biofilms during human pulmonary infections. A common stress faced by intra- and extracellular NTM inside activated immune cells, in avascular necrotic and caseous regions of granulomas, and within microaggregates or biofilms is the inhibition of aerobic respiration caused by O2 depletion or exposure to nitric oxide (NO) and carbon monoxide. M. tuberculosis (Mtb) is known to survive this stress by inducing a regulon of ~50 genes that drives the entry of the bacterium in a non-replicating state while adapting its metabolism to maintain energy levels and a redox balance compatible with survival in the absence of respiration. Accordingly, inhibitors of the regulator which controls the expression of this regulon are actively being sought for their potential to shorten tuberculosis treatment and lower relapse rates when used in combination with standard-of-care antibiotics. Our recent studies indicate that the orthologous regulators of MABSC and MAC play a similar function as in Mtb. Genetic and pharmacological disruption of this regulator in MABSC led to inhibition of biofilm formation in addition to decreasing bacterial viability and reversing drug tolerance under hypoxia. Most importantly, two inhibitors of this regulator in MABSC which we identified showed significant bactericidal activity in MABSC-infected mice in addition to potentiating the activity of standard-of-care antibiotics used in combination. Since these two inhibitors are either clinically-used or in phase II clinical trial, they offer repurposing opportunities that could be a short route to the clinic. These exciting findings stimulated the submission of this grant application in which we propose to thoroughly decipher the mechanisms underlying the therapeutic and adjunct therapeutic benefits of these inhibitors in MABSC (Aim 1) and to determine whether the same therapeutic strategy may be applied to MAC (Aim 2).

NIH Research Projects · FY 2026 · 2022-06

MARC at Colorado State Univeristy The training mission of the MARC at Colorado State University is to identify promising underrepresented (UR) biomedical science students and provide them with rigorous, evidence- based courses, mentored research laboratory experiences, synergistic social networks, stable financial support and structured individualized training activities to develop a cohort of highly qualified trainees who will matriculate into advanced biomedical sciences degree programs. To achieve this, we will conduct strategic outreach and engagement to recruit students who represent the diversity of our national population. Individualized academic success plans will ensure academic preparedness to attain 90% retention and graduation rates of MARC students. We will reach this goal by creating cohorts with a sense of empowered community in a supportive training environment. All MARC students will engage in hypothesis-driven laboratory research for 3 academic years and will complete two summer research experiences. Each trainee will be supported by synergistic Mentoring Teams of faculty and peers. Oral and written communication skills will be developed and success will be evidenced through participation in scientific meetings, outreach to the lay public, manuscript publication and funded fellowship proposals. The MARC at CSU program will cultivate career-long professional development, guided by an Individual Development Plan and Mentoring Plan. Collaborative mentors who are committed to diversity and inclusion will encourage, critique and help trainees to think critically; identify important biomedical research questions; and design and perform research ethically, responsibly and with rigor. The CSU Institute for Research in the Social Sciences will evaluate the MARC program’s Context, Input, Process and Product (CIPP model), to provide evidence-based assessment that will guide decision-making and program improvement. Our overarching goal is for 70% of MARC at CSU students to matriculate into advanced research training programs and continue on to careers in biomedical research.

NIH Research Projects · FY 2026 · 2022-05

Project Summary/Abstract The essential multi-subunit RNA polymerases are regulated at each stage of the transcription cycle to control gene expression in each Domain. In many cases the rate limiting step of gene expression is during transcription elongation, but major knowledge gaps remain in our understanding of post-initiation regulation of transcription. Our studies directly address outstanding questions of transcription regulation in archaeal and eukaryotic cells. Our overarching goals are to establish molecular mechanisms that regulate post-initiation activities of RNAP, and establish the regulation imposed by histone-based chromatin on gene expression. How does altering the chromatin-landscape alter gene expression? How, in molecular detail, do factors that modify the activities of RNA polymerase accelerate transcription on histone-bound DNA? How can the otherwise extremely stable transcription elongation complex be disrupted to terminate transcription accurately? These complex challenges demand continued attention to define the foundational mechanisms underlying gene expression and the aberrant gene expression associated with disease states and cancer. We defined that archaeal transcription and chromatin systems are closely related, yet minimal versions of the component complex eukaryotic transcription systems. We have described the complete archaeal transcription cycle and defined three mechanisms that control transcription termination decisions, each of which reveals similarities with bacterial and eukaryotic termination mechanisms and demands continued experimentation to delineate conserved mechanisms to disrupt the transcription elongation complex. Our understanding of how chromatin structure regulates gene expression is also incomplete. We will leverage the simplicity of single-histone chromatin formed with native histones and histone-variants to establish the regulation imposed by extended chromatin structures on a genome-wide level. We will also describe the molecular activities of conserved archaeal-eukaryotic transcription factors that modify RNA polymerase and accelerate transcription through histone-bound DNA.

