University Of Rochester

NIH Research Projects · FY 2024 · 2015-07

PROJECT SUMMARY/ABSTRACT The primary objective of the Rochester Partnership to Advance Research and Academic Careers of Deaf (RPP-DEAF) Scholars is to rigorously train and support the career advancement of deaf and hard of hearing (D/HH) post-doctoral Scholars for independent academic careers that support the biomedical research enterprise. This is an important unmet national need, because the D/HH community is greatly underrepresented in the biomedical sciences academic workforce. In order to address this disparity, we have partnered the faculty and programs of the University of Rochester (UR) with those of the Rochester Institute of Technology (RIT) and its National Technical Institute for the Deaf (NTID). To achieve our program goals, we will pursue four Specific Aims; 1) To provide outstanding program leadership that “mentors the mentors”, builds community for D/HH Scholars, and successfully coordinates program recruitment. This goal will be achieved through (A) special training specific to this IRACDA program (including training in Deaf-aware mentoring practices and Deaf culture), (B) training in mentoring diverse learners and (C) advanced mentoring training (for research mentors) or leadership mentoring training (for program leaders).; 2) To provide individualized, mentored teaching experiences and career development activities for all Scholars. Teaching experiences will be provided principally at RIT/NTID, the teaching-intensive partner institution.; 3) To provide individualized, mentored research experiences for all Scholars. Research experiences will be provided principally at UR, the research-intensive partner institution.; and 4) To rigorously, comprehensively and continuously evaluate the RPP-DEAF program. We will ensure that RPP-DEAF meets its goals by conducting comprehensive and ongoing program evaluation of: Scholar Achievement, Faculty Mentorship & Program Leadership; and Institutional Impact. We will recruit three new D/HH scholars per year, with a maximum of a three-year appointment, in order to maintain a steady state cohort of 9 D/HH Scholars. Overall, this unique IRACDA program will equip our Scholars with the comprehensive set of knowledge and skills in research, teaching and professionalism necessary for successful academic careers - with the goal that all of our Scholars will acheive full-time academic appointments in higher education and/or research institutions. This innovative inter- institutional partnership is the first of its kind in the world and will begin to address the dearth of D/HH faculty at academic institutions serving the nation's biomedical research needs. A long-term outcome will be the attraction of more D/HH Scholars into careers in the biomedical sciences, a field in which D/HH persons are greatly underrepresented.

NIH Research Projects · FY 2025 · 2015-07

ABSTRACT The current SARS-CoV-2 pandemic has made it readily apparent that infectious diseases are a major threat to U.S. health. The goal of this predoctoral T32 program is to train the next generation of researchers in microbiology, and to prepare them with the skills necessary to address the nation’s critical needs in the battle against infectious disease. To do this, we have developed a training program that takes a student-centric, autonomy-supportive approach with the goal of training self-motivated, independent research scientists who are well prepared for diverse research careers. Innovative program features include: an autonomy-supportive educational approach that encourages self-directed exploration of diverse career options; instruction in the “soft skills” necessary for research career success; and the establishment of faculty mentoring practices that support student autonomy and self-directed learning. The program’s renewal expands and enhances our 6 overarching objectives. First, it will provide outstanding interdisciplinary research training in microbial pathogenesis, in part by leveraging unique opportunities created by signature institutional NIH-funded centers that include the Center of Excellence for Influenza Research and Surveillance, Environmental Health Science Center, Vaccine and Treatments Evaluation Unit, and the University of Rochester’s Clinical and Translational Sciences Institute. Second, it will provide outstanding education in microbiology and pathogenesis, which incorporates four major components: 1. a streamlined required core didactic curriculum; 2. required program-specific experiences (including courses, workshops and career/professional development activities); 3. optional elective courses and experiences designed to encourage student exploration, and 4. hands-on thesis research. Third, it will train self-directed, autonomous scientists who are prepared for diverse research career options. To facilitate this, all trainees will complete a personality inventory and an annual Individualized Development Plan (IDP), participate in “Microbiology Career Stories” and other activities offered by the UR’s Broadening Experiences in Scientific Training program which provides avenues for diverse career exploration, and offered the opportunity to participate in extramural internships. Fourth, we provide training in the “soft-skills” necessary for success through participation in a “Leadership Academy” program and supervised training in mentoring. Fifth, we will continue to improve faculty mentoring practices through required instruction in autonomy supportive mentoring practices involving IDPs and “Mentor our Mentors” workshops. Sixth, we will develop a new UR Student Lifecycle program to longitudinally track program alumni, obtain experiential feedback to further optimize the program, assess career outcomes, and establish a formal portal to facilitate trainee networking and career advancement.

