University Of Colorado

NIH Research Projects · FY 2025 · 2024-09

Project Summary The mammalian cell cycle is commonly conceived as a well-understood, hardwired, invariant pathway. Emerging work, however, indicates that the cell cycle is much more plastic than generally believed, with multiple adaptive routes through the cell cycle under different conditions. This plasticity makes the cell cycle robust to environmental perturbations, but also drives adaptive drug resistance to targeted cell-cycle inhibitors. A fresh look at the dynamics and pliability of cell-cycle progression will reveal new principles that predict dependence on a particular cell-cycle node and new strategies to suppress adaptive cell-cycle rewiring. Cyclin-Dependent Kinases (CDKs) are key enzymes that drive cell proliferation, and consequently, multiple CDK inhibitors are in development to suppress unwanted cell proliferation. However, cells eventually find a way around these drugs to resume proliferation. A plausible hypothesis is that cells leverage cell-cycle plasticity to pursue alternative paths through the cell cycle. In one striking example, inhibition of CDK2 leads to rapid loss of substrate phosphorylation as expected, but then CDK2 substrate phosphorylation rebounds within several hours. This rebound depends on CDK4 and CDK6, which insulate the cell from fluctuations in CDK2 activity by maintaining Rb hyper-phosphorylation and E2F transcription. This enables CDK2 re-activation and eventual cell-cycle completion, even in the presence of potent CDK2 inhibitors. My lab has pioneered the development of a set of powerful time-lapse microscopy tools to visualize rapid drug responses in single, living cells. Here, we will apply our technology to determine the mechanisms driving this unusual rebound in CDK2 activity observed upon CDK2 inhibition. First, we will test the role of the p16 CDK4/6 inhibitor protein on the CDK2 activity rebound. Second, we will test whether CDK2-dependent degradation of Cyclin D modulates the CDK2 activity rebound. Third, we will identify additional mechanisms underlying the robustness of Rb phosphorylation to inhibition of CDK2 and show how long-term drug pressure unleashes the plasticity of CDKs. Since our findings are likely to be broadly applicable beyond CDK2 inhibitors, our proposed work will fill long-standing gaps in our understanding of how the cell cycle is wired for success in the face of perturbations.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY The healing of large bone defects resulting from traumatic injuries, fracture nonunion, and tumor resection remains a significant clinical challenge. Approximately 500,000 patients undergo bone transplants each year in the US alone and bone diseases and their complications account for half of chronic disease among individuals greater than 50 years old. Common surgical interventions such as autologous, allogenic, or xenogeneic bone grafts can suffer from serious limitations (e.g., availability of grafts, donor site morbidity and pain, incompatibility, immunogenic reactions, and infectious disease). Therefore, researchers have explored tissue engineering approaches to develop suitable bone replacements or regeneration strategies; however, these approaches can be complicated by a heavy, persistent immune response and inadequate vascularization throughout large constructs in vivo. We aim to use a multifactorial approach to regenerating bone tissue by harnessing the regenerative potential of the MSC secretome, specifically extracellular vesicles (EVs), in combination with controlled released from porous biomaterial scaffolds. We hypothesize that controlled release of anti- inflammatory and pro-angiogenic EVs from granular hydrogels will create a pro-healing microenvironment and promote scaffold vascularization, improving overall bone formation by endogenous cells in critical-sized defects. First, we will systematically identify granular hydrogel properties (e.g., porosity, stiffness, bioactive molecule presentation) that promote MSC secretion of EVs enriched with anti-inflammatory and pro-angiogenic factors (Aim 1). Next, we will use glycoengineering approaches to produce modified EVs that can be conjugated to our granular scaffolds via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistries before encapsulating cells within EV-laden scaffolds to investigate the influence anti-inflammatory and pro-angiogenic EVs on macrophage polarization and vascularization, respectively, in vitro (Aim 2). We will then fabricate granular hydrogels using heterogeneous populations of microgels to vary in vivo degradation and enable temporal control over EV release profiles. We will conduct a short-term (7 days) rat subcutaneous implant study to test the capacity of EV-laden granular scaffolds to modulate early inflammation and vessel invasion, providing iterative feedback on scaffold design. Finally, we will interrogate the ability of these complex granular scaffolds to modulate inflammation, vascularization, and osteogenesis to promote bone regeneration by endogenous cells in a rat critical-sized calvarial defect model (Aim 3). This proposal will utilize sophisticated experimental techniques to explore the complexities of cell-cell, cell-matrix, and cell-EV interactions. The contributions of this proposal would significantly impact the wide array of regenerative medicine strategies focused on granular hydrogels, extracellular vesicle therapeutics, and bone tissue engineering.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY / ABSTRACT The objective of this proposal is to establish a new microphysiological ‘joint-on-chip’ system, with structural biomimicry and biomechanical function, that can support mechanistic study of arthritic diseases and rapidly test candidate treatments. Recent advances in the development of on-chip technologies have shown potential to miniaturize musculoskeletal tissues and emulate key aspects of a healthy joint. One primary goal of on-chip technologies is to create robust and reliable human models of the joint so that the study of disease pathogenesis and screening of promising drug treatment candidates is possible with high throughput. No disease modifying treatments are available to address osteoarthritis (OA), a health burden that afflicts millions of people in the United States. With the development of on-chip technologies, pharmaceutical companies would be positioned to ‘fail fast’, and advance or accelerate only the most promising candidate drug therapies toward clinical trials. Unfortunately, realistic joint-on-chip models currently lack minimal essential functionality that is necessary for the study of arthritic diseases and evaluation of treatment candidates. Challenges include the need for human- derived biomaterial inks that support tissue-specific mechanical and cellular responses, and the need to recapitulate the complexity of the human joint, including crosstalk between multiple tissue types, and movement- induced biomechanical stimuli like frictional sliding that mimics the in vivo environment. To improve realistic joint- on-chip models, our lab has developed human-derived biomaterial inks – particulated allograft extracellular matrix with a unique crosslinking technology – to enable 3D bioprinting of tissues that more closely mimic the natural structure of cartilage, bone, and synovium. We have additionally demonstrated that differential biomechanical stimuli (e.g., compression and frictional sliding) promote distinct cellular responses that are characteristic of healthy tissue and needed in the engineering of a miniaturized version of the human joint. We now plan to develop a joint-on-chip with minimal essential functionality using human-sourced tissues and cells to study arthritic diseases and drug treatment candidates. We will optimize human biomaterials to recreate essential tissue structure and function, and engineer necessary biomechanical stimulation that is currently lacking in on-chip technology. We will pursue three related specific aims. In Aim 1, we will optimize a library of particulated and human-derived biomaterial inks for 3D joint-on-chip printing. In Aim 2, we will establish a human joint-on-chip platform with minimal essential functionality. In Aim 3, we will quantify the joint-on-chip pathogenic response to biomechanical injury and inflammatory challenge. If successful, we will for the first time create a realistic joint-on-chip model with essential functionality that is useful to study arthritic diseases and evaluate treatment candidates.