NIH Research Projects · FY 2026 · 2022-04

Abstract As a board-certified veterinary radiation oncologist and radiobiologist, I am committed to, and excited for, a career in translational cancer research as a physician scientist. My long-term career goal is to develop into an independent veterinary clinician scientist, proficient in designing and performing innovative radiation research, with a focused interest in tumor microenvironmental effects of radiation therapy and immunotherapy to improve treatment outcomes for patients with head and neck cancer. Head and neck cancer (HNC) is common in the United States and Europe and the prognosis is poor for patients with advanced disease. Stereotactic body radiation therapy (SBRT), which allows delivery of high dose, high precision radiation in a few fractions, is a novel therapy that can be used to treat HNC patients. Evidence exists that SBRT is a more potent activator of anti-tumor immune responses compared to conventional radiotherapy. Emerging preclinical and clinical data suggest SBRT combined with immunotherapy has the potential to convert immunologically “cold” (immunosuppressed) tumors into “hot” (inflamed) tumors. SBRT and IO combinations can stimulate effector T cell responses to each patient’s tumor. HNC patients with high risks for lymph node metastasis typically receive RT targeted to their primary tumor and regional lymph nodes (RLN) in order to eradicate latent metastatic tumor cells; however, RLNs are critical sites for generating immune responses, and RLN irradiation is likely to destroy the immune cells responsible for anti-tumor responses. Based on my preliminary data that SBRT caused depletion of T cell density and expansion of immunosuppressive immune cell populations in RLNs compared to RLNs spared from RT, we propose to study how RLN irradiation affects local and systemic anti-tumor immunity when combined with RT and IO. We will test our hypotheses with orthotopic murine head and neck cancer models and in canine cancer patients who have developed oral carcinoma. For the study, we will use the local tumor immunotherapy combination of agonistic OX-40 monoclonal antibody + TLR9 ligand, which has demonstrated positive tumor microenvironmental immune effects in mice and dogs. If we demonstrate RT+IO and RLN sparing improves outcomes in translational preclinical models of advanced HNC, the results of this project would challenge the current standard of care and clinical paradigm surrounding radiation, immunotherapy, and elective RLN irradiation for patients with advanced HNC. Through the K01 career development program, I will have the opportunity to delve deeper into radiation and immunology research and grow as an independent translational scientist through the direct influence, support, and guidance of my strong mentorship team, Dr. Steven Dow, Dr. Xiao-Jing Wang, and Dr. Sana Karam.

NIH Research Projects · FY 2026 · 2022-04

Project Summary/Abstract There has been rapid escalation in adolescent-onset type 2 diabetes (T2D), particularly in females from historically disadvantaged racial/ethnic groups. Prevention is critical because adolescent-onset T2D often shows a more aggressive disease course than adult-onset, and effective treatment options remain elusive. Standard- of-care for T2D prevention includes exercise training to ameliorate insulin resistance, a key physiological precursor to T2D. Despite short-term benefits, exercise training shows insufficient effectiveness in adolescents at-risk for T2D. Depression may be explanatory in a considerable subset of teenagers. Adolescence is notable for increases in depression and decreases in physical activity, especially in females with obesity. Youths' depression symptoms contribute to worsening insulin resistance over time, independent of BMI (kg/m2), likely through stress-mediated pathways such as reduced physical activity and fitness. Also, adolescent depression is associated with decreased physical activity and cardiorespiratory fitness, even after accounting for adiposity, and depression predicts greater non-adherence to exercise training. The central theme of this proposal is that an intervention sequence of delivering cognitive-behavioral therapy (CBT) first, followed by intervening with exercise training second, will offer a targeted, efficacious strategy for improving insulin resistance and consequently, lowering T2D risk in adolescent females at-risk for T2D with depression symptoms. In a prior NIH/NIDDK K99/R00 randomized controlled trial (RCT), we found that 6-week group CBT decreased depression at 6-week follow-up in adolescent females at-risk for T2D with moderately elevated depression, compared to a 6-week didactic health education control group. Adolescents with elevated depression who were randomized to CBT had lower fasting and 2-hour insulin at 1-year vs. controls. Our preliminary data suggest that CBT's focus on enhancing frequency/enjoyment of physical activity to combat depressed mood partially explained why decreasing depression lowered T2D risk. It is not known if CBT is just as efficacious as standard-of-care exercise training, or whether CBT followed by exercise training results in a maximally potent alleviation of T2D risk in adolescent females at-risk for T2D with depression symptoms. To address these gaps and directly build on our prior work, we propose a four-arm RCT to: (1) Compare the efficacy of four 6-week-->6-week sequences for improving insulin resistance in N=300 adolescent females at-risk for T2D with elevated depression symptoms: (i) CBT-->exercise, (ii) exercise-->CBT, (iii) CBT only (CBT-->continue CBT), and (iv) exercise only (exercise-->continue exercise); (2) Evaluate physical activity/fitness as mediators underlying the depression- insulin resistance association; and (3) Evaluate underlying mechanisms by which decreasing depression increases physical activity and improves fitness and insulin resistance using a mixed-methods process evaluation. Findings will support our long-term goal to identify feasible, cost-effective intervention strategies with high potential for effective dissemination to adolescents at-risk for T2D with elevated depression symptoms.