NIH Research Projects · FY 2025 · 2015-07

Project Summary Reactive oxygen species (ROS) contribute to pathology, but conversely, in limited measure they can also act as second messengers, whereby they contribute to beneficial cellular signaling. Similar to calcium signaling or other second messengers, the precise location, timing, and duration of ROS production likely determine divergent signaling outputs. The mechanism underlying this functional dichotomy in redox biology is currently under studied. An intriguing example of an apparent paradoxical impact of ROS occurs at complex I of the mitochondrial electron transport chain. In the case of detrimental effects of oxidation, mitochondrial complex I ROS production is mechanistically linked to oxidative damage in ischemia reperfusion (IR) injury, the pathology of stroke. In the case of beneficial signaling, complex I ROS production is implicated in protective hypoxic signaling. Indeed, the fact that some ROS production is a normal consequence of mitochondrial respiration supports the idea that ROS contribute to normal physiology. Therefore, describing the nuances of complex I ROS production and its context- dependent metabolic effects is necessary to fully determine the mechanisms of mitochondrial redox signaling, both damaging and physiologic. To achieve that goal, precise experimental control of ROS production is required. Until recently, controlling ROS production as an independent variable has been difficult. This renewal leverages an optogenetic approach championed by our lab to overcome this barrier, and isolates ROS production at complex I in the genetic model organism C. elegans. Previously, we have shown that ROS production at the complex II microdomain differentially affects redox-sensitive outcomes in models of IR injury, depending on whether the ROS were produced inside the mitochondrial matrix or in the intermembrane space. Using our published novel CRISPR/Cas9 technology optimized for rapid use in C. elegans, we will target well-characterized light-activated ROS generating proteins (RGPs) to endogenous complex I in order to precisely control the location, timing, and duration of complex I ROS production with light. This will provide a model of either complex I redox signaling, or oxidative damage, depending on the light-titration of RGP activation, where more light will produce more ROS. Combined with tissue-specific expression, we will determine the effects of each of these spatiotemporal parameters on normal mitochondrial function, neuronal function, and stress-resistance signaling programs in response to simulated IR injury. We will focus on the neuronal outcomes of complex I ROS production, both in response to strong literature support for the importance of neurons in mediating hypoxic stress signaling, and to determine neuronal circuits that could be targeted for translation to mammalian models of stroke. This approach is perfectly suited to the powerful C. elegans genetic system. We expect that completion of our aims will provide novel, fundamental insights with clear answers to questions about how the mitochondrial complex I ROS microdomain controls diverse outcomes in both disease and physiology.

NIH Research Projects · FY 2026 · 2015-06

Project Summary One out of seven adults in the United States suffers difficulty in hearing. Most hearing loss is related to inner ear dysfunction. A prominent signature of hearing loss is damage of outer hair cells, which can result in 40-60 dB of hearing loss. Outer hair cells are known as cellular actuators that are needed for cochlear amplification. Outer hair cells are situated in the cochlear sensory epithelium called the organ of Corti. The organ of Corti is sandwiched between two collagen-fiber-rich matrices called the basilar and tectorial membranes. Traditionally, studies on cochlear amplification were focused on motion of the basilar membrane, but new evidence is suggesting that we have been seeing only one-side of the story (basal-side of the outer hair cells). Recent advancement in imaging and velocimetry techniques enables scientists to ‘see-through’ the cochlea so that they can capture organ of Corti motion beyond the basilar membrane. New observations using recent techniques are both exciting and puzzling. For example, at the apical side of the outer hair cell, vibrations are broadly tuned as compared to basilar membrane motion or neural responses. Functional consequences of the incongruence between mechanical and neural tuning are unclear. Two challenges are impeding the progress of hearing science regarding cochlear amplification. First, despite recent progress, the resolution of velocity measurement techniques is insufficient to specify individual outer hair cells. Second, in experiments with live animals, there are very limited means to control outer hair cell physiology. This project resolves those two challenges. We have developed novel in vitro methods that enable us to measure the motion of individual outer hair cells under electrically, chemically and mechanically controlled conditions. Experimental outcomes are explained and extended by our Virtual Cochlea—a set of computer models to analyze cochlear mechano-transduction. By combining these experiments and the computer models, we can ensure scientific rigor of our research while minimizing animal use. For transparency, acquired data and computer programs will be made available to public. The three aims of this proposal are designed to quantify the most needed biophysical attributes needed to address open questions regarding cochlear amplification and tuning. They are 1) the deflection of stereocilia (mechano-receptive organelle of the hair cells) due to outer hair cell motility, 2) mechanical properties of the tectorial membrane and the Deiters cell (structures in series with the outer hair cell), and 3) the operating range of stereocilia mechano-transduction (stereocilia deflection to saturate hair cell mechano-transduction). The Virtual Cochlea is validated by comparing with the measurements of three aims before testing our overarching hypothesis—the organ of Corti must be as compliant as the outer hair cells for the outer hair cells to generate power for cochlear amplification.