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY / ABSTRACT. Substance use disorders affect millions of Americans, with substance use often beginning in adolescence. There is a pressing need to identify targets for interventions that reduce substance use initiation and misuse, especially for those at high risk. Our primary goal is to examine adolescent music engagement as a potential protective factor for early substance use behaviors. Music engagement is promising because of its many prosocial benefits, including for quality of life, social connectedness, and emotional competence. However, existing studies have not evaluated whether associations between music engagement and substance use are driven by correlated genetic or environmental influences versus direct causal effects. Beyond this, gene-by-environment interactions (GxE) are highly relevant to both music engagement and substance use, and music engagement may be especially protective for individuals at high genetic or environmental risk for substance use. Establishing these associations and understanding whether they are specific to music engagement and/or may be observed for other activities (e.g., art, sports engagement) will inform translational and intervention efforts aimed at preventing or reducing adolescent substance use before problematic use. We will conduct secondary data analyses of large longitudinal twin/family studies with multiple waves of assessment of music engagement, substance use, and genotyping on most subjects. Datasets include the Adolescent Brain Cognitive Development Study (ABCD) and the Colorado Adoption/Twin Study of Lifespan Behavioral Development and Cognitive Aging (CATSLife). Analyses include multiple twin/family and genomic approaches that inform whether associations are due to a combination of genetic, environmental, and causal effects (e.g., Mendelian Randomization Direction of Causation, Aim 1a), longitudinal analyses examining whether music engagement predicts later substance use behaviors (Aim 1b), and a detailed comparison of music engagement with other activities (Aim 1c). We will also test competing theoretical models of GxE (diathesis stress versus differential susceptibility) using polygenic scores (Aim 2a), environmental risk variables (e.g., peer use, Aim 2b), and socio-demographic measures (Aim 2c). The study team is well-versed in longitudinal structural equation modeling approaches to study substance use and music engagement, including polygenic score and twin/family approaches leveraged here (and combinations of both). The research supported by this R01 award will be critical for understanding how music engagement relates to adolescent substance use initiation and progression, and to quantify the nature of these associations. It will lay groundwork for music intervention studies targeting individuals at highest risk for substance use problems (e.g., polygenic scores, environmental risk factors). This work responds directly to NIDA’s mission to identify factors leading to the prevention of substance use disorder and is directly relevant to the Music and Health initiative’s goal to examine how music engagement relates to important health traits such as substance use.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY/ABSTRACT While consolidated sleep is crucial for healthy cognition and mood in older individuals, many suffer from sleep- wake fragmentation, a risk factor for developing Alzheimer's disease. One countermeasure of sleep-wake fragmentation is exposure to bright light ("phototherapy"). Studies using morning phototherapy, which targets circadian phase, to restore sleep-wake fragmentation have reported mixed results. However, both mathematical models of the circadian pacemaker and data from our lab suggest that afternoon light exposure, targeting circadian amplitude, will have greater effects on sleep-wake consolidation. Since phototherapy can be administered without significant adverse effects, it is a promising tool to reverse sleep-wake fragmentation and slow cognitive decline. Therefore, the overarching goal of the proposed studies is to slow cognitive deterioration in older individuals with mild cognitive impairment (MCI) by investigating the utility of afternoon phototherapy. The Research Training Plan will leverage state-of-the-art artificial intelligence techniques on big datasets (specific aims 1 and 3) and an intervention clinical trial (specific aim 2). In aim 1 (K99), the applicant, Dr. Lok, will train with Dr. Kochenderfer as she applies state-of-the-art machine learning techniques to determine underlying factors contributing to sleep-wake fragmentation and cognitive decline. During this time, Dr. Lok will also learn to conduct neurocognitive testing in individuals with mild cognitive impairment. Dr. Lok will conduct a clinical trial (R00), investigating the utility of afternoon phototherapy in a stepped care approach to reduce sleep- wake fragmentation and improve cognition. Finally, Dr. Lok will use machine-learning techniques to develop a personalized phototherapy model to create a prediction score calculator. These endeavors ensure that these projects' outcomes benefit the scientific and medical communities. Dr. Lok has the requisite training in machine learning and clinical trials to undertake the proposed projects. The career development plan is intricately designed to empower Dr. Lok with enhanced machine-learning skills and to facilitate a deeper understanding of gerontology and the social determinants of aging. Mentor Dr. Zeitzer is a leading expert in human translational chronobiology. Co-mentors Drs. Kochenderfer (machine learning), Fairchild (neuropsychology), and advisors Drs. Jo (statistician) and Yesavage (Alzheimer's disease) offer complementary expertise. Dr. Lok proposes to pursue these development goals and begin the proposed research with the support of the Department of Psychiatry and Behavioral Science at Stanford University, which provides an ideal environment of research support and resources to attain her training and research goals. In summary, the solid mentoring team, environment, and proposed training plan anticipate fully launching Dr. Lok's independent career. The proposed study will increase knowledge about contributory factors to sleep-wake fragmentation and cognition, as well as a scalable intervention with the potential to ameliorate cognitive decline and other concomitants of fragmented sleep, prevent Alzheimer's disease onset, delay institutionalization, and improve quality of life in older individuals.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract The proposed project will leverage 16 twin samples from the Interplay of Genes and Environments across Studies (IGEMS) Consortium, and incorporate four additional samples, to clarify which modifiable risk factors may be most influential in increasing risk for Alzheimer’s disease and related dementias (AD/ADRD) and at which developmental stages their risks are greatest. The overarching goal of this project is to apply twin models to strengthen or refute causal hypotheses and test gene-environment interplay among modifiable factors for AD/ADRD, considering risk and resilience profiles within and across developmental periods of the lifespan. Given the aging of the populations worldwide, AD/ADRD are expected to show substantial increases over the next few decades, making the proposed study especially timely. We focus on modifiable risk factors in the hope that clarifying the mechanisms and timing of their effects may help to guide prevention and intervention to reduce AD/ADRD. Many of our genetically- and environmentally-informed samples include longitudinal data and polygenic scores (PGS). Drawing from samples in Sweden, Denmark, Australia, and the United States, we will (a) create multi-dimensional risk scores for early life contexts and (b) evaluate PGSs for neurodevelopmental disorders and educational attainment in addition to PGSs for Alzheimer’s disease (AD), AD resilience, and modifiable factors. We will leverage within-between models of twins and siblings of exposures and genetic risk to test causal hypotheses that control for confounding, and explore gene- environment (GE) interplay of modifiable risk factors for AD/ADRD and qualities of resilience to address our aims: (SA1) evaluate mid- and late life physical health and health behavior factors that alter AD/ADRD risk; (SA2) evaluate midlife and late life socioemotional factors that alter AD/ADRD risk; and (SA3) examine how early life risks work together with midlife and late life health and socioemotional factors to influence AD/ADRD risk while also incorporating GE interplay. The proposed research study will extend the life course model of AD/ADRD by implementing a systems-level approach, guided by the NIH disparities framework, to investigate key environmental factors that contribute to social inequities, including multiple early life adversities and risk factors and measuring them at different times. Indeed, we will evaluate how mid- and late life risk act in combination with early life risks and genetics to create differential profiles of AD/ADRD risk across sex, cohorts, and countries. Our novel approach of leveraging both twin and genomic data will provide converging evidence to inform clinical and policy recommendations with regard to the genetic interplay among risk and protective factors that create differential vulnerabilities for AD/ADRD. The IGEMS Consortium is uniquely positioned to advance the study aims using powerful approaches, rich phenotypes, high variability across exposures and socioeconomically diverse samples, and lifespan coverage.