NIH Research Projects · FY 2026 · 2022-02

Initiatives to Maximize Student Development in Translational Medicine (IMSDTM) Summary Statement Over the last 20 years many professional organizations have documented that an insufficient number of underrepresented students are being trained in biomedical sciences, including those trained for research in translational medicine (TM) despite the significant increase in job opportunities for well-trained TM scientists. Lack of exposure and training opportunities for historically underrepresented populations (URPs) severely hampers their capacity to fill this national need. The College of Veterinary Medicine & Biomedical Sciences (CVMBS) at Colorado State University (CSU) proposes to create a unique training opportunity for URM Pre-doctorates to earn a PhD or DVM/PhD in TM. CVMBS has a strong history of T32 pre-doctoral training programs as well as T35 summer research experiences for DVM, DVM/MS, and DVM/PhD students interested in TM, but has seen just a moderate increase in URPs entering graduate programs. We seek to enhance our efforts in training and recruiting URPs by leveraging pre-existing training programs; as well as develop new pre- doctorate programs specifically designed to provide URPs with a an inclusive, supportive and comprehensive training environment for research, communication, and career development in TM. The proposed plan provides comprehensive and flexible TM training to facilitate basic biomedical and clinical research. To achieve these goals, five objectives are proposed: (1) Increase the number of PhD students from under-represented backgrounds with interest and aptitude for TM research to enter a mentored training program. (2) Enhance existing programs by building a flexible and rigorous research training program for students in basic and clinical research. (3) Encourage, support and enhance TM training and collaboration through CSU’s new Translational Medicine Institute (4) Facilitate entry into the workforce by providing training in team research, science communication and professional development experiences. (5) Build social and learning networks to provide a supportive and inclusive environment, enabling timely completion of the PhD degree. These frameworks will support our IMSDTM students through individualized counseling, mentoring and advising relationships at multiple levels. Success will be determined by a comprehensive assessment by the CSU Institute for Research in the Social Sciences. We will recruit historically underrepresented students to our IMSDTM training program from CSU’s NSF CO-WY AMP program; Fort Lewis College, a non-Tribal College Native American serving institution; CSU-Pueblo, a Hispanic Serving Institution with well-established undergraduate training program for Hispanic students; CVMBS undergraduates from three Departments of Microbiology, Immunology, Pathology, Biomedical Sciences, Environmental Radiological Health Sciences, and the Cellular & Molecular biology program. This comprehensive training approach will prepare IMSDTM scholars to successfully complete their PhD degrees and enter careers in the multiple discipline areas found in translational medicine The IMSDTM cohort will enhance the experience of all our students by increasing diversity of experiences, thought and perspectives in our graduate programs.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Organic Photoredox Catalysts for Synthetic Method Development Garret M. Miyake, Colorado State University, Department of Chemistry Catalysis is arguably the most important chemical contribution to society as it enables the synthesis of medicines and materials that are critical to enhancing human lives. To address current and future health needs, the development of catalyst systems that accelerate the discovery and manufacturing of medicines through new and more efficient chemical reactions are necessary. However, many classic catalytic methodologies employ precious metals, hazardous reagents, or forcing conditions. By contrast, photoredox catalysis has emerged as a powerful approach to access unique reaction pathways and drive chemical reactions using light. Advantages of photoredox catalysis include the ability to perform reactions under mild conditions, obviation of hazardous and non-selective reagents, achievement of new selectivity, increased functional group tolerance, increased reaction efficiencies, and access to unprecedented reaction intermediates and manifolds. Much of the work in photoredox catalysis has applied well- studied precious metal complexes or organic dyes that possess oxidizing excited states as the catalysts. As such, to address metal contamination, sustainability, and the need for expanded and novel reactivity, new organic photoredox catalysts with diverse photophysical and electrochemical properties must be developed. The long-term goal of the proposed research activities is to develop organic photoredox catalyst systems that enable novel and improved syntheses of small molecules and materials that address human health needs. This proposal describes the development of organic photoredox catalyst systems with unprecedented redox potentials that enable new chemical reactivity. We will employ a combination of experimental and computational approaches to gain a fundamental understanding into the catalysts and reaction mechanisms. Through mechanistically guided catalyst design, we seek to engineer systems that can access the extremely reducing or oxidizing chemical potentials necessary to enable challenging reactivity. Ultimately, this research will contribute to human health through the development of catalysts with unique reactivity to enable new and improved syntheses of medicines and medically important materials.

