Colorado State University

NIH Research Projects · FY 2026 · 2016-09

Project Summary/Abstract At the heart of cellular identity and function lies gene expression, the dynamic flow of information from DNA to RNA to protein. Our lab aims to understand how this process is orchestrated in real time, at the resolution of individual molecules and within the native context of living cells. We are particularly focused on uncovering regulatory mechanisms that remain hidden using conventional techniques, what we refer to as cellular “dark matter.” To achieve this, we develop and apply fluorescent intrabodies – engineered antibodies that function inside cells – to visualize key molecular events without genetic editing or fixation. These intrabodies allow us to track native protein modifications, RNA dynamics, and translation in living cells with unprecedented spatiotemporal precision. In the next phase of our research, we will integrate these intrabodies with advanced single-molecule imaging and targeted perturbation to address three fundamental questions in gene regulation. First, we will test whether specific histone modifications causally drive gene activation or silencing by directly imaging epigenetic editing events in living cells. Second, we will investigate how cells regulate ribosome traffic on highly translated transcripts to maintain efficient protein synthesis and avoid ribosome collisions. Third, we will explore how cells coordinate transcription and translation, focusing on mechanisms of translational buffering that prevent protein overproduction during persistent transcriptional upregulation. By merging novel intrabody technologies with live-cell imaging and quantitative modeling, our work will shed light on how cells control gene expression with speed, precision, and flexibility. These insights will enhance our ability to interpret and manipulate gene regulatory networks in development, disease, and synthetic biology.

NIH Research Projects · FY 2025 · 2016-08

Structural genomic variation has only recently come into focus as a major source of genetic diversity in humans, and in biology in general. Despite its critical importance, we still have a very limited understanding of the processes that cause the structure of genomes to change over time, and of the consequences of these large-scale changes to living organisms. Over the last four years of MIRA support, my laboratory has been using two parallel and integrated approaches to study this problem, taking advantage of the unique research tools available in the budding yeast model system. Our work has been fruitful. We have significantly advanced the boundaries of our research field, while also contributing to the development of a new generation of rigorously trained, creative young scientists. In the next funding cycle (1), we will continue to investigate the forces that cause chromosomes to break, and the cellular mechanisms that are responsible for preventing, surveying, and repairing this damage. To do so, we will use custom and highly sensitive cell-based assays to measure the rate of gene copy number variation (CNV), Loss-of-Heterozygosity (LOH), and whole chromosome gains and losses (aneuploidy), both in mitotic and meiotic cells. We will also deploy advanced genomic analysis tools to characterize the associated structural changes. In addition, we will continue to expand on a brand-new investigation front we opened through work carried out during the current funding cycle. Specifically, we recently reported on a new form of structural mutagenesis (systemic genomic instability, SGI), through which cells can acquire multiple rearrangements simultaneously, and thus radically reconfiguring their genomes. In addition (2), we will also investigate the phenotypic consequences associated with chromosomal rearrangements in a diploid yeast strain that shares many of the properties that characterize the complex human genome. These include a high degree of heterozygosity, structural chromosomal polymorphisms between homologs, gene redundancy, and CNVs; all the while retaining the small and manageable genome of S. cerevisiae. I strongly believe that by opening these new and integrated avenues of investigation, in close partnership with the talented junior colleagues I mentor in my laboratory, we will contribute much needed insight into how structural genomic variation arises and how it affects all aspects of life, from the evolution of species to human health.

NIH Research Projects · FY 2025 · 2016-08

PROJECT SUMMARY Despite their diminutive length, 20-30-nucleotide-long small RNAs affect nearly all developmental and disease processes, from fertility in flies to cancer in humans. Small RNA associate with Argonaute proteins to direct sequence-specific degradation or translational repression of matching mRNAs in a process called RNA interference (RNAi). Small RNAs can also function in an alternative mode to promote gene expression. Within the germline of the tiny nematode worm, Caenorhabditis elegans, two broad classes of small RNAs – piwi- interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – interact with nearly all genes, silencing some and promoting the expression of others. Remarkably, some piRNAs and siRNAs are transmitted from one generation to the next, providing a heritable mechanism for regulating gene expression without changes to the underlying DNA. In C. elegans, piRNAs and siRNAs are required for optimal fertility and germline immortality. We and others identified a role for maternally-derived piRNAs and siRNAs in protecting essential genes from silencing. We also showed that these small RNAs have a role in establishing proper gene expression in the embryo that is crucial throughout development, although the mechanism underlying this phenomenon requires further study. To identify the roles of piRNAs and siRNAs in ensuring proper gene expression from one generation to the next, we will address two related questions: 1) How do piRNAs and siRNAs regulate gene expression to promote fertility and germline immortality? and 2) What are the molecular roles of maternally deposited piRNAs and siRNAs? A second area of my research centers on gene regulatory mechanisms involving a third class of small RNAs, called microRNAs (miRNAs). We recently uncovered a distinct branch of the miRNA pathway required for proper developmental timing and optimal fertility in the germline. How this pathway regulates gene expression is an important area of future research. This relates to the third question we will address: 3) How do miRNAs regulate developmental timing in the germline? miRNAs are processed from a limited number of transcripts that form hairpin-like secondary structures. The hairpin itself is not sufficient to mark a transcript for processing, and in many species, including humans, primary transcripts contain additional sequence elements that promote miRNA formation. Not all miRNA transcripts contain these elements and in some animals, including C. elegans, they are completely lacking. We developed a sensor that reports on miRNA transcript recognition. Using the sensor, we will address a fourth question: 4) How are miRNA transcripts distinguished from other RNAs in C. elegans? The ease in which genetics, genome editing, and genomics assays can be done in C. elegans makes it an ideal system to address these four questions. The mechanism of miRNA formation and the molecular roles of miRNAs, piRNAs, and siRNAs are highly conserved in animals. The knowledge gained through this study will have important implications in our understanding of the counterparts in humans and how their dysfunction contributes to sterility and disease.