NIH Research Projects · FY 2025 · 2024-08

As cannabis legalization increases, there have been concurrent increases in use. A common reason for use is the mitigation of anxiety and stress, which has been exacerbated with the COVID-19 pandemic. However, cannabis use for coping purposes is associated with greater quantity and frequency (Q/F) of cannabis use, increased risk for cannabis-related problems, and greater likelihood for cannabis dependence. In turn, greater Q/F of cannabis use and dependence can lead to safety risks, mental/physical health issues, and other problems like greater Q/F of alcohol use. Thus, developing a greater understanding of cannabis use for coping purposes is a critical research endeavor. There are several important avenues of research that can inform our understanding of this use pattern. The first is examining if quantity of cannabis is actually increased when used for coping purposes, which has not yet been experimentally tested, as well as exploring factors that may moderate this effect (e.g., social anxiety and inhibitory control). A second avenue is investigating whether cannabis use actually mitigates stress. A third avenue is exploring the biological role of the endocannabinoid system as a mechanism by which cannabis use may relate to acute stress reduction as well as the role of cannabinoid content in this process. Specifically, research shows that the endocannabinoid, arachidonoyl ethanolamide (AEA), is negatively associated with anxiety and stress such that it may mediate the relationship between cannabis use and stress reduction. Because the two main cannabis constituents, 9-delta tetrahydrocannabinol (THC) and cannabidiol (CBD), are associated with disparate effects on AEA, they may differentially influence how cannabis use relates to stress. In particular, CBD may actually decrease stress compared to THC via greater effects on AEA production. This study proposes to examine these research questions with four aims. The first will experimentally test a causal relationship between cannabis use for coping purposes and quantity of cannabis use (i.e., if more cannabis is used after stress induction compared to a control condition among individuals who endorse cannabis use for coping purposes). The second will test if the relationship between stress and cannabis use is stronger for individuals with social anxiety and/or poorer inhibitory control. The third will test if cannabis use after stress is related to decreases in subjective and objective stress. The fourth will ask whether decreases in stress are mediated via increased AEA, and if this indirect relationship is stronger with greater CBD to THC product ratios. Knowledge gained from this study will have significant public health impact including aiding in intervention and prevention efforts for cannabis misuse and contributing data on the harm reduction potential of CBD.