NIH Research Projects · FY 2026 · 2022-01

Modified Project Summary/Abstract Section The X-chromosome linked Neuroligin-4 (NLGN4) is a postsynaptic cell-adhesion molecule (CAM) abundantly expressed in human cerebral cortex, however, its cellular function and molecular properties remain relatively unclear. Human NLGN4 consists of a unique amino-acid sequence that is not evolutionarily well-conserved in conventional rodent models, limiting our ability to investigate how this human-specific gene impacts synapse organization. This inherent species differences between diverse NLGN4 orthologs underscore the immediate need to generate a human model system to uncover its human-specific mechanisms. Recent technological advances in the fields of genetic engineering and epigenetic reprogramming of pluripotent stem cells provide us with a unique opportunity to examine the mechanistic properties of NLGN4, while maintaining the fidelity of human cellular context. In this proposal, we aim to utilize neuronal subtypes derived from human stem cells to assess our central hypotheses that NLGN4 plays an instructive role in defining the input-output parameters of excitatory vs. inhibitory synapses. We anticipate that NLGN4 establishes molecular interactions with a subset of synaptic proteins via its intra- and extracellular domains, which collectively regulate its proper maturation, trafficking, and function. Both the amino-acid sequence of different NLGN4 motifs as well as post-translational modifications at some those critical residues might play significant roles in determining its functional specificity. In aim 1: To inquire how NLGN4 can modulate synaptic network activity, we will either completely eliminate its endogenous expression in human neurons or introduce loss-of-function mutation, and inspect adverse effects on synaptic morphology and transmission using confocal imaging and electrophysiological recording. In Aim 2: We will determine how distinct amino-acid residues of NLGN4 can differentially regulate its characteristics, by performing systematic structure-function and biochemical analyses. In Aim 3: We will investigate how NLGN4’s binding to other synaptic proteins may define its functional identity, using rigorous co-immunoprecipitation, cell-aggregation, and proximity-dependent biotinylation assay. This project will essentially provide a comprehensive knowledge about NLGN4 function, its similarities and differences with other NLGNs. Using NLGN4 as a model, this extensive set of complementary approaches would also allow us to acquire fundamental information about human synaptic environment and how pre- or postsynaptic CAMs modulate its composition and activity.

NIH Research Projects · FY 2025 · 2022-01

Protein misfolding diseases, or proteinopathies, are a group of invariably fatal neurodegenerative disorders affecting more than 6.8 million Americans. In multiple system atrophy (MSA) and other synucleinopathy patients, the protein α-synuclein (α-syn) misfolds into a self-templating conformation that spreads via a prion- like manner throughout the body, including the central nervous system (CNS). It is hypothesized that the conformation, or strain, that α-syn misfolds into encodes information about the clinical symptoms and neuropathologies a patient will develop. While previous studies focused on the biochemical differences between α-syn strains, the mechanism of how those differences encode distinct biological phenotypes of disease is poorly understood. The long-term goal of our research is to identify the agent and host factors that contribute to the varied clinical presentations observed across synucleinopathies. In this proposal, we will test the hypothesis that strain-specific differences in aggregate transport and neuroanatomical spread contribute to disease pathogenesis. In Aim 1, we will use alexa fluor-labeled α-syn aggregates to investigate the rate and direction of axonal transport in vitro and in vivo. To determine the molecular mechanisms responsible for α-syn transport, we will use chemical and genetic tools to disrupt microtubule polymerization, dynein motor activity, and dynein cargo adaptor binding, and quantify the strain-specific effects on α-syn axonal transport. In Aim 2, we will determine the role of trans-synaptic spread on α-syn strain pathogenesis. To rigorously perform these studies, we will first determine the titer of three different α-syn strains both in vitro and in vivo. We will then use the sciatic nerve injection model, with and without nerve transection, to determine if α-syn neuroinvasion relies exclusively on trans-synaptic spread, of if extraneural pathways contribute to disease pathogenesis when the same titer of each strain is injected. Finally, we will perform a thorough disease pathogenesis study to establish a temporal-spatial map of strain-specific α-syn spread. This work is innovative because it is the first study to investigate how interactions between the host and strain impact disease progression, and to establish between in vitro and in vivo α-syn titers. This work is significant because it is the first to investigate how interactions between host and strain contribute to the mechanisms underlying axonal transport and trans-synaptic spread of disease. Critically, by identifying the cellular and molecular machinery responsible for α-syn propagation, the results of these experiments will lead to new areas of promising investigation.

