University Of Texas At Austin

NIH Research Projects · FY 2025 · 2016-09

SUMMARY Cilia are essential organelles, with functions ranging from cell-cell signaling to the generation of homeostatic fluid flow in tubular organs. Consequently, an array of human congenital diseases has been characterized as “ciliopathies,” because they share an etiology of defective cilia structure or function. Despite clear roles in the development of the central nervous system, limbs, axial skeleton, kidneys, airway, brain, and reproductive tracts, our understanding of the mechanisms that govern ciliogenesis and cilia-mediated developmental patterning remain incomplete, not least because hundreds of different proteins are required for proper cilia biogenesis and function, acting via an extensive interaction network containing diverse proteins of unknown function. We propose here to study several large multi-protein assemblies that are essential for proper cilia formation in order to determine the roles of these complexes in key steps in ciliogenesis, including recruitment of proteins to the basal body, intraflagellar transport and recruitment of specific intraflagellar cargoes. This grant combines directed mechanistic experiments, proteomics, 3D modeling, in vivo cell biology, and testing of human disease alleles in model organisms to understand mechanisms by which key ciliary proteins and their interaction partners effect proper cilia formation, and how specific mutations in these genes lead to birth defects. By focusing on proteins with demonstrated importance in development and disease, but for which no mechanism of action is yet known, experiments proposed here will provide important new breadth and depth to our understanding cilia-mediated developmental patterning and novel cell processes in ciliary biology. In turn, these findings should provide greater insight to a range of congenital diseases ranging from the relatively mild Oral-Facial-Digital syndrome to the wholly lethal Short Rib Polydactyly.

NIH Research Projects · FY 2025 · 2016-08

Project Summary / Abstract Genomic methods have catalyzed a renaissance in the way that microorganisms are being studied. Previously, most knowledge of microbial processes and functions was obtained from a very limited number of tractable genetic systems. In contrast, current focus on microbiomes has shifted emphasis towards understanding a broad diversity of lifeforms that are only known from sequences and whose functions are inferred by homology to known or characterized organisms. Among the primary goals of our research program is to bridge these two approaches by adopting genomic, computational and experimental methods that directly test the roles, functions and adaptations of the non-model organisms that predominate in microbiomes. The first of the three Subject Areas proceeds from our findings that not only is there a high degree of host species-specificity in the contents of gut microbiomes but certain bacterial lineages have been co-diversifying with their hosts over millions of years. We will investigate how this co-diversification has progressed within the human population, and determine if and how these co-diversifying bacteria interact with hosts by testing their adaptive role in a dietary trait that is subject to strong selection in humans. We will also gain insights into the evolutionary history of the human microbiome by analyzing the new resolved strain-level variation detected in microbiome in a population genetics framework. The second Subject Area asks how new genes and functions originate in bacterial genomes. Most models of new gene formation are based on the duplication and modification of existing genetic information, and ignore the more fundamental question about how completely new genes can arise de novo. Whereas in eukaryotes, non-coding sequences seem to serve as templates for de novo gene origination, in bacteria there is strong, though indirect, evidence that phage may be the inventive source of new genes. The proposed research will determine the mechanisms by which new genes originate by testing the functional relevance and beneficial effects conferred by new viral sequences integrated into bacterial genomes. The third Subject Area proposes to address several questions concerning the role of viruses in microbial communities using a newly developed experimental system derived from viruses known previously only from metagenomic sequences. This system will allow us to resolve issues relating to viral host ranges, lifestyles and interactions—all questions that would remain as inferences, or wholly unanswered, if only genome sequences are available. Finally, we our plan to define the limits of gene exchange among viruses both to establish uniformity in their classification into meaningful biological units and to understand the potential for strains with new etiologies, distributions, and host tropisms to emerge.

NIH Research Projects · FY 2025 · 2016-05

Virus-host interactions and microbial ecology This proposal encompasses two very different aspects of microbiology, both at cellular and group levels. (1) Probing E. coli genome organization and chromosome dynamics using phage Mu transposition as our tool. Mu transposition is unique not only in its high efficiency and lack of target specificity, but also in its transposition mechanism, which occurs by a nick-join rather than a cut-and-paste pathway. In the last grant period, we exploited these properties to measure in vivo rates of interactions between genomic loci in E. coli, and studied their proximity using new statistical tools. In a complete reversal of the current view of the E. coli genome, our analysis has revealed an uncompartmentalized, well-mixed genome, where transpositions occur freely between all measured loci. The analysis also revealed that several gene families (for example, six widely distributed ribosomal RNA operons) show `clustering' i.e. strong 3D co-localization regardless of linear genomic distance. The activities of the SMC/condensin complex MukBEF and the nucleoid-compacting protein HU-α are responsible for these properties. We propose to explore these phenomena to obtain a high- resolution view of genome organization, and to understand how it influences gene expression in bacteria. (2) Dissecting the mechanism of antibiotic tolerance under two specific growth conditions: swarming (moving as a collective), and c-di-GMP synthesis catalyzed by the diguanulate cyclase YfiN. Swarming bacteria can withstand exposure to antibiotics at concentrations that are lethal to their planktonic counterparts. We call this swarming-specific (non-genetic) resistance, SR. In the last grant period, we discovered that death of a sub- population as a result of antibiotic-induced killing, is beneficial to the swarm in promoting SR. Introduction of pre-killed cells into a swarm indeed enhanced SR, allowing us to purify the SR factor from killed cells of both E. coli and Salmonella. We identified the SR factor to be AcrA, a periplasmic component of a tripartite RND efflux pump; the outer membrane component of this pump, TolC, is also a constituent of multiple drug efflux pumps. We showed that AcrA stimulates drug efflux in live cells by interacting with TolC from the outside, activating efflux in the short term, and inducing the expression of other classes of efflux pumps in the long term, thus amplifying the response and establishing SR. We have called this phenomenon `necrosignaling', and discovered species-specific necrosignaling in both Gram-positive and Gram-negative bacteria. We also discovered that production of c-di-GMP by the specific cyclase YfiN, arrests cell growth to promote an antibiotic-tolerant persister-like state. We propose to explore both these responses further. Given that non- genetic resistance is a known incubator for evolving genetic resistance, our findings are relevant to the current widespread emergence of genetic resistance to antibiotics, and may be relevant to chemotherapy-resistant cancers, which efflux the drugs prior to acquisition of genetic resistance.