NIH Research Projects · FY 2024 · 2024-08

PROJECT SUMMARY/ABSTRACT Thirteen percent of adults over 75 suffer from aortic valve stenosis (AVS), a progressive disease that leads to aberrant collagen deposition, leaflet stiffening, and eventual valve calcification at late stages. As it stands, surgical or transcatheter valve replacement is the only treatment option for severe AVS and is limited in durability and can require indefinite anticoagulation therapy. AVS is also sexually dimorphic with men having a two-fold higher risk for developing direct calcification, whereas women with equal disease severity tend to have more valvular fibrosis prior to calcification. The molecular mechanisms underlying sexual dimorphism in AVS progression remain poorly understood, but growing evidence suggests AVS is an inflammation-dependent disease. Recent studies have focused on the role that infiltrating immune cells such as macrophages play in AVS, with a growing appreciation that men and women undergo different immune responses and show differences in compositions of inflammatory cytokines. Combined, these observations suggest not only the need for alternative treatments for AVS (e.g., pharmaceutical therapies), but also the opportunity to develop personalized treatments based on sex and inflammatory state. Based on the extant evidence, we propose to investigate the role of inflammation in sex-specific valve disease progression. We hypothesize that AVS sexual dimorphism is perpetuated by: 1) sex differences in valve cell secreted factors promoting differential polarization of macrophages, 2) differences in infiltration behavior of male and female macrophages, and 3) direct macrophage-valve cell contact eliciting distinct phenotypic differentiation dependent upon sex, leading to the formation of a pro-fibrotic or pro-calcific niche. We will test these hypotheses with the following specific aims (1) Delineate how secreted factors from valve cells effect macrophage phenotype and vice versa in a sex-dependent manner, (2) Elucidate sex-specific differences in AVS disease initiation within an in vitro 3D co-culture model. The applicant, Dr. Alex Khang, will work with sponsor, Dr. Kristi S. Anseth, collaborating investigators with expertise in immunology (Dr. Laurel Hind) and sex-specific differences (Dr. Leslie Leinwand), and microscopy consultant Dr. Joseph Dragavon to carry out the experimental studies. All studies will be performed at the University of Colorado Boulder. All members of the research team and the required equipment for the proposed research are housed in the same building (Jennie Smoly Caruthers Biotechnology Building, JSCBB) which will facilitate constant interactions as well as frequent opportunities for mentorship, while eliminating logistical issues. The JSCBB also houses numerous faculty that are domain-experts and word-leaders in their respective field who Dr. Khang will interact with frequently at local seminars and events. During the funding period, Dr. Khang will gain expertise in engineering in vitro co-culture models, immunology, sex-specific differences, and advanced light microscopy. The funding period will also enable Dr. Khang to expand his network and attend new conferences, which will further add to his knowledge and training toward an independent research career.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Optical coherence tomography (OCT) is an emerging biomedical imaging technology that provides label-free and depth-resolved images with micron-scale spatial resolution and sub-millisecond temporal resolution. Since its inception in 1991, OCT has revolutionized eye disease diagnosis with over 32 million ophthalmic OCT procedures performed world-wide annually. OCT-based technologies have also been exponentially adopted in a wide range of clinical applications, including cardiology, endoscopy, urology, dermatology, and dentistry. Traditionally, OCT only provides tissue-level morphological information. Recently, there is a surge in extending this label-free technology to also delineate the physiological information (e.g., cellular viability, necrotic regions, and growth dynamics) at the cellular level. The so-called dynamic contrast microscopic OCT (DyC- µOCT) or dynamic contrast optical coherence microscopy (DyC-OCM) is distinguished from traditional scattering-based OCT by its emphasis on dynamic fluctuations: the motions of viable cells are accented against the motionless regions in the OCT images, enhancing the image contrast and revealing both cellular morphological and physiological information. Today, there are two dominating DyC-OCM architectures: spectral-domain OCM (SD-OCM) and full-field OCM (FF-OCM), each optimized for temporal analysis of different 2D images. Unfortunately, none of the two dominating DyC-OCM architectures can support the 3D volumetric dynamic contrast analysis even though organelles and cells are naturally organized in 3D. This limitation mainly comes from the fact that both SD-OCM and FF-OCM can only provide a voxel rate of ~100 Mvoxel/s but a voxel rate exceeding 1 Gvoxel/s is necessary for 3D DyC-OCM. Such a high voxel rate has ever only been demonstrated with another OCM architecture, swept source OCM (SS-OCM). Even though SS-OCM can break through the voxel rate barrier, it suffers from poor axial resolution, and thus its ability to image cellular structure has been severely limited. In this program, we aim to develop the first 3D DyC-OCM technology that simultaneously breaks through the voxel rate and axial resolution barriers. We will accomplish the goal by introducing several key innovations in photonic integrated circuit technology to develop a novel swept source architecture (Aim 1) and a scalable parallel imaging platform (Aim 2). A dual-modality imaging system consisting of a widefield fluorescence microscope and the 3D DyC-OCM will be developed (Aim 3). Validation experiments will be conducted using in vitro 3D human heart and intestinal organoids (hHO and hIO, Aim 4). Given the non-invasive nature of 3D DyC- OCM, together with its high penetration and resolution, we expect to obtain a host of new information on the dynamics of hHO and hIO development over time. This information will be valuable to evaluate how similar (or dissimilar) in vitro organoid development is to embryonic and fetal heart and intestine development and guide new interventions to improve organoid modeling of human development.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Vascular endothelial dysfunction is a key antecedent to CVD in patients with CKD. A primary mechanism of endothelial dysfunction in CKD is a decline in the vasoprotective molecule nitric oxide (NO). Reactive oxygen species (ROS)-related oxidative stress, a key source of which is mitochondria, mediates reductions in NO bioavailability in CKD. Establishing new therapies to enhance NO bioavailability and lower ROS-related oxidative stress to improve endothelial function in patients with CKD is a biomedical research priority. Targeting the nitrate-nitrite-NO pathway represents a promising approach for enhancing NO bioavailability and improving endothelial function in CKD. I have shown in older adults without CKD that targeting the nitrate- nitrite-NO pathway with inorganic nitrite (sodium nitrite) improves NO-mediated endothelial function. The improvements are associated with changes in circulating factors in plasma that reduce endothelial cell total and mitochondria-specific ROS bioactivity. Sodium nitrite also alters the plasma metabolome, and changes in select metabolites with treatment are associated with lower plasma-induced endothelial cell ROS bioactivity. To translate these findings to CKD, I am completing a randomized clinical trial testing the effects of 3 months of treatment with nitrate-rich beetroot juice vs. placebo (nitrate-depleted beetroot juice) for improving endothelial function in individuals with CKD (K01DK115524). My preliminary findings suggest that nitrate-rich beetroot juice improves NO-mediated endothelial function. My preliminary results also suggest nitrate-rich beetroot juice may change circulating factors in plasma to improve endothelial cell function, as shown by an increased acetylcholine (ACh)-stimulated NO production and reduced ROS bioactivity in human aortic endothelial cells (HAECs) exposed to plasma from subjects taken before/after active treatment vs. placebo. The purpose of this R03 application is to leverage samples from my K01-supported clinical trial to show changes in ‘circulating factors’ as a novel mechanism contributing to improvements in endothelial function with nitrate-rich beetroot juice supplementation in patients with CKD and to identify the specific circulating molecular transducers of the benefits of nitrate-rich beetroot juice on endothelial function in CKD. Aim 1: To assess before/after 3 months of nitrate-rich beetroot juice or placebo (double-blind, randomized) in men and women ≥50 years of age with stage II-IV CKD: i) ACh-stimulated NO production; and ii) total and mitochondria-specific ROS bioactivity in HAECs exposed to plasma from subjects before/after the intervention. Aim 2: a) To determine the effects of nitrate-rich beetroot juice on metabolites in plasma via targeted metabolomics analysis of plasma samples taken before and after nitrate-rich beetroot juice treatment vs. placebo; and b) determine NO production as well as total and mitochondrial-specific ROS bioactivity in HAECs cultured with subject plasma with vs. without normalization metabolites changed with treatment.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY. Fungal infections caused by dimorphic fungi Coccidioides immitis and Coccidioides posadasii are endemic to the southwestern parts of the United States and the selected areas in the Western Hemisphere. While most infections are mild and, symptomatic pneumonia can be extremely difficult to treat and requires lifelong azole therapy underscoring the need for new therapeutic options. In this respect, targeting the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway, and, specifically, hybrid histidine kinases (HHKs), represents a promising approach because HHKs are not present in humans but are critical components of cellular responses to oxidative stress in pathogenic fungi. We have identified a natural product candidate that shows potent fungicidal activity against Coccidioides in a murine model. The overarching goal of this collaborative project is to optimize antifungal activity of synthetic analogs as a potential lead for valley fever treatment. In Aim 1, we will perform rational SAR study on a lead candidate by introducing modifications in three distinct parts of the molecule. In Aim 2, we will study the mechanism of action of the lead candidates and fine-tune in silico model to guide rational analog preparation. In Aim 3, we will perform PK/PD studies and validate the activity of the optimized candidate in a murine model of coccidioidomycosis. Collectively, this study will result in a small molecule fungicide with optimized properties for future pre-clinical work.

NIH Research Projects · FY 2026 · 2024-07

ABSTRACT Thirteen percent of adults over 75 suffer from aortic valve stenosis (AVS), a progressive disease that leads to aberrant collagen deposition, leaflet stiffening, and eventual valve calcification at late stages. AVS is also sexually dimorphic with men having a two-fold higher risk for developing direct calcification, whereas women with equal disease severity tend to have more valvular fibrosis prior to calcification. The molecular mechanisms underlying sexual dimorphism in AVS progression remain poorly understood, but growing evidence suggests AVS is an inflammation-dependent disease. Sex is known to influence the magnitude of the immune response and function, so we propose to investigate the role of inflammation in sex-specific valve disease progression. We hypothesize that three major variables contribute to differences in valve calcification in male and female patients: (1) heightened myofibroblast activation in female valve cells when exposed to inflammatory cues, (2) elevated expression of bone mineralization inhibitors, such as osteopontin, in female valve cells, and (3) sex- specific differences in epigenetic machinery. To test these hypotheses, we will develop sex-specific in vitro models of fibro-calcification that are comparable to diseased valve tissue. We will then investigate the sex- specific role that macrophage and valvular interstitial cell (VIC) crosstalk has on AVS (Aim 1) and determine the role of epigenetics in sex-specific regulation of calcification regulators (Aim 2). Ultimately, we aim to identify sex- specific therapies targeted to VIC populations to slow or halt AVS disease progression (Aim 3).