NIH Research Projects · FY 2025 · 2021-12

Project Abstract. The goal of this project is to introduce a new synthetic strategy to functionalize pyridine heterocycles. Pyridines are the second most common nitrogen heterocycle found in FDA approved drugs, an unsurprising fact because of their propensity for hydrogen bonding and suite of valuable physiochemical properties such as aqueous solubility, net polarity, and resistance to oxidative metabolism. Synthetic methods to directly and selectively functionalize pyridines are critical in drug discovery efforts because drug-developers require variation in positional selectivity, various carbon- and heteroatom bearing groups on the scaffold a wide variance in their steric and electronic properties. However, general approaches to transform pyridine C–H bonds into valuable derivatives are lacking. In this proposal, we outline a dearomatization strategy by converting pyridines into Zincke imine and iminium adducts. In effect, these adducts make pyridines react like a series of alkenes rather than an aromatic system and thus open up the plethora of reactions associated with olefins that were previously ineffective on pyridines themselves. We will show that pyridines can now participate in numerous processes, such as halogenation, reaction with sp2-carbon electrophiles radical reactions that were either not viable or operated under extreme reaction conditions. The processes will occur with exclusive control of regioselectivity for the 3-position of Zincke intermediates and are also switchable to the 5-position based on the Zincke adduct's structure. The ring-opening-functionalization-ring-closing is sequencable into one-pot processes in many cases. We will also use the Zincke platform to access pyridine isotopologs for ADME studies and new methods to form N–oxides. We plan to employ this strategy for simple building block pyridines, drug-like intermediates and for late-stage functionalization of complex pyridine-containing drugs.

NIH Research Projects · FY 2025 · 2021-09

Infectious Disease Research and Response Training Program (IDRRTP) The Infectious Disease Research and Response Network (IDRRN) was established in 2016 and has been recognized as a Program of Research and Scholarly Excellence (PRSE) at Colorado State University. The IDRRN promotes the development of interdisciplinary teams to investigate, study, and develop mitigation strategies for diseases locally, nationally and internationally. The IDRRN uses team dynamics to focus on infectious and neurodegenerative diseases and maximize the impact of its expertise in disease transmission and pathogenesis, development of diagnostics, vaccines and therapeutics, vector biology, epidemiology, and bioengineering. The IDRRN has a strong commitment and a proven track record in training the next generation of research scientists who will continue to face pandemics while also confronting novel emerging infectious agents. In addition to the rich scientific environment, there are vast, state-of-the-art facilities featuring BLS2, BSL3, ABSL3 and GMP capabilities. The parent grant provides trainees with personalized coursework in microbiology, immunology, pathology, statistics and bioinformatics to build foundational knowledge, critical thinking skills, and mastery of literature interrogation. With this competitive revision, we propose a comprehensive approach to transition from what is currently self-directed data science training that often occurs after data generation to an intentional, structured approach that prepares trainees prior to data generation. Trainees will be co-mentored by faculty from both infectious disease and data science disciplines. Projects are expected to bridge the research of the co-mentors and bring together students from each discipline to create peer to peer collaborative interactions. Faculty from the Data Science Research Institute, a recently formed, university-wide initiative, have been added as training faculty. These data science experts represent four colleges and have a track record of engaging in interdisciplinary research. A Graduate Certificate in Applied Data Science consisting of structured courses (10 credits) has been created to specifically address the needs of infectious disease pre- doctoral students and will provide recognition of their achievements. Required data management and sharing workshops along with interactive data science seminars on a variety of topics will meet students where they are to improve their comfort and competence in computational approaches while preparing them for the certificate program. The data science training positions provided through the competitive revision will serve as a catalyst to increase engagement of infectious disease trainees in data science, enhance recruitment to the new certificate program, and serve as a segue for a competitive renewal of the parent grant that will have data science as an ongoing emphasis.