NIH Research Projects · FY 2026 · 2016-01

ABSTRACT The essential metal manganese (Mn) accumulates in the basal ganglia at elevated levels and induces motor disease. Mn-induced motor disease is historically associated with occupational Mn exposure in adults. However, environmental exposure to elevated Mn has emerged as a more recent public health problem in infants, children and adolescents. Environmental Mn exposure in early-life substantially impairs motor function. But, the biology of Mn-induced motor disease in early-life is poorly understood, and there are no treatments for this condition. The fundamental question of how early-life, environmental Mn exposure impacts dopaminergic (DAergic) or GABAergic neurons in the basal ganglia that control movement to induce motor deficits is unanswered. This major knowledge gap exists because prior mechanistic work focused on the effects of Mn in adults. But, results from adults cannot be directly applied to early-life periods because environmental exposures are vastly different from occupational, early-life stages are more sensitive to Mn, and the impact of occupational Mn exposure on basal ganglia neurons is itself controversial. Our goal is to establish the effects of early-life, environmental Mn exposure on basal ganglia DAergic and GABAergic neurons that lead to motor disease. We will use innovative mouse models developed in the previous cycle that provide the feasibility, for the first time, to alter Mn levels specifically in DAergic or GABAergic neurons and isolate the role of the targeted neurons in Mn-induced motor disease. Our models are based on the neuron-specific knockout or knockin of the critical Mn efflux transporter SLC30A10. Pan-neuronal Slc30a10 knockouts had increased basal ganglia Mn levels and exhibited early-life motor deficits. Notably, Mn levels were elevated in targeted neurons of DAergic-specific or GABAergic-specific Slc30a10 knockouts, but only the DAergic-specific knockouts phenocopied the pan-neuronal strain and developed early-life motor deficits. These novel results lead to the hypothesis that Mn induces motor disease in early-life by targeting DAergic neurons. We will test our hypothesis through three specific aims. In Aim 1, we will use neuron-specific Slc30a10 knockout mice and test whether increasing Mn levels in DAergic, but not GABAergic, neurons enhances sensitivity to Mn-induced motor deficits. In Aim 2, we will use neuron-specific Slc30a10 knockin mice and test whether reducing Mn levels in DAergic, but not GABAergic, neurons protects against Mn neurotoxicity. We will use a combination of behavioral, microscopy, and neurochemical assays to distinguish between dysfunction or degeneration of DAergic or GABAergic neurons as the cause of early-life Mn-induced motor disease. In Aim 3, we will use a pharmacological approach and test whether dopamine agonists rescue early-life Mn-induced motor deficits. In totality, our studies (1) will establish a definitive neuronal mechanism of motor disease induced by environmental Mn exposure in early-life; and (2) may identify dopamine agonists to be a potential treatment for pediatric Mn-induced motor disease.

NIH Research Projects · FY 2025 · 2015-07

PROJECT SUMMARY/ABSTRACT The Center for Learning and Memory (CLM) at the University of Texas at Austin brings together researchers whose goal is to identify the neural mechanisms that support learning and memory. The CLM has grown to include eighteen faculty since its inception in 2004. The highly interdisciplinary and collaborative nature of the CLM faculty provide the key elements for an innovative and active training program in learning and memory. The CLM faculty represent a broad range of approaches to the mechanisms of learning and memory – from the molecules of neural signaling and synaptic plasticity to cellular and systems level studies to functional neuroimaging in humans. We will leverage the strengths of this collaborative faculty to provide structured training in rigorous research design and methodologies, state-of-the-art quantitative approaches, and professional development training activities to prepare graduate students and postdoctoral trainees to become innovative leaders in the field of learning and memory research. The proposed training will support four predoctoral and two postdoctoral trainees each year. Trainees are expected to be appointed to the training program for no more than 3 years before transitioning to individual grants and fellowships. The proposed training has four important components. First, we will provide our trainees with hands-on instruction in the implementation of interdisciplinary approaches to the study of learning and memory that cross levels of analysis. Second, we will provide extensive training in advanced statistical and computational neuroscience methods that are increasingly necessary to understanding the neural mechanisms that support learning and memory. Third, the proposed activities will provide our trainees with the ability to place their research in a biomedical context, with an emphasis on the role of learning and memory processes in disorders of mental health. Finally, the proposed activities and resources provide our trainees with many opportunities for career development, including the skills necessary to obtain a tenure track position (e.g., presentation and grant writing skills) as well as research-related careers outside of academia. These components will prepare our trainees to be the next generation of leading-edge researchers dedicated to understanding the neurobiological mechanisms of learning and memory and how they are impacted in disorders of mental health.

NIH Research Projects · FY 2025 · 2015-06

PROJECT SUMMARY Depression and falls are significantly higher in low-income, racially diverse homebound seniors than in the general older-adult population; however, the existing systems of care are not equipped to address disparities in mental health and fall prevention services for these vulnerable older adults. The long-term objective of the proposed study is to improve access to depression treatment and fall prevention for growing numbers of low- income homebound seniors. Specific aims are to compare clinical and cost effectiveness of integrated tele- delivered behavioral activation (Tele-BA) and fall prevention (FP) by bachelor’s-level lay counselors/coaches to Tele-BA or FP alone and attention control (AC). The current and projected shortages of licensed clinicians and the costs of deploying highly trained professionals pose barriers to providing services to older adults in general and low-income homebound seniors in particular. A more scalable option is to utilize lay counselors/coaches, and our recent clinical trial (1R01MD009675) and a FP pilot study show that lay counselors/coaches are as effective as licensed clinicians. The study participants will be 320 low-income, racially diverse homebound seniors who are served by a home-delivered meal (HDM) program and other aging-service agencies in Central Texas. The lay counselors/coaches will be co-located in the HDM program for seamless referral and care coordination. In a 4-arm, pragmatic clinical trial with randomization prior to consent (a preferred public health approach), the participants in the integrated Tele-BA and FP (TBF hereafter) arm will receive 5 Tele-BA sessions and 4 in-home FP sessions. Those in the Tele-BA or FP alone arms will receive the respective intervention and 4 bimonthly telephone check-in (booster) calls, and those in the AC arm will receive 5 weekly telephone check-in calls followed by 4 bimonthly follow-up calls. Study hypotheses are: At 12, 24, and 36 weeks after baseline, (1) TBF will be more effective than Tele-BA or FP alone, and Tele-BA or FP alone will be more effective than AC in reducing depression (the 24-item Hamilton Rating Scale for Depression), falls, and fall injuries; (2) TBF than Tele-BA alone or FP alone will be more effective in reducing disability (WHODAS 2.0) and healthcare and social service use; and (3) TBF will be more cost effective than Tele-BA alone or FP alone. Cost-effectiveness analysis (CEA) will be based on depression free days, prevented falls, and health-related quality adjusted life-year measured by EuroQol-5 (EQ-5D). We will also conduct budget impact analysis (BIA) of TBF relative to Tele-BA or FP. Both CEA and BIA will employ a hybrid public program perspective of the Administration for Community Living and the Centers for Medicare and Medicaid. Public health significance of this study is that it will provide empirical data needed for real-world adoption of an intervention delivery model that targets to intervene for the two most frequent sources of disability acceleration and healthcare use among a rapidly growing, underserved population. (We use the terms older adults and seniors interchangeably because the latter term is frequently used in aging services.)