NIH Research Projects · FY 2026 · 2007-05

Arthropod-borne viruses (arboviruses) adapt to local conditions, maximizing their potential to perpetuate and emerge as health threats. The adaptive potential of arboviruses is driven by error-prone replication, which creates a genetically diverse pool of competing virus genotypes within each host. This proposal examines some of the ways that temperature may impact arbovirus evolutionary biology. Our previous research has allowed us to make clear predictions about the outcome of each proposed aim, and generated molecular and computational tools, and methodological approaches that we propose to combine in this project. Global temperatures are changing at an unprecedented rate, and RNA viruses such as WNV continue to emerge at a frightening pace. Our preliminary studies have shown quite clearly that temperature is a key factor that dictates how natural selection affects arboviruses within mosquitoes. Therefore, Aim 1 will address how temperature, both constant and fluctuating, with varying means and amplitudes, alters natural selection on WNV within mosquitoes and the strength of bottlenecks. Our predictions (in general) are that fluctuating temperatures will increase the strength of purifying selection, that diversity will be maximized at optimal constant temperatures, and that bottlenecks will become wider as temperature increases. Our results have demonstrated that flavivirus infections are most frequently initiated by aggregates of virus particles. Aim 2 will address the extent that this occurs in a host- and temperature-dependent manner, bringing our previous work into a more ecologically relevant, realistic context. In the second phase of Aim 2, we will ask whether these genome aggregates can help to facilitate the maintenance of genetic diversity in the WNV population. Birds that generate high WNV viremia and are highly infectious to mosquitoes have significantly more unique WNV genomes per cell than those that have lower viremias. Aim 3 will assess whether something similar may occur in mosquitoes. We will use barcoded WNV to infect mosquitoes with a range of vector competence and assess the number of unique WNV genomes per cell. As above, we also will assess the degree to which this phenomenon may allow for the maintenance of low fitness viral genotypes while preventing those of high fitness from gaining dominance. This work will provide comprehensive data on the ways that changing environmental conditions may alter the fundamental population biology of arboviruses. Our work is also significant because it will provide mechanistic data on how viruses may maintain genetic diversity in the face of both selective and stochastic reductions in genetic diversity. The proposed studies are technically and conceptually innovative because of the ways that we can combine realistic transmission systems in the lab with barcoded viruses, single cell approaches, and other new molecular tools.

NIH Research Projects · FY 2026 · 2004-04

The picornaviruses are a family of small positive-sense single-stranded RNA viruses that cause a wide range of diseases at an annual cost well into the hundreds of million dollars. Members include paralyzing poliovirus and enterovirus D68, the heart disease causing coxsackie B3 virus, and rhinoviruses that cause more than half the occurrences of the common cold. These viruses share a life cycle where RNA replication and viral assembly occurs in large membrane anchored replication complexes assembled and the replication process is driven by a virally encoded RNA dependent RNA polymerase (3Dpol) that is responsible for the synthesis of all viral RNA. This research project is focused on the structure and assembly of the viral replication centers and on structure-function studies of the viral polymerases to understand mechanisms that control elongation rates and replication fidelity. We will use biochemical and structural biology approaches to study how the fidelity checkpoint used in 3Dpol was rearranged as these polymerases evolved to support large-genome coronavirus replication, providing novel information about functional constraints to help us understanding pathways for virus evolution. In a second aim we will elucidate the mechanisms whereby RNAs interact with and stimulate viral proteases, a finding that suggests viral RNA elements can regulated polyprotein processing the context of a viral replication center. Last, we will solve the structure of the picornaviral uridylylation complex that generates the VPg-pUpU primers used for all RNA synthesis by the viral 3Dpol polymerase.