NIH Research Projects · FY 2025 · 2021-09

Abstract The covalent modification of prokaryotic cell envelope glycans, namely lipopolysaccharide (LPS) and (lipo)teichoic acids, with discrete substituents such as sugars, amino acids, phosphates or acyl groups, is a well-known strategy used by Gram-negative and Gram-positive bacteria to modulate their cell surface properties, the way they interact with their environment, their resistance to biocides and host defenses, and pathogenicity. Although evidence exists that Mycobacterium tuberculosis (Mtb) similarly decorates its major cell envelope glycans, arabinogalactan (AG) and lipoarabinomannan (LAM), with various tailoring substituents, little is known of their biological significance. The discovery by our laboratories of the biosynthetic machineries responsible for the synthesis and transfer of these discrete motifs to AG and LAM, and the generation of the first Mtb knock-out mutants deficient in their biosynthesis have opened the way to studies aimed at understanding the function of these motifs in the physiology and immunopathogenesis of Mtb. We hypothesize that Mtb has evolved to modify its cell-envelope glycans with a distinct array of strategically placed substituents to promote its survival in the host environment. Accordingly, a multidisciplinary team of investigators with complementary expertise in mycobacterial cell envelope genetics and glycobiology, TB immunopathogenesis, and carbohydrate chemistry here proposes to investigate how simple (amino/thio)sugars or other charged groups strategically placed within the Mtb cell envelope landscape affect not only the physiology of this microorganism (Aim 1), but also the course of pulmonary infection, pathology and development of innate and adaptive immunity in infected C3HeB/FeJ mice (Aim 2), and the interactions of Mtb with host macrophages and dendritic cells thereby promoting survival within the host (Aim 3). Ultimately, these studies are expected to lead to significant new knowledge about the biological significance of understudied aspects of the unique cell wall of mycobacteria.

NIH Research Projects · FY 2025 · 2021-09

Project Summary This proposal aims to train a dual-degree, DVM-PhD student for a career as a lab animal veterinarian and independent scientist. The research outlined in this proposal will develop a pre-clinical platform to evaluate CAR-T therapy in a canine model. Chimeric antigen receptor (CAR)-T cells have induced up to 90% remission rates for treatment relapsed/refractory B cell malignancies. While mice have been instrumental to CAR-T progress, CAR-T therapy for solid tumors have been hampered by this inbred, immunodeficient model. Pet dogs are a higher fidelity translational model due to their outbred genetics, intact immune system, high incidence of cancer, and similar cancer biology. A CAR is a fusion protein comprised of a T cell receptor signaling domain, costimulatory domain, and an antibody based binding domain. CARs are introduced to patients’ T cells ex vivo, enabling the T cells to directly recognize tumor antigen. CAR-T cells are a “living therapy” wherein the efficacy of the treatment relies not only on the design of the CAR, but also how the CAR-T cells are able to home to the tumor and elicit anti-tumor effects. The CAR helps T cells to “recognize” the tumor, but the trafficking, persistence, and effector function of these cells relies heavily on intrinsic T cell biology. To adequately assess CAR-T cell function in vivo, design of the CAR (Aim 1) and patient T cell biology (Aim 2) will be evaluated. Aim 1 – Determine optimal CAR design for targeting tumor associated antigens GD2, FolR1, and CD20. CAR constructs will be designed for the tumor associated antigens GD2, FolR1, and CD20. These CARs will be introduced to primary canine T cells via a lentiviral vector. The CAR-T cells will be evaluated for efficacy against antigen positive tumors by IFNγ ELISA, IL-2 ELISA, and Incucyte live cell videomicroscopy. Each of these constructs will be tested with CAR costimulatory domains 4-1BB and CD28. The CAR constructs with the strongest reactivity will be further evaluated with an NOD scid gamma (NSG) mouse xenograft model, measuring tumor growth inhibition and CAR-T expansion in vivo. Aim 2 – Determine which subset of CAR-T cells preferentially traffic and persist in the tumor in vivo. To evaluate the respective contribution of CAR-T cell subsets to anti-tumor efficacy in vivo, semi-random nucleotide barcodes will be added to the CAR constructs allowing for the tracking of clonal lineage during CAR-T production, infusion, and post- engraftment in mice. Using single cell sequencing, clonal diversity of the CAR-T infusion product will be compared to clonal diversity intratumorally. Subsets of CAR-T that preferentially home to and expand in the tumor will be identified. Together, these aims will set the basis for future studies of CAR-T therapy in a canine model. Aim 1 will provide a candidate CAR construct to be evaluated in a canine model. Aim 2 will provide a method for understanding how CAR-T cells traffic to and persist within a tumor in vivo. This platform will be used to screen and refine novel approaches to CAR-T therapy in a high-fidelity, high-throughput animal model.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY / ABSTRACT Healthy nutrition habits are key to managing type 2 diabetes (T2D). However, American Indian and Alaska Natives (AI/ANs) often lack access to healthy food and disproportionately experience food insecurity. Food insecurity, defined as lack of consistent access to enough food for an active, healthy life, negatively impacts one’s ability to manage blood sugar and body weight. I am pursuing a K01 Mentored Research Scientist Development Award to fill critical training gaps in the areas of quantitative data analysis of intervention studies and effectiveness and pragmatic study design. In order to effectively bridge my prior training and research experience to obtain my first R01, I have carefully developed a mentored training plan that strategically supports my research aims for this K01 award. I am currently Co-Investigator on a study team that developed a culturally responsive diabetes nutrition education curriculum for AI/AN adults with T2D, entitled “What Can I Eat? Healthy Choices for AI/ANs with T2D” (WCIE). WCIE includes interactive, hands-on nutrition education learning activities, including portion control, decreasing sugary beverages and saturated fat, and problem solving for barriers to healthful eating. In January 2020 we launched a tribally-supported, 6-site, waitlist control design test of WCIE. In June 2021, we will complete data collection with 150 AI/AN adults who have T2D. Data will include 3 timepoints for immediate intervention and 5 for waitlist intervention participants over 6 months with surveys (nutrition knowledge, behavior, self-efficacy) and clinical outcomes (BMI, blood pressure, and HbA1c). Though WCIE does not include a specific food insecurity intervention, we included a household food insecurity measure at all data collection timepoints. In Aim #1 of this proposed K01 I will examine the relationship between food insecurity and clinical WCIE outcomes in the existing dataset through secondary analysis to utilize my new intervention quantitative data analysis skills and advance understanding of the moderating effect of food insecurity on diabetes nutrition education and T2D outcomes among AI/ANs. In Aim #2 I will design/pilot test WCIE with added service to improve healthy food access and evaluate its feasibility/impact on T2D outcomes at an urban AI/AN- serving clinic in Oklahoma City, OK. We will work with the Oklahoma City clinic to identify their need for a healthy food access service in the following domains: transportation, availability of healthy food, or affordability of healthy food. Aim #2 will provide me the opportunity to utilize my new training in effectiveness and pragmatic trials study design and quantitative data analysis of intervention studies. My detailed training plan includes formal coursework at the University of Colorado Anschutz Medical Campus, seminars, workshops/conferences on AI/AN health research, and support from my mentors who are experts and leaders in the area of AI/AN diabetes health, AI/AN health interventions, T2D and food insecurity interventions, and quantitative/study design methodology. The proposed training plan and aims will provide me with a set of skills and expertise needed to successfully obtain R01 funding and become a leader in diabetes health disparities intervention science.