NIH Research Projects · FY 2025 · 2014-08

Project Summary Intracellular recording from sensory cortex provides a window into the synaptic inputs that shape spiking responses of individual cortical neurons, but until recently, this powerful technique has been limited to anesthetized animals. By combining the unique expertise from our laboratories, we have developed a novel technique that allow us to conduct, on a routine basis, reliable, whole-cell intracellular recording in primary visual cortex (V1) of awake, behaving macaque monkeys. We combine intracellular recording with an array of concomitant measurements that provide access to the state of the local network in which the neuron is embedded as well as to the internal state of the animal. Using these techniques, we have access to both subthreshold (membrane potentials, representing input) and suprathreshold (spikes, representing output) responses of individual cortical neurons, while also utilizing the precise control of visual stimulation and the subject’s behavioral state afforded by behaving primates. Our ability to perform intracellular recording in awake, behaving primates opens the door to addressing three fundamental questions with respect to the circuit-level mechanisms that mediate visual perception: (1) what are the nature, sources, and behavioral consequences of the large neural variability of sensory cortical neurons, (2) what is the contribution of internal state fluctuations to this variability, (3) what circuit models can account for the observed neural variability during spontaneous and evoked responses? To address these questions, in Aim 1 we will study the quantitative relationship between sub- and suprathreshold activity during spontaneous and stimulus-evoked responses in V1 of fixating monkeys. This will allow us to test the generality of previous findings from anesthetized animals. In Aim 2, we will examine the relationship between the activity of single V1 neurons and perceptual decisions in monkeys that are engaged in a demanding visual detection task. Specifically, we will examine how sub- and suprathreshold responses are altered by changing the attentional and motivational states under which the stimulus is presented. Finally, in Aim 3 we will test a novel set of circuit models that can account for our observed results and guide future experiments.

NIH Research Projects · FY 2025 · 2014-03

ABSTRACT As T cells develop in the thymus, VDJ recombination generates diverse T cell receptors (TCRs) needed to recognize the myriad pathogens encountered throughout life, but also yields TCRs that recognize self-antigens and could thus induce autoimmunity. Central tolerance limits autoimmunity by enforcing negative selection of thymocytes expressing TCRs with high affinity for self-antigens or diverting them to the regulatory T cell (Treg) lineage. To undergo self-tolerance, thymocytes must enter the thymic medulla, where they encounter dendritic cells (DCs) and medullary thymic epithelial cells (mTECs), key antigen presenting cell types that collectively display most self-antigens. Recent data suggest that thymic DCs express proteins induced during inflammation, while mTECs display tissue-restricted self-antigens (TRAs) expressed at steady state by differentiated cell types. Establishing T-cell tolerance to inflammation associated self-antigens, such as cytokines, is critical for mounting productive immune responses to pathogens, while establishing tolerance to TRAs is essential for preventing tissue-specific autoimmunity. Our findings over the previous project period demonstrate that the chemokine receptors CCR4 and CCR7 cooperate to promote thymocyte medullary entry and negative selection, but also suggest a new model in which CCR4 promotes early-stage tolerance to inflammation-associated self-antigens expressed by DCs, while CCR7 promotes late-stage tolerance to TRAs expressed by mTECs. We recently found that CCR4 and CCR7 are sequentially expressed by and promote medullary entry and negative selection of immature versus mature post-positive selection thymocyte subsets, respectively. In keeping with a cooperative role in supporting thymocyte medullary entry, double deficiency in CCR4 and CCR7 more severely impairs negative selection. However, distinct roles for these receptors in regulating central tolerance is suggested by the finding that CCR4 and CCR7 ligands are expressed by activated thymic DCs versus mTECs, respectively. Also, CCR4 deficiency enhances autoreactive T-cell responses to activated DCs, while CCR7 deficiency yields lymphocytic infiltrates in differentiated tissues. These findings support a new model in which CCR4 and CCR7 promote central tolerance to inflammation-associated antigens expressed by DCs and TRAs expressed by mTECs, respectively (Aims 1-2). Also, CCR4 is expressed by recirculating Treg, which suppress new Treg selection, while CCR7 is expressed by and supports survival of thymic DCs that present TRAs. Thus, CCR4 and CCR7 may promote central tolerance not only by impacting location and cellular interactions of thymocytes, but also by altering the activity of recirculating thymic Treg and DCs (Aims 1-2). Finally, autoimmunity in CCR4 and CCR7 deficient mice may reflect failed central and/or peripheral tolerance mechanisms of T cells and/or Treg (Aim 3). Altogether, the proposed experiments will test key tenets and mechanisms of the model that CCR4 and CCR7 respectively enforce thymic tolerance to inflammatory versus tissue-restricted self-antigens.

NIH Research Projects · FY 2026 · 2013-08

Abstract: The mucociliary epithelium plays a key role in both normal and pathological airway biology, as it provides the first line of defense against inhaled agents. Defects in ciliary beating in multiciliated cells (MCCs) in the airway contribute to the progression of both genetic and acquired airway diseases. Here, we will study the molecular mechanisms controlling development and function of MCCs. The motor proteins that drive ciliary beating are assembled in the cytoplasm, and under the previous version of this award, we discovered a novel organelle in which these motors are concentrated with various factors that direct their assembly. The first Aim of this project will explore the hierarchy of interaction among proteins in these novel organelles; the second will explore the mechanisms by which motors are transported into cilia, and the third will use large-sale proteomic approaches to identify novel protein-protein interactions important for motor assembly and function. By rapidly determining the functions of several new protein involved in distinct processes in MCC development, the Aims in this proposal will provide critical new depth to our understanding of these essential cell. Moreover, by linking these the disparate processes in MCCs, the experiments here will add crucial new breadth to our understanding as well. Impact: Experiments proposed here will lead to a more detailed understanding of the cell biology and genetics of MCCs and ciliary beating. The results will aid in the development of regenerative therapies aimed at repairing or restoring damaged tissue and improving mucus clearance in patients with airway disease.