NIH Research Projects · FY 2026 · 2024-07

Project Summary / Abstract Ventral tegmental area (VTA) dopamine neurons are essential for associative learning of reward and aversion- related cues, updating learned associations by prediction errors, as well as supporting a value of rewarding and aversive outcomes. While it is well established that VTA dopamine neurons show heterogenous signaling patterns related to these motivationally-relevant events, as well as express diverse molecular characteristics, our understanding of how dopaminergic molecular heterogeneity contributes to VTA dopamine neuron functions are unclear. A primary distinction between subtypes of VTA dopamine neurons is whether they release dopamine alone or co-release dopamine with glutamate. We propose to test the hypothesis that VTA dopamine neuron signaling patterns and roles in motivated behavior are genetically-determined by whether they co-release dopamine with glutamate or they release dopamine without glutamate. In AIM 1, we will determine the neuronal activity signaling patterns of VTA glutamate-dopamine co-releasing neurons, nonglutamate-dopamine neurons, or glutamate-only neurons that do not co-release GABA or dopamine, in a variety of reward and aversion-based motivated behavior tasks involving associative learning, outcome prediction and errors, and outcome values. In AIM 2, we will causally determine the roles of these same neurons in reward and aversion-based motivated behavior tasks involving associative learning, outcome prediction, reward, and aversion. Finally in AIM 3, we will causally determine the roles of dopamine and/or glutamate release from these distinct VTA neuronal phenotypes in reward and aversion-based tasks involving associative and nonassociative learning as well as other motivationally-relevant behaviors. Together these studies will comprehensively identify how the molecular heterogeneity of the midbrain dopamine system contributes to associative learning and motivated behaviors.

NIH Research Projects · FY 2026 · 2024-07

Project Summary Alzheimer's disease (AD) and related neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are characterized by neuroinflammation and neuronal cell death. Also associated with these diseases is an abnormal deposition of the TDP-43 protein. This protein has multiple roles in RNA metabolism, and we have previously demonstrated that one of its functions is to limit the accumulation of double-stranded RNA (dsRNA). dsRNA rarely accumulates in normal cells (particularly in the cytoplasm), but commonly occurs during viral infection. Thus, abnormal dsRNA is recognized by cells as a sign of infection, leading to the induction of an antiviral innate immune response and the induction of cell death pathways. The central hypothesis we will test in this proposal is that in AD and ALS/FTD the accumulation of dsRNA induces inappropriate activation of antiviral responses that contribute to the observed neuropathology. Our goals are to determine: 1) the underlying molecular mechanisms that lead to dsRNA accumulation, and 2) how dsRNA accumulation leads to glial and neuronal dysfunction. Characterization of pathways that lie both upstream and downstream of dsRNA accumulation in the context of AD, ALS/FTD, and other neurodegenerative diseases has the potential to generate novel therapeutic targets for these currently untreatable pathologies. Our preliminary studies demonstrate that dsRNA accumulates in the hippocampus of Alzheimer's patients and the motor cortex of ALS patients. We will immunopurify and sequence this dsRNA from human brain to determine its origins. Transgenic mouse models with conditional depletion of TDP-43 will be characterized to define the temporal relationships between TDP-43 loss, dsRNA accumulation, gliosis, and neuronal cell death. Infection of human iPSC-derived neural cultures with engineered lentivirus and infection of mouse models with engineered adenovirus will be used to artificially generate dsRNA to investigate the pathways by which dsRNA leads to disease-relevant neuropathology.

NIH Research Projects · FY 2025 · 2024-06

PROJECT SUMMARY Biofabrication is an emerging field that uses the controlled three-dimensional (3D) processing of materials to produce structures (e.g., scaffolds, microparticles, microfluidic platforms) for use in biomedicine. Technologies such as 3D printing, electrospinning, and photopatterning guide material structures to alter cellular behaviors, with impact on the fields of tissue engineering, regenerative medicine, drug delivery, 3D cell culture, and in vitro tissue models, across various tissue and disease systems. In order to capitalize on these advances and to train a future workforce in biofabrication for academia, start-up companies, and mainstream industries, organized educational programs are needed. We propose unique training that connects education in the fundamentals of biofabrication techniques and quantitative methods to clinical applications and product-oriented design to address major challenges in medicine. Our University of Colorado Boulder Biofabrication (CU BioFab) Trainees will experience rigorous training in biofabrication via a newly developed lecture and hands-on course, industrial engagement through coursework product design or participation in an industrial internship, co-mentoring by clinical faculty to provide appropriate context to their PhD projects, an annual retreat to provide opportunities to present their work across all stages of the program, and a bi-weekly seminar course that will harness webinars, interactions with international biofabrication experts, soft skills development, and engagement with entrepreneurship programs. We expect to develop a distinct trainee phenotype that exhibits: (a) decision-making capabilities that balance innovative thinking with practical considerations (clinical applications, entrepreneurship) and (b) leadership skills to make a meaningful impact on society. CU BioFab Trainees will become engineers familiar with biofabrication techniques that are uncommon in traditional or even mainstream biomaterials and bioengineering education, as well as industry and entrepreneurship interactions that are often missing in training programs. CU Boulder is the place for such a training program, due to our extensive expertise across relevant topics through our knowledgeable Preceptors, which include faculty in the Departments of Chemical and Biological Engineering and Mechanical Engineering, as well as interdisciplinary graduate programs in Materials Science & Engineering and Biomedical Engineering. Additionally, the infrastructure on campus, as well as the extensive and growing biotechnology industry within the Rocky Mountain region, will guide the success of this new training program.

NIH Research Projects · FY 2026 · 2024-06

PROJECT SUMMARY Cell-cycle exit can be either transient (quiescence) or permanent (senescence), and these different states are thought to have distinct molecular characteristics. Further, quiescence itself is not a single homogeneous state as cells that remain quiescent for longer durations of time move progressively “deeper” into quiescence, taking longer to return to the cell cycle upon stimulation. While senescent cancer cells are tumor suppressive as they limit the proliferation of damaged cells and recruit the immune system, quiescent cancer cells are a source of drug resistance as they evade chemotherapeutic treatments targeted at cycling cells and retain their ability to proliferate in the future. The mechanisms controlling quiescence depth and the relationship between deep quiescence and senescence are still poorly understood due to the lack of robust biomarkers and necessity for single-cell assays to study questions of reversibility. However, there is a critical need for such studies as chemotherapeutic treatment results in a mix of these cell fates. By using a functional readout that gets at the core of what it means to be senescent: the long-term total lack of cell-cycle entry or progression, we found that a subset of cells pushed into deep quiescence fail to return to the cell cycle after release from treatment and that cells induced to deep quiescence by four different treatments for 2-12 days showed increasing levels of SA-βgal, the gold standard marker of senescence. Thus, we hypothesize that 1) senescence is a deep quiescence from which cells will never reawaken and 2) the transition between deep quiescence and senescence is marked by a decrease in the ratio between pro-proliferative signaling (Cyclin D) and anti-proliferative signaling (p21/p27), mediating the progressive decrease in probability of cell-cycle re-entry. Experiments proposed in Aim 1 will use time-lapse microscopy paired with post-hoc immunofluorescence imaging to measure canonical markers of senescence in cells that remain permanently arrested after release from deep quiescence. In Aim 2 we will perform RNA-sequencing on the subset of permanently arrested cells from Aim 1 and compare the transcriptomes to existing gene signatures for senescent cells. Finally, we will use time-lapse microscopy of biosensors for Cyclin D and p21/p27 to determine if the ratio of these signals predicts future cell-cycle re-entry. Collectively, these aims will help define the relationship between quiescence and senescence and contribute to a much needed quantitative and mechanistic understanding of how cells transition between transient, prolonged, and permanent cell-cycle withdrawal. If successful, the proposed work will aid in the development of more targeted and effective chemotherapy treatments and provide more accurate biomarkers to determine which cells will and will not proliferate in the future.