NIH Research Projects · FY 2025 · 2021-09

It is well established that structured moderate to vigorous physical activity (i.e., aerobic and resistance exercise) improves many physical and psychosocial health outcomes for cancer survivors. However, it is estimated that less than half of cancer survivors are achieving the cancer-specific exercise guidelines. Interventions that are supervised, and include theory-based behavior change strategies are effective for increasing moderate to vigorous physical activity (MVPA) among cancer survivors; but following an intervention, many survivors return to previously inactive, or insufficiently active lifestyles. To achieve the many benefits associated with MVPA or ‘exercise’, cancer survivors must not only adopt or begin a program, but also be able to maintain these PA levels long-term. In addition to individual behavior change strategies, interpersonal, and environmental support for PA can extend the success of PA interventions. This can be achieved by increasing the accessibility of tailored, evidence-informed cancer-specific exercise programs at community-based locations. Thus, we propose to engage in a research-practice partnership to deliver an adaptive PA maintenance intervention for cancer survivors. In the R21 phase, we will examine acceptability of maintenance intervention components, and conduct a needs assessment for intervention delivery at three- community partner locations. In the R33 phase, we will utilize an adaptive intervention design to determine the optimal level of support needed to maintain PA following a community-based exercise program. We will enroll cancer survivors who are not currently achieving aerobic and resistance exercise guidelines in a three-month, supervised, group-based exercise and PA behavior change program at one of three community fitness facilities. Upon completion of the program, participants will be encouraged to continue exercising during a three-month free-living follow-up period, during which time there will be no active intervention. After this follow- up period, exercise levels will be assessed. Those who are not achieving aerobic and resistance exercise guidelines for cancer survivors will be classified as ‘incomplete responders’ and randomized to one of two subsequent interventions: (a) monthly PA behavior change discussion sessions, or (b) monthly PA behavior change discussion sessions plus bi-weekly, exercise sessions. Responders will be randomized to either: (c) no further intervention, or (d) monthly PA behavior change discussion sessions. After three-months of the subsequent PA maintenance intervention, exercise levels will be assessed again (i.e., 6-months after completion of the initial exercise program). This project is significant because it aims to develop a tailored approach to enhancing PA maintenance, by identifying non-responders and providing them with the additional support necessary to engage in MVPA long-term. Delivering the intervention in community-based facilities will increase potential for scalability and widespread dissemination. Findings from this study will prepare our team to test this intervention in a full-scale adaptive trial, powered for efficacy.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract: The overall goal of this proposal is to investigate the poorly understood mechanisms controlling long distance AMPAR transport in delivery and removal of receptors for synaptic maintenance and plasticity. Excitatory neurotransmission mediated by glutamate and ionotropic glutamate receptors of the AMPA subtype (AMPAR) at synapses plays a central role in cognition. Tight regulation of the number and function of these receptors is, therefore, essential. Since synapses are often far away from the neuronal cell body, they are critically dependent on long-distance transport by microtubule-dependent molecular motors to provide a steady supply of AMPARs. The field of excitatory synaptic transmission has a detailed understanding of how cell- signaling pathways control local synaptic AMPAR trafficking but almost no understanding of how these synaptic signaling events control long-distance AMPAR transport. The major reason for this lack is technical: transport studies require powerful, high-speed microscopy in intact neuronal circuits. Direct observation and informative manipulation of transport in vivo is extremely difficult in vertebrates. We have pioneered real-time in vivo studies of AMPAR transport in intact neuronal circuits using the transparent model organism, C. elegans. Here we will test a new mechanistic framework for the regulated cellular distribution of AMPARs to synapses centered on the long-distance transport of receptors by molecular motors. Our model predicts that Kinesin-1 scaffolds (JIP1 and 3) are necessary for AMPAR transport and their assembly onto Kinesin-1 is dependent on neuronal activity, calcium and calcium calmodulin-dependent kinase 2 (CaMKII). In addition, we identify a modulator of transport, PTP-3A, that modifies export from the cell body and synaptic delivery ultimately affecting memory. Specific Aim 1 will determine how synaptic inputs at cell bodies and at dendrites modify calcium and AMPAR transport. Specific Aim 2 tests the hypothesis that synaptic activity leads to modification of the AMPAR transport complex conferring different export and synaptic delivery properties. Specific Aim 3 tests the hypothesis that PTP-3A the longest isoform of the receptor tyrosine phosphatase PTP-3, regulates AMPAR somatic export and synaptic delivery using 2 domains released by cleavage induced by neuronal activity. The experiments described in these aims will combine genetics, in vivo spinning disk dual channel microscopy, optogenetics, photobleaching and photoconversion, biochemistry and behavior analyses to elucidate the mechanisms of long- distance AMPAR transport regulation by synaptic signaling. Our studies will: 1) provide a new model for understanding the cellular mechanisms regulating synaptic function, 2) have broad impact on the understanding of cargo delivery and removal mechanisms by molecular motors applicable to multiple biological systems, and 3) improve understanding of AMPAR transport that could reveal novel therapeutic targets for modulating excitatory synaptic transmission in the context of human disease.