NIH Research Projects · FY 2025 · 2000-07

ABSTRACT/SUMMARY Multiple studies by domestic workshops and committees have concluded that the number of veterinary scientists trained in biomedical and infectious disease research falls far below national needs. The goal of this training program is to continue to address this personnel gap by providing PhD training in molecular and mechanistic research methods to enable post-DVM/VMD candidates to conduct translational research. The training program is built upon our strong history and experience in post-DVM research training at Colorado State University in the College of Veterinary Medicine and Biomedical Sciences (CVMBS) and melds molecular, multidisciplinary methodologies with translational application. Critical thinking in experimental design and data interpretation, manuscript and grant writing, publication, communication skills, and ethical conduct of research are stressed in this balanced and well-mentored program. New to this application is: (1) integration with the University of Colorado Anschutz Medical Campus CTSA (Colorado Clinical Translational Science Institute, CCTSI) TL-1 training program; (2) greater emphasis and goals to increase diversity, equity, and inclusion; and, (3) additional emphasis on mentoring and professional development, with a particular focus on trainee NIH K award preparation. An exceptional and diverse External Advisory Board has been assembled to assist in strategy to further enhance the goals of this renewal application. The program action plan is to recruit rigorously selected, diverse, post-DVM/VMD candidates and provide training in translational research applications emphasizing experimental and/or natural disease animal models. The Program is fueled by an abundant supply of talented candidates and a large, elite faculty of well- funded mentors representing 18 research concentrations and all four departments of the CVMBS. Mentor faculty comprise 18% of CVMBS faculty and were awarded $22.8M in direct cost dollars in the most recent fiscal year. Ninety-four percent of completed trainees since grant inception (34/36) are currently employed in research related positions; 7 of 8 trainees (88%) who have submitted NIH K Series Career Development Awards have been funded; and 7 of 39 completed or enrolled trainees are from under- represented minority groups. All 15 appointees during this grant cycle have been women. The targeted outcome of the program is to continue to produce DVM/VMD-PhD scientists who emerge prepared as successful, funded principal investigators and contribute to translational biomedical research that addresses pressing and emerging problems and challenges in human, animal, and environmental health (One Health). The intent is that this training program represents a targeted action in response to a demonstrated need for more translational scientists to fill an alarming deficiency in the biomedical workforce and contribute to national and global research demands.

NIH Research Projects · FY 2026 · 1983-09

Project Summary This ongoing integrated epidemiologic and etiologic research study (Our Youth, Our Future) is the only source of nationally-representative substance use data for reservation-area American Indian (AI) adolescents, a population that is at-risk and experiences large health disparities. Substance use rates among AI adolescents have been greater than national rates for decades, and our current findings point to complex changes since the onset of COVID-19. Each year, 3,000 or more 6th - 12th grade reservation-area AI students will be surveyed to provide nationally-representative rates of substance use for this population. In collaboration with Monitoring the Future researchers, these rates will be compared with national rates to identify areas of special concern for reservation-area AI youth. In addition, age, period, and cohort effects will be distinguished by combining our past data with data gathered in the upcoming cycle, emphasizing substance use before, during, and after the onset of COVID-19, to identify changes in substance use and related constructs due to historical (e.g., COVID- 19), regulatory, and normative factors. Finally, we will examine heterogeneity in SU disparities among reservation-area American Indian youth across intersections of sociodemographic identities and contextual variables using intersectional multilevel analysis of individual heterogeneity and discriminatory accuracy. Collecting data from multiple reservations across the country yields a unique opportunity to conduct the first systematic assessment of Social Determinants of Health (SDOH) impacts on substance use and related constructs for this population. A wide range of SDOH factors specific to reservation communities will be compiled in order to identify how SDOH factors either contribute to or shield against use, providing a comprehensive perspective on the interplay of vulnerabilities and strengths within AI communities. Building etiologic evidence for substance use prevention efforts is also a key part of this project. Although AI youth are influenced by broader society, unique individual, cultural, and contextual factors influence substance use. An Ecosystemic Resilience Framework (ERF) will guide examination of the ecosystemic resilience processes involved in the relationships of adversity to substance use and its comorbidities in reservation-area AI youth, with particular emphasis on unique cultural and community factors. This will provide actionable insights for prevention efforts. Additionally, we will expand the ERF to incorporate an intersectional analytic approach to examine how ecosystemic risk and resilience processes impact SU among AI youth occupying different socio-demographic intersections and social contexts. Project findings will be disseminated to key stakeholders, with special emphasis on tribal and non-tribal entities involved in policy and resource-related decisions that affect AI youth substance use.