NIH Research Projects · FY 2025 · 2013-07

A renaissance in the field of modular polyketide synthases has begun. New tools and paradigms are enabling deeper insights into the architectures and activities of these enzymatic assembly lines and are facilitating our long-term goal of applying the synthetic power of modular polyketide synthases to the development and production of new medicines. Using the updated definition of the “module”, our lab has engineered diverse tri- /tetra-/pentaketide synthases that are functional both in vivo and in vitro. While these short assembly lines are uncommon in nature, they are ideal for our structural and functional studies. In Specific Aim 1 we propose to plunge-freeze these assembly lines as they are synthesizing their polyketide products and investigate them by cryo-electron microscopy. Since this approach has enabled us to capture high-resolution, dynamic information of the priming ketosynthase and acyltransferase of a model triketide synthase, we will apply it to synthases that contain other regions of interest. One objective is to learn how the ketoreductase, dehydratase, and enoylreductase processing enzymes are oriented relative to one another and the neighboring ketosynthase+acyltransferase didomains to understand how acyl carrier protein domains move between these enzymes during the extension and processing of polyketide intermediates. Thus, we will investigate at least thirteen engineered tri-/tetraketide synthases and two natural synthases functionally validated in our lab that contain different sets of these processing enzymes. In Specific Aim 2 we propose to elucidate interactions between processed polyketide intermediates and the ketosynthases that gatekeep for them. We have strong hypotheses for how sets of substrate tunnel residues interact with intermediates closest to the reactive thioester to ensure they are properly modified by upstream processing enzymes. Thus, we will appropriately mutate the gatekeeping residues of ketosynthases in less active model synthases as well as model synthases with inactivated upstream processing enzymes and determine whether their productivities improve as predicted. Since our data indicate that polyketide intermediates rigidify the ketosynthase dimer and dimeric ketosynthase+acyltransferase didomains can be readily identified in cryo-electron microscopy studies, we will also perform electron microscopy on stalled synthases to solve structures of polyketide-bound ketosynthase+acyltransferase dimers. In Specific Aim 3 we propose to determine key domain-domain interfaces. We have evidence that interfaces between processing enzymes and downstream KSs drive the ordered self- assembly of synthase polypeptides more than the small interface observed between the C- and N-terminal docking domains and seek structures of representative complexes. We also aim to determine how acyl carrier protein domains dock with ketosynthases during the transacylation reaction. If we are successful in these projects, it will greatly inform the rational engineering of modular polyketide synthases that synthesize new molecules and, ultimately, new medicines.

NIH Research Projects · FY 2026 · 2013-04

PROJECT SUMMARY/ABSTRACT Hippocampus (HPC) structure and its connectivity with frontoparietal regions continue to develop through adolescence, a developmental period associated with substantial gains in memory and reasoning. While such structural changes are well documented, we know less about the functions that HPC and frontoparietal development confer, fundamentally limiting our understanding of the mechanisms through which individuals learn and reason about the world at different ages. From very early in life, children can learn simple associations that they directly experience. However, with age, our memories become more complex, reflecting not only directly observed information, but also knowledge derived across multiple episodes. Such derived knowledge might represent commonalities among experiences while simultaneously exaggerating important differences between them, thus forming hierarchical knowledge structures that can support inference decisions about event relationships, while also preserving detailed memory for when and where those relationships vary by context. The overarching goal of this proposal is to test the hypothesis that representational capacity within HPC and frontoparietal cortex undergoes qualitative changes during adolescence. We will use a serial cohort design to collect data from adolescents (13-18 years) at three timepoints, each 1.5 years apart, as well as data from adults (19-25 years) at a single timepoint. This design will allow us to test longitudinal predictions about how changes in neural representation within individual adolescents, over time, predict corresponding changes in memory and inference behaviors, as well as cross-sectional predictions about how HPC and frontoparietal cortex representation differs between adolescents and adults. We will use high-resolution functional magnetic resonance imaging (fMRI), representational fMRI analyses, and computational modeling, to test three Aims. Aim 1 will test the prediction that HPC and ventromedial prefrontal (vmPFC) representations will transition from coding simple, individual associations to extracting hierarchical knowledge about the relationships among experiences. Aim 2 will test the prediction that lateral parietal cortex (LPC)-mediated memory reactivation during new learning and inference will have different consequences for HPC—vmPFC representation and inference behavior at distinct points in adolescence. Aim 3 will test the prediction that emerging vmPFC control will influence what memories are available in LPC during learning and inference, as well as directly mediate the impact reactivated memories have on the trajectory of HPC representation during adolescence. The results from this project will provide a key test of fundamental theories of cognitive development and substantially advance our knowledge of the representational capacities of the HPC—frontoparietal memory system in typically developing adolescents. In doing so, the findings may have important implications for our eventual understanding of how the onset of mental health disorders (e.g., affective disorders and schizophrenia) during adolescence impact neural representation as well as memory and reasoning ability.

NIH Research Projects · FY 2025 · 2012-08

PROJECT SUMMARY The human functional neuroimaging literature has experienced sustained and explosive growth, making it difficult to effectively glean insights from this vast knowledge base. Previously, we introduced Neurosynth, a web-based framework for automated fMRI synthesis that supports meta-analysis at scale across over 12,000 studies. We subsequently extended the ecosystem by developing a powerful platform for expert-driven data annotation, enabling researchers to define and execute custom image-based meta-analyses. Although the Neurosynth framework has become a widely used resource in the neuroimaging community, the laborious challenge of annotating studies at scale remains. We previously addressed this problem by using frequency-based text-mining techniques to extract meaning from the abstracts of articles, but these methods were unable to make fine-grained distinctions between neuroscientific concepts or encode methodological details. Recent breakthroughs in natural language processing and artificial intelligence promise to extract accurate information from unstructured texts with little to no labeled data using Zero Shot Learning. However, the application of state-of-the-art language models to the neuroimaging literature requires a systematic framework to ensure machine comprehension accurately captures known concepts, and robust infrastructure to retrieve information from the literature at scale. The overarching goal of the present project period is to bridge the gap between manual annotation and fully automated meta-analysis, by extending the Neurosynth framework to leverage state-of-the-art natural language processing models to enable precise large-scale automated meta-analysis. In Aim 1, we will develop an extensible framework for information retrieval and validation from neuroimaging articles, with a focus on features relevant to neuroimaging meta-analysis such as study participant demographics (e.g. sample size, age, phenotype), methodological details, and semantic concepts mapped onto expert-defined ontologies. In Aim 2, we will develop and deploy infrastructure for continuous retrieval and processing of the neuroimaging literature, ensuring the timely acquisition of new articles and transforming our meta-analytic study database into a comprehensive knowledge base. In Aim 3, we will develop an interactive researcher-in-the-loop platform meta-analysis, enabling users to use extracted study information to find and identify studies, create gold-standard meta-analyses, and browse hundreds of pre-generated large-scale meta-analyses, enabling discovery-driven science. Meta-analyses will be automatically updated, transforming them from a static to a “living” scientific product that remains relevant and up-to-date. Realizing these objectives will introduce powerful and flexible tools for synthesizing the neuroimaging literature at a large scale and with unprecedented precision and ease. These tools will be freely and publicly available to anyone with an internet connection, enabling rapid and efficient meta-analysis across a broad range of clinical and basic research questions.