NIH Research Projects · FY 2026 · 2024-05

Project Summary / Abstract Our strongest memories often stem from our most rewarding experiences, allowing us to learn what features of experience predict reward and to use these predictions in the future. The ability to remember rewarding spatial locations, such as food sources, is crucial for survival. In humans, reward memory can become dysfunctional in memory disorders and mental illnesses like drug addiction, highlighting the need to understand how the brain amplifies information associated with rewards. The hippocampus and medial entorhinal cortex (MEC) comprise a potential neural circuit for this amplification process. Neurons within these regions construct and update a neural map of spatial experience, notably "overrepresenting" reward locations within the neural activity. However, it remains unclear exactly what aspects of the rewarding experience the overrepresentation encodes and how this information is learned. The goal of this proposal is to understand the neural dynamics of how the reward overrepresentation develops in both the hippocampus and MEC and how this process is synchronized within the hippocampal-MEC circuit. To achieve this goal, the proposal combines powerful neural recordings technologies across rodent species, using innovative behavioral tasks that disentangle the reward itself from sensory stimuli, movement dynamics, and the cognitive demand of remembering a specific location which predicts reward. Aim 1 (K99) will use calcium imaging to determine whether changing cognitive demands shape the hippocampal reward overrepresentation over learning. Aim 2 (K99/R00) will use high-density electrophysiology to disentangle reward and cognitive demand in the MEC code, tightly controlling for motor correlates around goals. Aim 3 (R00, pilot K99) will combine simultaneous recordings in the hippocampus and MEC with inactivation of each region to dissect their reciprocal contributions to spatial reward memory and decision-making. This work will build on the candidate’s extensive expertise in electrophysiology and behavior by providing training in three key scientific domains, under lead mentor Lisa Giocomo: (1) computational modeling and statistical analysis with the guidance of co-mentor Scott Linderman, to illuminate how neural coding properties change in different task states; (2) calcium imaging with advisor Jun Ding, to solidify a toolkit to monitor the circuit dynamics underlying flexible coding; and (3) hippocampal and cortical population dynamics with advisors William Newsome and André Fenton, to understand how population activity is structured and coordinated across regions at moments of decision-making. The training plan will build professional skills in inclusive mentorship, lab management, and scientific communication to propel a transition to independence. Stanford University offers a collaborative, interdisciplinary, and supportive environment to pursue cutting-edge science and launch an independent career. The proposed work will provide key insights into how and when spatial reward information is amplified in the brain, building a foundation for the candidate’s career goal: to investigate how neural circuits flexibly shape the information stored in memory according to behavioral demands.

NIH Research Projects · FY 2026 · 2024-05

PROJECT SUMMARY Alzheimer's disease (AD) is the most common form of dementia, and its association with type 2 diabetes is well established. However, the molecular mechanisms underlying this link are not fully understood. Hyperglycemia and dysregulated glucose metabolism have been suggested as factors contributing to the increased risk of AD. This project aims to investigate the role of glycated tau and advanced glycation end products (AGEs) in AD pathology, neurotoxicity, aggregation, and neuroinflammation. The proposed research will pursue the following aims: Aim 1: Investigate how tau proteoforms with specific saccharide modifications control aggregation and neurotoxicity. Aim 2: Expand in vitro studies to other reactive dicarbonyl species operating through a mechanism similar to glycation. Aim 3: Evaluate the effects of glycation, AGE structure, and attachment of DOPEGAL on interneuronal transmission, inflammation, and mitochondrial dysfunction/oxidative stress. These studies will use hiPSC-derived models and will be accompanied by extensive biophysical and biochemical characterizations to develop a molecular understanding of the role of non-enzymatic post-translational modifications in AD. Successful completion of these aims will enable a more precise diagnosis of AD and can potentially lead to novel therapeutic approaches based on the perturbation of non-enzymatic modifications of tau and other proteins implicated in neurodegenerative diseases. Overall, this research will provide important insights into the molecular mechanisms of AD and may lead to the development of new treatments for this devastating disease.

NIH Research Projects · FY 2025 · 2024-05

ABSTRACT The evolution of PC (layer of proteins/biomolecules that adsorbs at the surface of NPs upon contact by biological fluids) in vivo still remains highly challenging due to the complexity of human physiology, the complex nature of the biomolecules adsorbs on the surface, which is one of the main barriers of clinical translation of nanomaterials. The ex vivo approaches for preparation of PC on the surface of the model NPs do not provide homogenous corona formation as commonly perceived in the literature and the composition of the PC varies even across identical NPs with the same sizes in the same batch and using the same biological fluid. The current available PC characterizations or isolation techniques are not able to detect small variations in the composition of the PC and heterogeneity of the PC structure. Therefore, non-specific extraction and pool analysis of the PC in ex vivo condition may cause error and/or misinterpretation of the PC outcomes which significantly influence the clinical translation of NPs. We show in the preliminary data that although the standard MagLev systems are able to separate PC coated and uncoated NPs however, it is not able to discriminate among PC coated NPs with various PC composition at the surface of the NPs due their very similar density and low resolution of the standard MagLev systems. In this exploratory research we aim to develop a higher sensitivity MagLev system to show the heterogeneity and variation of PC composition on the surface of the NPs, a critical ignored factor in clinical translation of nanomaterials and failures of nanomedicine. In such highly sensitive MagLev configuration, the resolution enhances 1 to 3 orders of magnitude (compared with sensitivity of standard MagLev system (i.e., 10-3 g/cm3)) which is high enough to separate PC coated NPs with different PC coverages/compositions (e.g., number and type of the proteins at the surface) and very slight differences in the density (ranging from 0.001 g/cm3 to 0.00001 g/cm3 which is not detectable using standard MagLev system). Our preliminary data using a prototype high sensitivity MagLev system as proof-of-concept, confirms that how identical objects (i.e., commercial identical nylon particles) with the same density and levitation heights in the standard MagLev system have significant different levitation heights in a high sensitivity (prototype) MagLev system indicating low resolution of the standard of the MagLev systems to detect such small variations/changes. The current state-of- the-art in this type of separation is that there are simple proofs-of-concept of many of its foundational concepts, but little use of fully developed methods by biologists and clinicians. This project will both solidify the fundamental biophysical science of MagLev as a simple analytical tool in biochemistry and provide demonstrations of uses for nanomedicine applications.