NIH Research Projects · FY 2026 · 2021-07

PROJECT SUMMARY Organization is a fundamental and defining feature of life at all degrees of scale. On the cellular level, molecules and organelles must be arranged with appropriate temporal and spatial precision such that processes may proceed according to the needs of the cell. Similarly, cells are arranged into appropriate layers to define underlying tissue organization, which provides the basis for organ function, and supports the health of the organism. Major determinants of subcellular and cellular organization are molecular motors that transport diverse cargoes throughout the cellular environment. One such motor is cytoplasmic dynein, which transports numerous types of cargoes along microtubule tracks during all cell cycle stages and within many cell types. In addition to orchestrating appropriate subcellular organization, dynein plays a major role in the establishment and maintenance of tissue architecture. For instance, a major determinant of cell fate and consequent tissue organization is the orientation and position of the mitotic spindle with respect to the boundaries of the cell. In addition to localizing to the membrane of small vesicular cargoes, dynein motors are anchored at the plasma membrane from where they orient and position the spindle through precisely tuned interactions with microtubules. During such processes as organismal development and tissue homeostasis, spindle orientation and position dictate the plane of cell division, and thus whether a cell divides symmetrically or asymmetrically. Symmetric stem cell divisions result in two identical stem cells, whereas a switch to asymmetric division results in one stem cell and a differentiated cell, which promotes tissue stratification. Thus, dynein is a critically important molecule that dictates biological organization on many levels of scale. The precise mechanisms by which dynein performs all these disparate functions with appropriate spatial and temporal control are unclear. The lack of such information presents an impediment towards the development of effective therapies that may prevent or reverse defects in cellular and tissue organization that can lead to various devastating disorders (e.g., malformations of cortical development, motor neuron diseases). In the proposed studies, we will use a combination of in vitro and cell biological approaches to determine the mechanisms by which dynein is regulated to perform its cargo transport functions. Specifically, we will: (1) determine the mechanisms by which dynein is autoinhibited, and the means by which this is regulated; (2) understand how the lissencephaly-related protein Lis1 initiates dynein- mediated cargo transport; (3) identify how assembly and activation of motile dynein transport complexes are tuned by phosphorylation; (4) investigate the molecular basis for diseases that arise as a consequence of mutations in dynein and its many regulators; and, (5) determine how dynein mediates spindle movements with precise directional precision. Our studies will provide critical insight into fundamental mechanisms that dictate transport of numerous cargoes with spatial and temporal precision such that cellular and ultimately organismal health is established and maintained.