NIH Research Projects · FY 2026 · 2012-08

Dysregulation of Hedgehog (HH) signaling is linked to catastrophic birth defects in infants and is an underlying cause of the structural defects in a range of syndromes, including ciliopathies. The effects of HH signaling are primarily mediated through de-repression of target genes, but the mechanisms by which GLI proteins act as transcriptional repressors remain poorly understood. Recently, we found that GLI repressors prevent transcription of target genes by deactivating their own enhancers through chromatin-based histone deacetylation. We then found that GLI3 is present but apparently inert in early limb buds before the onset of HH signaling. We hypothesize that GLI repression is normally regulated by the expression of co-repressors rather than being the default state occurring in the absence of HH signaling. In Specific Aim 1 we will determine how GLI3 repressor activity is inactive prior to the onset of Hedgehog signaling. We will determine if GLI repression is inert because of sub-threshold protein kinase A activity or, alternatively, if it is triggered by the onset of HH expression. In parallel, we will determine if the lack of GLI3 repression is due to an unexpected early redundancy with GLI2. These findings will determine whether HH pathway members trigger GLI transcriptional repression. In Specific Aim 2 we will determine how GLI transcriptional repression regulates chromatin accessibility. GLI transcriptional repression causes both reductions in H3K27 acetylation as well as reductions in ATAC-seq accessibility at GLI enhancers. We will determine if the reduced accessibility occurs because of changes in nucleosomal density or because of reduced histone acetylation. Additionally, we hypothesize that GLI3 transcriptional repression occurs through the NuRD complex, which uniquely contains both the HDAC and chromatin remodeling activities that are observed with GLI3 repression. Our preliminary data supports this hypothesis, indicating that GLI3 binds to CHD4, a component of the NuRD repressor complex that mediates nucleosomal sliding. The results will provide a mechanistic understanding of how GLI transcriptional repression controls chromatin accessibility to regulate HH target gene expression. In Specific Aim 3, we will identify co-factors regulating GLI repressive activity. We hypothesize that GLI repression occurs through the recruitment of an unknown co-repressor complex containing HDAC activity. We will test several ranked candidate co-factors by determining if they bind to known GLI enhancers and if this binding is reduced in limb buds lacking GLI3. In a complementary, unbiased approach, we will identify factors that bind to GLI3 on chromatin using an endogenously biotinylated GLI3 allele. The identification and subsequent validation of a co-factor(s) mediating GLI repression will fill a major gap in our understanding of how HH signaling regulates gene expression. Overall, these results will be impactful in illuminating a novel layer of HH pathway regulation as well as enabling a mechanistic understanding of GLI transcriptional regulation that will be applicable to understanding the full biological spectrum of HH-mediated responses.

NIH Research Projects · FY 2025 · 2012-07

Project Summary/Abstract The Center for Perceptual Systems (CPS) is an interdisciplinary program at the University of Texas that provides a focal point for research and training in sensory systems. Continued growth in the Neurosciences at UT, as well as in CPS, particularly in the area of vision research, has lead to the development of a distinctive group in the Center that has a broad interdisciplinary research program in vision, representing psychophysical, neurophysiological, imaging, and computational approaches. Additionally, we have distinctive expertise and resources for the investigation of vision in the context of natural environments, and the development of computational tools for neural modeling and for modeling visually guided behavior. Because of both our broad expertise and our special strengths in these areas we are well positioned to train new scientists who will be at the forefront of vision research in the future. There are three important components to our training focus. First, we take advantage of the highly interdisciplinary and collaborative structure of CPS to provide broad cross- disciplinary training, which we consider essential for students of vision and visual performance. Second, we believe that training in computational methods is an essential component of research in vision and we take advantage of our particular strengths in this area. Third, we take advantage of our strengths in the analysis of natural behavior and environments to provide a unique training opportunity in an area that is becoming increasingly important for a broad understanding of vision. These components lie at the core of our program, which includes basic courses, specialized seminars, training in advanced methodologies, attendance at the CPS colloquium series and the CPS symposium on Natural Environments, Tasks, and Intelligence, ethics training, and the development of professional skills.

NIH Research Projects · FY 2025 · 2012-05

Coordinated regulation of developmental transition by protein and long noncoding RNA components Project Summary Epigenetic mechanisms enable organisms to adapt to developmental and environmental cues. Plants have evolved intricate regulatory networks to control development in response environmental stimuli such as temperature and light. For example, prolonged cold triggers vernalization, a process that accelerates flowering through epigenetic changes in genes involved in development. Therefore, the vernalization response in Arabidopsis is an excellent model system for study of complex epigenetic regulation of gene expression triggered by environmental cues in eukaryotes. Vernalization-mediated epigenetic changes include the formation of chromatin loops and alterations in chromatin modifications and in expression of long noncoding RNAs (lncRNAs). Changes in the three-dimensional structure of the genome, such as formation of local chromatin loops, are increasingly recognized as important gene regulatory events in eukaryotes; however, how chromatin modifications and lncRNAs coordinate to influence gene expression through changes in the three-dimensional structure of the genome is not well understood. Here we seek to understand the mechanistic details of how the three-dimensional structure of the genome influences gene regulation in vivo using developmental changes in flowering and photomorphogenesis as phenotypical read- outs. Our overriding goal is to elucidate structural and regulatory components governing protein- and lncRNA-mediated epigenetic gene regulation in Arabidopsis through three specific aims designed to: 1) elucidate the detailed mechanisms of chromatin structure-mediated gene regulation focusing on two groups of chromatin architectural proteins that we showed coordinate gene regulation through the formation of chromatin loops, 2) characterize mechanisms underlying light signaling-mediated chromatin conformation changes, and 3) characterize genome-wide changes in chromatin structure induced by environmental stimuli. Our approach will reveal the mechanistic details of chromatin structure-based epigenetic regulation by both protein and lncRNA components during flowering and photomorphogenesis. These findings will further our understanding of the mechanism of epigenetic regulation of gene expression, which has a deep evolutionary root among eukaryotes.