NIH Research Projects · FY 2026 · 2024-04

Project Summary / Abstract Social avoidance and exaggerated threat responding are transdiagnostic constructs related to stress-linked disorders such as anxiety and post-traumatic stress disorder. We have found that these behaviors are recapitulated in mice following exposure to uncontrollable, but not physically identical, controllable stress. Further, we have found that the newly identified group of neurons within the ventral tegmental area (VTA) that release the excitatory neurotransmitter glutamate mediate social avoidance and enhanced fear that result from uncontrollable stress. However, VTA glutamate neurons are diverse in their projection targets and are capable of releasing GABA or dopamine, in addition to glutamate, to distinct targets. We aim to determine which downstream pathways of VTA glutamate neurons are affected by and control the behavioral consequences of uncontrollable stress as well as identify the neurotransmitters within these pathways that control the outcomes produced by uncontrollable stress. In Aim 1 we will determine the responsivity of VTA glutamatergic axons in their two major projection targets, nucleus accumbens shell and lateral habenula, to inescapable or escapable stress treatment, as well as during the outcomes produced by uncontrollable stress. Our preliminary data suggests that inescapable stress potentiates VTA glutamatergic axons in lateral habenula and accumbens shell following fear-inducing stimuli, but in different ways. We will also determine which downstream target of VTA glutamate neurons is responsible for the development and maintenance of the behavioral consequences of inescapable stress via select optogenetic photoinhibition of VTA glutamate axons. Preliminary data support a role of the VTA glutamate pathway to LHb in the development of both social and fear-related consequences of inescapable stress. Given that VTA glutamate neurons are capable of releasing glutamate, dopamine, or GABA, we will identify which neurotransmitters released by VTA glutamate neurons are required for the social and non-social consequences of inescapable stress. Additional studies will monitor how the loss of these neurotransmitters from VTA glutamate neurons affects habenula and accumbens stress-induced activity. Together the studies will identify at the intersection of cell-type, pathway, and neurotransmitter-specificity, how stressor experience alters social, fear, and anxiety-like behavior.

NIH Research Projects · FY 2025 · 2024-04

Project Summary The overall aim of this project is to determine the contribution of the rostral region of the posterior hypothalamic nucleus (rPH) in stress adaptation. More specifically, stress habituation is defined as a progressive reduction in responses upon repeated exposures to the same (homotypic) stressor. Psychological or “emotional” stressors elicit a range of behavioral, autonomic, and neuroendocrine responses that normally help organisms cope with perceived or actual threatening situations. However, prolonged, unabated and chronic stress exposure is frequently associated with the development, precipitation, or exacerbation of several psychopathologies and physical disorders. The sustained activation of stress responsive systems can negatively affect physiologic functions and ultimately, survival. Thus, the reduction or inhibition of stress-elicited responses afforded by habituation is likely a vital mechanism serving to reduce the cumulative impact of the same stressors experienced repeatedly. Importantly, impaired habituation has been reported in several human clinical populations including anxiety and mood disorders. Unfortunately, the neural mechanisms responsible for stress habituation are still ill- defined, which makes it currently impossible to evaluate the contribution of habituation in psychopathologies. In this application, robust habituated neuroendocrine, autonomic and behavioral responses will be obtained to repeated homotypic stress paradigms that will test the response specificity and generality of the proposed brain manipulations. Based on detailed anatomical and functional results from our laboratory, emphasis will be placed on the role of the rPH in the acquisition of stress habituation. Anatomically, the rPH is located at a focal intersection between several sensory, limbic forebrain, and effector response systems activated by various stressors, placing it in a unique position to undergo habituation-related plasticity that can regulate coordinated responses to stress. The first aim is therefore directed at evaluating the novel hypothesis that the rPH region, and some of its specific cell populations, contribute to the acquisition of stress habituation. Aim 2 seeks to evaluate the possibility that intrinsic rPH neural activity is modified by repeated homotypic stress. Throughout these aims, both female and male rats will be evaluated for putative rPH-related differences in stress adaptation due to the differential prevalence of gender to anxiety and mood disorders and the widely reported sex differences in habituation to stress. The proposed anatomical, functional and behavioral analyses of the rPH in stress adaptation through habituation is novel and will provide converging evidence implicating this brain region in stress adaptation and its sequelae.

NIH Research Projects · FY 2026 · 2024-04

Project Summary In the current paradigm of protein degradation by the ubiquitin-proteasome system, ubiquitination of protein substrates has been considered as a major regulatory step. Hundreds of ubiquitinating enzymes regulate polyubiquitination, to target proteins to the proteasome for degradation. This paradigm has led to remarkable discoveries on how protein degradation is required for fundamental cellular processes, and also for destroying misfolded or aberrant proteins, which can lead to cancers and neurodegenerative conditions. However, a significant knowledge gap exists in the current paradigm, as to how the cell ensures the proper number of functional proteasomes. Our research goal is to overcome this gap, by elucidating mechanisms of proteasome assembly. We discovered multiple evolutionarily conserved chaperones that are dedicated to proteasome assembly. In our model of proteasome assembly, chaperones hinder subunit addition until a given step of assembly occurs correctly. This model suggests a new concept, that assembly is not simply a series of subunit additions—it is also integrated with extensive quality control (QC). We will pursue this new direction of research—unraveling mechanisms of chaperone-mediated QC during proteasome assembly. For QC, the assembly process is a “moving target” as one step progresses to the next. How can these moving targets be monitored? Chaperones can “mark” all of them, using a fundamental mechanism—specifically binding to assembly intermediates. We will investigate how chaperones serve as components (e.g. adaptors) of extensive QC of the proteasome, via three aims. First, we will examine chaperones’ new connections to proteasomal nuclear localization signals (NLSs), which have been known for ~20 years to exist on proteasome subunits, but their functions are poorly characterized. We hypothesize that proteasomal NLSs drive chaperones into the nucleus, as a QC mechanism, to prevent defective proteasomes from forming in the cytoplasm. Second, we will investigate the non-canonical role of ubiquitination via an E3 ligase, Not4, during proteasome assembly. Bulky polyubiquitin sterically prevents the addition of the next subunits, blocking proteasome assembly, for QC. We hypothesize that this non-canonical role of polyubiquitin depends on a specific chaperone, Nas6 (an oncoprotein, Gankyrin in humans). Third, we will interrogate how chaperones help form proteasome storage granules, which preserve functional proteasomes during nutritional stress. We hypothesize that chaperones help distinguish functional proteasomes—via binding to sub-complexes—but in this case, resulting from proteasome disassembly during nutritional stress. We will interrogate these hypotheses using our well- established biochemical, cell biological, and proteomics strategies, together with our powerful yeast model to assess proteasome functions in vivo. Proteasome assembly chaperones are dysregulated in many cancers, altering proteasomal activities. Since the proteasome is a proven therapeutic target, we envision exploiting these chaperones to modulate cellular protein degradation, to broaden the application of the proteasome as a target.