NIH Research Projects · FY 2026 · 2011-09

Project Summary/Abstract Neuroimmune signaling is strongly implicated in the development and progression of alcohol use disorder (AUD). Innate immune molecules are upregulated in human postmortem brains from alcoholics and are correlated with lifetime alcohol use. Human genome-wide association studies (GWAS) also link immune/inflammatory genes to alcohol dependence. We hypothesize that alcohol consumption initiates a positive feedback loop involving neuroimmune activation and proinflammatory responses that promote excessive alcohol consumption. Neuroimmune signaling also regulates voluntary alcohol consumption in rodent models and activation of specific pathways results in escalation of drinking. Our recent work shows that immune signaling in glial cells have emerged as key regulators of alcohol drinking. Using single cell and spatial transcriptome technologies, we are now able to precisely define the role of individual types of brain cells in the context of excessive alcohol consumption. Our overarching hypothesis is phenotypic changes characteristic of escalated alcohol consumption and preference are due, in part, to spatially distinct and cell-specific molecular mechanisms that significantly reshape the neuroimmune transcriptome. Multi-omic genetic changes at the cellular level from human and animal models will reveal new gene candidates and therapeutic targets.

NIH Research Projects · FY 2025 · 2011-05

SUMMARY Human ISG15 is a ubiquitin-like protein (Ubl) that functions in innate immune responses, and it is remarkable for possessing three distinct biochemical activities. As a ubiquitin-like modifier it is conjugated to hundreds of cellular and viral proteins. The E1, E2, E3, and de-conjugating enzymes for ISG15 (Ube1L/Uba7, UbcH8/Ube2L6, Herc5, and Usp18, respectively), are, like ISG15, induced at the transcriptional level by Type I interferon (IFN-α/β) signaling. A second function of ISG15 is as an extracellular signaling protein. ISG15 is released into the extracellular space from many cell types and signals to Natural Killer and T cells to secrete IFN-γ, which plays a major role in the response to pathogen infections. The cell surface receptor for extracellular SG15 is LFA-1, an immune cell-specific integrin. A third function of human ISG15 is that it negatively regulates Type I IFN signaling, as revealed by the Type I interferonopathy that occurs in some ISG15-deficient patients; this function of human ISG15 is not shared with mouse ISG15. A challenge in the field is to understand how the activities of ISG15 are regulated and temporally controlled and which activities are most closely associated with responses to specific pathogens, including Mycobacterium tuberculosis and SARS-CoV-2. To further our understanding of ISG15 in innate immune responses to these and other pathogens, this proposal will focus on the basis of substrate and lysine selectivity of the Herc5/Herc6 family of ISG15 ligases, the ISG15-induced signaling pathway downstream of LFA-1 that leads to cytokine secretion, and the mechanism by which ISG15 released into the extracellular space.

NIH Research Projects · FY 2024 · 2009-08

7. Project Summary/Abstract The over-arching theme of this proposal is to train “comprehensive imaging scientists” in the skills necessary to identify clinically relevant problems; develop instrumentation, sensors, and contrast agents to form images appropriate for the problem; and analyze the resulting imaging data using signal processing, mathematical modeling, visualization, and informatics techniques to improve the prevention, detection, diagnosis, and treatment of human diseases. The program spans from molecular to cellular to tissue to organ. In order for imaging scientists to be knowledgeable of the full trajectory from image formation to analysis and decision-making, they must be trained in four core areas: Instrumentation, Devices, and Contrast Agents; Image processing; Modeling and Visualization; and Data Mining and Informatics. All students in the program are trained in the core concepts of these areas. The current training program is a two-year pre-doctoral portfolio program. A total of 41 students have been admitted to the program. The proposed renewal will train another 20 students. The program includes off-campus externship research experiences; in-depth clinical engagement; and a wide-ranging professional development component. Imaging Science is an integral element of basic science research and clinical medicine. Imaging cell trafficking and receptor pharmacology in vivo have already led to targeted drug and gene therapies and an understanding of cellular biochemical pathways will contribute to new advances in medicine. Individualized medicine relies heavily on imaging techniques to select the best therapies and monitor progress. Although structural in situ human imaging is already a critical component of clinical medicine, many advances are needed in functional imaging of the brain and other organs to improve healthcare. Brain mapping which is a core focus of NIH research relies heavily on imaging. We have identified a critical need for imaging scientists to develop new imaging instrumentation and apply that instrumentation with appropriate methods from image processing; modeling and visualization; and informatics and data mining. In recognition of the potential of artificial intelligence to transform medical imaging, our program emphasizes applications of machine learning. This training program fills a critical niche by providing highly skilled scientists who are trained in the broad trajectory of imaging science. Understanding the interplay between instrumentation and image analysis, including machine learning methods, is important for designing the next generation of hardware and software tools for quantifying complex biological systems and providing robust clinical tools. A key outcome of the program is that trainees gain the skills necessary to identify clinically relevant problems.

NIH Research Projects · FY 2026 · 2009-04

The Research Society on Alcohol (RSA) Annual Meeting is the premier annual scientific meeting involving all areas of alcohol research. In addition to biological/preclinical aspects, it also covers medical/clinical, and psychosocial research. The RSA meeting is held annually, and previous meetings (42) have been held mostly in the continental US, although some meetings have also been held in Hawaii and Canada (Vancouver, Montreal). Also, the 2021 meeting is being planned as a joint meeting between RSA and the International Society for Biomedical Research on Alcoholism. The location of these meetings will provide an attractive opportunity for scientists from around the U.S.A. along with others around the world to present and discuss their research. The meetings will have a large impact on alcohol research in the U.S., and the outcome of the research presentations and discussion will advance the field. The program for each meeting will feature topics that are highly relevant to the alcohol research community and provide opportunities for discussion of existing and new collaborative research. The research presentations will be in the form of symposia, roundtables, and workshops that will be proposed by the membership of RSA. The RSA Program Committee will select the proposals with the highest scientific merit and innovation. In addition, numerous poster sessions will be held. The RSA has made an effort to recruit and retain new investigators in the field by highlighting their work as well as providing professional development activities, and this will continue for the 2021-2025 meetings. Since RSA is a stable, established organization with annual meetings, this application is for 5 years of funding (support for the 2021-2025 meetings). This application specifically requests funds to offset expenses for young scientists (predoctoral and postdoctoral within 4 years of their terminal degree) to attend these important meetings. Funds are requested for travel and registration fees. Some administrative costs are also requested. The specific aim of this grant is to provide travel and registration assistance for students and young investigators to attend the annual RSA meeting. The overall goal is to provide information to those individuals who are interested in learning more about current research in the alcohol field with the possibility of pursuing alcohol research as a career.

NIH Research Projects · FY 2025 · 2009-02

Project Summary  Hispanic populations in the United States and Latin America will confront critical health issues in the 21st century. In the United States, poverty among older Hispanic individuals is twice as high as that for non-Hispanic whites. Similarly, Latin American countries have parallel issues, including rapid population aging and high poverty rates. Social determinants (e.g., social class, social behaviors, and social networks) profoundly shape the health and quality of life of large numbers of populations, necessitating robust responses from social institutions. Moreover, the interplay of contextual exposures, biological factors, and social dynamics shapes health outcomes across the lifespan. The proposed fifth competing renewal application extends a successful annual conference series on Aging in the Americas (ICAA) that started in 2001. Previous ICAA installments have had a distinctive focus and each resulted in peer-reviewed books and special journal issues as published on the CAA website. The next five installments at The University of Miami, Coral Gables, Florida (2025), University of Illinois, Chicago, Illinois (2026); Cornell University, Ithaca, New York (2027); University of Southern California, Los Angeles, California (2028), and University of Texas Medical Branch, Galveston, Texas (2029) build upon the same very high quality of work as previous meetings to address a new theme that is a priority for the national aging and health agenda. This will be accomplished by commissioning 50 papers, including five keynote speakers from sociology, psychology, demography, social policy, medicine, gerontology, and economics to address three primary goals: First, to provide a vehicle for reviewing and analyzing contemporary social research on disparities in “healthy aging” examining the major dimensions of determinants of which are contextual, social, behavioral, and biological as they relate to supporting the health of aging Hispanics; second, to further the development of emerging scholars through their increased exposure to this body of knowledge, developing their research, and career mentoring; and third to foster high-impact publications on healthful aging, extending our research focus to nations in Latin America through cross-national comparisons using the longitudinal Health and Retirement Study (HRS) family surveys in Latin America. The ICAA will foster research using new methods, data, cross-national comparisons, and analyses of the effects of policies and interventions. This installment will emphasize the role of country-specific economic and social policies, as well as institutional contexts, in shaping the health and well-being of older Hispanic population. The new ICAA installments will aim to create an agenda of emerging themes regarding healthy Hispanic aging.

NIH Research Projects · FY 2024 · 2007-05

PROJECT SUMMARY The development of compensatory behavioral strategies that circumvent impairments to enable life activities is a predictable response to the onset of functional disability. Increased reliance on the less-affected, "nonparetic", hand and arm for the performance of daily activities after stroke is a prominent example due to the prevalence of stroke and of upper limb hemiparesis as a chronic post-stroke impairment modality. This compensatory strategy involves major behavioral changes which start early after stroke and have strong potential to impact post-stroke neural reorganization patterns via mechanisms of experience-dependent plasticity. The focus of this project across periods has been on understanding neural changes that are driven by compensatory experiences of the nonparetic forelimb and their impact for functional outcome, as studied in rodent models of chronic upper extremity impairments resulting from primary motor cortical (M1) infarcts. We have previously established that learning to rely on the nonparetic forelimb interacts with post-ischemic regenerative responses in both hemispheres to influence behavioral function bilaterally. Its impact for the paretic side is to exacerbate disuse and lessen the efficacy of motor rehabilitative training of the paretic limb. In the most recent project period, we have firmly linked these deleterious effects of learning with the nonparetic forelimb to its disruption of functionally relevant reorganization of peri-infarct M1 and its promotion of synaptic changes in the same region. We have also uncovered that learning compensatory ways of using the two limbs together is beneficial for unimanual function of the paretic side. These and related findings lead us to hypothesize that unimanual experiences of the nonparetic forelimb promote synaptic changes that compete, whereas bimanual experiences promote those that cooperate, with patterns of synaptic change mediating improved paretic forelimb function. We lack detailed knowledge of the time course of the synaptic changes that are promoted by experiences of either forelimb and clear evidence that they compete, and how they vary with bimanual experiences is untested. One major goal for this project is to efficiently tackle these knowledge gaps by capitalizing on in vivo imaging approaches to monitor synaptic structural responses to forelimb experiences in peri-infarct cortex as they unfold over time. Another is to probe the efficacy of bimanual skill training as a strategy for competing with and countering deleterious influences of compensating with the nonparetic side. The specific aims are to test the hypotheses that: (Aim 1) skill learning with the nonparetic forelimb shapes synaptic connectivity in peri-infarct M1 via the activity of contralesional cortex and transcallosal projections to diminish its responsiveness to rehabilitative training of the paretic forelimb, (Aim 2) the same pathway mediates synaptic structural responses in peri-infarct cortex to bimanual skill training that improves paretic forelimb function and (Aim 3) bimanual skills have the capacity to compete with and overcome maladaptive behavioral and synaptic connectivity patterns resulting from unimanual experiences of the nonparetic forelimb.

NIH Research Projects · FY 2026 · 2006-09

PROJECT SUMMARY/ABSTRACT This Integrative Neuroscience Initiative on Alcoholism (INIA-Neuroimmune, INIA-N) consortium component is for a U01 research project that seeks to determine how neuroimmune signaling molecules and glia contribute to neuronal adaptations in the nucleus accumbens that promote excessive ethanol drinking. Prior work by this INIA- N component identified neuronal adaptations in the nucleus accumbens (NAc) that are associated with excessive alcohol consumption. In particular, we found that ethanol dependence and excessive intake are associated with plasticity in glutamatergic synapses on dopamine D1 receptor-expressing medium spiny neurons (D1MSNs), which comprise a major output pathway of the NAc that is heavily involved in reward-based behaviors. Recently we discovered that, although synaptic excitation is enhanced, the membrane excitability of these neurons not only is suppressed during acute withdrawal from ethanol, but is strongly, inversely, correlated with prior ethanol intake. We will employ mouse behavioral models of chronic and excessive drinking, genetically engineered mice, and brain slice electrophysiology to test the hypothesis that neuroimmune activity contributes to these synaptic and membrane adaptations of D1MSNs. Specific aim 1 will evaluate how neuronal physiology is altered by the cytokine interleukin-33 (IL-33) in an ethanol-dependent manner, and it will use genetically-engineered mice for inducible and conditional deletion of IL-33, or its receptor, from specific cell types (microglia, astrocytes, and/or neurons) to probe the role of this signaling pathway in the escalation of alcohol intake. Specific aim 2 will take a broader approach, using brain slice electrophysiology to ask whether NAc microglia exhibit functional adaptations that correlate with alcohol intake and its associated adaptations in D1MSNs. Specific aim 3 will be collaborative studies on other neuroimmune signaling and regulatory molecules identified by INIA-N investigators’ as playing a role in excessive alcohol intake. Here we will again use brain slice electrophysiology and will determine whether manipulations of these neuroimmune molecules may alter NAc D1MSN physiology to regulate alcohol consumption. These studies will serve the mission of NIAAA by generating new, fundamental knowledge about the effects of alcohol on brain health, and seeks to apply such knowledge to identify new targets for the treatment of excessive alcohol consumption.