NIH Research Projects · FY 2025 · 2024-04

Project Summary Transposons are ubiquitous genomic elements that can mobilize, replicate, and integrate within the host genome. Extensive research has focused on how transposons impact their host, revealing instances of both deleterious and selectively advantageous roles. While transposon exonization (defined as the inclusion of transposon-derived exons into cellular transcripts through alternative splicing) is common, the biological relevance of transposon-derived transcript isoforms remains unclear and controversial. Here, I propose to investigate transposon exonization in coding regions as an important, recurrent evolutionary mechanism that drives adaptive proteome diversification, specifically in the context of host-pathogen arms races. In preliminary studies, I re-analyzed long-read transcriptome data from human macrophages and discovered hundreds of poorly characterized isoforms derived from transposon exonization events for genes involved in immune and inflammatory responses. These include a transposon-derived isoform of the type I interferon receptor subunit 2 gene (IFNAR2), which I have experimentally shown to act as a novel immune decoy receptor. I will use alternative splicing of the IFNAR2 gene as a model to reconstruct the genomic events that can lead to the successful exonization of transposons to form functional protein isoforms (Aim 1), with the goal of reconstructing the genetic basis of the “road to co-option” of exonized transposons. Using IFNAR2 as a model, I will then investigate which mechanisms and factors are involved in the regulation of alternative splicing and expression of transcribed transposon-derived coding alternative exons (Aim 2). Finally, I will compare long-read RNA sequencing de novo transcriptome assemblies from multiple species of the vertebrate tree of life to evaluate the incidence of proteins that evolved through the inclusion of transposons in coding sequences (Aim 3). Taken together, these aims will establish an experimental framework to test the role of transposable elements in adaptive proteome diversification, and shed light on the process of how transposons can evolve novel functions beneficial for their host (transposon co-option) through their inclusion in coding regions. Additionally, they will provide me with new training in: 1) techniques for testing and validating mechanisms of mRNA and alternative splicing regulation; 2) how to design and perform immunological assays, including viral infections; and 3) the execution and troubleshooting of genome-scale screens for phenotypes of interest and training in primary cell culture and genome engineering. Additionally, I will learn how to perform high throughput long-read RNA sequencing and isoform-specific transcriptome assembly for model and non-model species. The research and training provided by this proposal will prepare me to launch my own research group studying how transposon co-option shapes the evolution of vertebrate proteomes.

NIH Research Projects · FY 2026 · 2024-04

PROJECT SUMMARY/ABSTRACT Insufficient sleep is associated with inflammation and adverse health. Inflammation modulates sleep and, conversely, insufficient sleep induces inflammation. A bi-directional relationship between disrupted sleep and inflammation represents a positive feedback loop that negatively impacts health; however, a substantial knowledge gap exists regarding the molecular mechanisms by which inflammation induces and/or contributes to sleep disturbances. Glia, specifically microglia, may facilitate the bi-directional interactions between neuroinflammation and sleep. Although microglia serve an overall beneficial role, prolonged microglial activation drives neuroinflammation and subsequent neurodegeneration. Interluekin-10 (IL10), an anti-inflammatory cytokine released by microglia, inhibits the synthesis of pro-inflammatory cytokines; hence IL10 may be an ‘off- switch’ for chronically activated microglia. Preliminary results from the PI’s laboratory demonstrated that depleting microglia (<0.5%) does not change central cytokine concentrations. However, under inflammatory conditions, induced by lipopolysaccharide (LPS) administration, mice with depleted microglia have elevated pro- inflammatory cytokines, a sustained increase in NREM sleep, and significantly lower levels of IL10 compared to control mice. These data suggest that microglia are necessary to modulate sleep triggered by inflammation. Importantly, preliminary results indicated IL10 administration normalizes sleep after an inflammatory challenge. We further show that IL10 administration after LPS in mice with depleted microglia, normalizes sleep and is linked to an immediate increase in IL10 in the brain, as well as rapid microglia repopulation/activation in the brain. This has led to the overarching hypothesis that microglia-mediated IL10 signaling is part of feedback mechanisms that are responsible for normalizing sleep after an inflammatory challenge. This hypothesis will be tested through the following specific aims: 1) Investigate the role of microglia and the IL10 axis in inflammation- induced sleep disturbances; 2) Determine the influence of microglia-mediated IL10 on other central nervous system cells after an inflammatory challenge; and 3) Investigate the extent to which IL10 replacement normalizes sleep after an inflammatory challenge. Impact: Successful completion of these studies will elucidate novel mechanisms that underlie inflammation-induced sleep disturbances and may open a new line of intervention to treat inflammation-induced sleep disturbances.

NIH Research Projects · FY 2025 · 2024-04

PROJECT SUMMARY/ABSTRACT Cell division requires spatial organization of cellular contents for its successful completion. The centrosome, which ensures appropriate chromosome segregation, is an example of a membraneless organelle which uses physical mechanisms other than segregation by lipid membranes to create spatial organization. Mutations of centrosomal proteins are associated with diseases such as ciliopathies, primordial dwarfism, neurodegeneration, and cancer. It is critical to understand the mechanisms that control the assembly of the centrosome to better understand the pathophysiology of related disorders. Liquid-liquid phase separation (LLPS) is thought to be the process by which centrosomes form, based on recent evidence that several centrosomal proteins, such as pericentrin in humans, are capable of this process. Centroso- mal proteins are enriched with both coiled-coil (CC) domains and disordered regions, but it is not yet known how these domains contribute to the LLPS of the centrosome. The central hypothesis of this proposal to be tested is that centrosomal proteins, such as pericentrin, use coiled-coil domains as the key drivers of LLPS. Our preliminary work with simulated and synthetic protein systems suggests that CC domains have physical features that enable them to drive protein LLPS. The ability of CC domains to mediate protein-protein interactions and the abundance of these domains in pericentrin makes them a good candidate for facilitating LLPS. However, CC-driven LLPS in biologically-relevant natural proteins systems has not yet been explored. This central hypothesis will be tested by pursuing two aims. In Aim 1, I will develop a chemically realistic simu- lation framework to predict likely CC domains that drive LLPS in natural proteins. I will use bioinformatics-based approaches to generate a sequence-specific interaction framework for molecular modeling. Simulations of natural CC proteins that phase separate will then be performed to identify possible interactions between CC domains that might support LLPS. In Aim 2, I will test the contribution of CC domains to the phase separation of peri- centrin, our model centrosomal protein, by using a combination of cellular and in vitro biophysical approaches. I will systematically mutate CC domains in pericentrin and find those that drive LLPS through in-cell visualization of deficient centrosome formation. The domains that drive LLPS will then be assembled into truncated protein constructs to assess the sufficiency of these regions to cause LLPS in vitro. Additional biophysical experiments will be used to characterize the contacts between CC domains in the synthetic protein to provide a mechanistic picture of pericentrin self-assembly. The outcomes of this proposal will contribute to our understanding of centrosome biogenesis and demonstrate how CC domains might mediate biological LLPS. This research will be performed as part of a fellowship plan with a carefully selected mentoring team which will provide me with individualized advanced training in computational methods and experimental approaches, as well as scientific and career guidance, positioning me for a successful career as an independent scientist.