NIH Research Projects · FY 2025 · 2004-08

This proposal seeks renewal of a Jointly Sponsored NIH Predoctoral Training Program in the Neurosciences (JSPTPN, T32DA018926). Our T32 program supports students in their first and second years of the PhD program of the Institute for Neuroscience (INS) at The University of Texas at Austin (UT). The overall objective of our T32 is to provide broad, quantitatively focused neuroscience training to students in their initial years of training. For two decades, our T32 has had a multi-dimensional impact in synergizing with other T32 programs, facilitating high-impact research, and promoting mentoring and outreach. The rationale for this T32 is that it raises the bar for excellence in graduate training not only in the INS but across the UT campus. Our specific aims are to (1) establish mentoring and community as core values that enable individuals from all backgrounds to succeed as leaders in neuroscience; (2) deliver rigorous, ethical, and quantitatively focused training that can be tailored to a variety of neuroscience research areas; and (3) provide a balanced and integrated training plan to accelerate the launch of trainees as top-flight neuroscientists. To support Aim 1 we have embedded regular mentor training as a requirement for our T32 faculty and trainees. To meet this requirement, we have formalized a mentor training workshop for faculty and created a variety of mentoring and outreach activities for our trainees to participate in. Aim 2 is accomplished through curriculum offerings that begin with a 2-week bootcamp that occurs prior to starting classwork and rotations. This bootcamp launches our student’s quantitative training and introduces them to experimental design and methodology. Subsequent required coursework includes fundamental neuroscience courses, and 3 semesters of statistics/quantitative literacy training. To accomplish Aim 3, we have developed an intentional plan that provides balanced training and accelerates our trainees’ progress. This includes streamlining the lab rotation process and integrating regular, required progress checks in the first several years to ensure students remain on track for graduation within 5 years. We take advantage of numerous UT-sponsored career development resources to ensure our students develop actionable plans for their future. In addition, we recognize that progress towards graduation is dependent on our trainees’ holistic wellbeing and therefore activities aimed at building community and supporting student wellness are embedded in all aspects of our training plan. Based on our large and highly qualified applicant pool, this application requests funding for 4 first-year trainees and 4 second-year trainees (totaling 8 per year). The expected impact of our T32 is the training of a new generation of rigorous and ethical neuroscientists who will apply their knowledge and skills as leaders in academia, industry, and other professions. The broader impact of this T32 is that it will establish UT as a beacon for innovative graduate training, exemplary mentoring, community-building, and excellence in neuroscience research.

NIH Research Projects · FY 2025 · 2002-07

OVERALL: PROJECT SUMMARY The Population Research Center (PRC) at the University of Texas at Austin (UT) seeks to renew our P2C infrastructure funding to continue successful programs and launch new initiatives that will catalyze the next generation of population dynamics research regarding the social, structural, and contextual processes underlying population health and differences in health outcomes across subpopulations. The activities and supports provided by the P2C infrastructure funding will allow PRC faculty scholars to continue to advance the field’s understanding of what factors promote population health and what factors undermine health and increase differences in health outcomes across subpopulations.

NIH Research Projects · FY 2026 · 2002-01

PROJECT SUMMARY Cancer incidence increases with age in a tissue-specific fashion. Genetic instability and epigenetic alterations (e.g., cytosine methylation) are hallmarks of both cancer etiology and aging, thus linking aging to cancer. Importantly, alternative DNA structure-forming sequences (i.e., non-B DNA) have been identified as endogenous mutation hotspots associated with cancer etiology in an age-related and tissue-specific fashion. Further, methylation can impact non-B DNA formation, its mutagenic potential, and DNA repair mechanisms; and the mutagenic processing of non-B DNA requires DNA repair proteins. However, the mechanisms involved in the age-related and tissue-specific generation of these mutation “hotspots” remain largely unknown. With the aging population increasing, there is a critical need to fill this fundamental gap in knowledge. Our long-term goal is to elucidate the mechanisms of age-associated, tissue-specific DNA structure-induced genetic instability to guide future studies to develop new strategies to prevent and/or treat cancer. Thus, the overall objective of this application is to determine the mechanisms involved in differential DNA structure-induced genetic instability with age and tissue type to inform on cancer etiology. We will test the novel hypothesis that the formation of non-B DNA and their mutagenic processing differ with age in a tissue-specific fashion due to alterations in DNA repair processing and cytosine methylation. The rationale is that determining the mechanisms associated with age-related, tissue-specific DNA structure-induced genetic instability will offer a novel scientific framework whereby new strategies to prevent and/or treat age-associated diseases, such as cancer, can be developed. The hypothesis will be tested in the following aims: 1) Measure the amount of non-B DNA formed and its mutagenic potential with age in mouse tissues; 2) determine age- and tissue-associated alterations in cytosine methylation that alter non-B DNA structure formation and mutagenesis; and 3) identify the DNA repair-associated mechanisms of mutagenic processing of endogenous mutation hotspots with age in mice. Novel mutation-reporter mice containing human non-B DNA sequences from cancer-associated mutation hotspots will be used to determine the effects of age and tissue type on the mutagenic processing of non-B DNA. This is innovative because it will test the novel hypothesis that DNA structure-induced genetic instability is altered with age and tissue type in mammals, dependent on age-related modulations in epigenetics and DNA repair. The expected contribution is the elucidation of the impact of age and tissue type on DNA structure- induced genetic instability, which is significant because the results will inform on the etiology of various cancer types with age. This is expected to have a significant positive impact because the results will help achieve our long-term goal to understand the mechanisms of age-associated, DNA structure-induced genetic instability in various tissues to guide future studies to develop new strategies to prevent and/or treat cancer.