NIH Research Projects · FY 2026 · 2015-06

ABSTRACT. The goal of Project 3 (P3) is to characterize, in OCD, white matter (WM) bundle and functional neural abnormalities among regions in a putative OCD neural network that are associated with persistent avoidance, a characteristic feature of OCD. Our overarching Center renewal hypothesis, building on our present findings, is that persistent avoidance in OCD is a manifestation of dysfunctional connections among specific hubs, i.e., subregions that integrate and distribute information from multiple regions, in the ventrolateral prefrontal cortex (vlPFC) and rostral anterior cingulate cortex (rACC), and other regions in the OCD network, including the insula, dorsal ACC (dACC), orbitofrontal cortex (OFC) and rostral striatum. These dysfunctional connections lead to impaired behavioral flexibility in response to changing contextual cues, specifically in situations with uncertain aversive outcomes. In P3, we will recruit and examine 50 unmedicated/ serotonin reuptake inhibitor/clomipramine medicated participants with OCD, and 50 healthy participants (18-35 yrs; to minimize effects of long illness history and medication on neural measures). We will use state-of-the-art diffusion imaging (dMRI), using tractography, segmentation and tract profiling - tractometry - to examine WM bundles in the network. We will use functional Magnetic Resonance Imaging (fMRI) to examine activity, functional and effective connectivity (FC, EC) among network regions during a novel probabilistic approach avoidance task (PAAT) that we developed to examine the influence of uncertain rewarding and aversive outcomes on choice behavior, and neural activity, FC and EC during evaluation and anticipation of these outcomes. We will examine relationships among WM, activity, FC and EC abnormalities in the OCD network in OCD participants and the severity of OCD symptom dimensions associated with persistent avoidance, e.g., harm avoidance. Biological sex will be a covariate in analyses. Aim (A)1 will compare the microstructure of WM bundles that connect vlPFC, rACC, insula, dACC, OFC and rostral striatum in OCD vs. healthy participants, using a novel combination of tractography, segmentation and tractometry. A2 will compare activity within and FC and EC among these OCD network regions in OCD vs. healthy participants during the PAAT, to determine relationships among PAAT performance and fMRI abnormalities in OCD vs. healthy participants. A3 will examine relationships among OCD symptom dimensions that are relevant to persistent avoidance: e.g., harm avoidance, contamination/ washing, responsibility for harm/checking symptoms, and: WM and fMRI abnormalities in A1-2. P3 will benefit from expertise in: NHP neuroanatomy and physiology in P1, 2 (A2); dMRI data analysis in P1, Core B (A1); clinical assessment and treatment of OCD in P4, 5 (A3); the study of individual differences in neuroanatomy in Core C (A2-3); and integration of analyses across projects in Core D. In close collaboration with other projects and cores, P3 will be the first study to elucidate the specific WM bundle, and functional, OCD network abnormalities that are associated with persistent avoidance, to inform interventions for disorders characterized by this behavior.

NIH Research Projects · FY 2025 · 2014-05

The overarching goal of this Program Project Grant (PPG), “Comparative Genomics of Longevity and Alzheimer’s disease” is to identify mechanisms responsible for health and longevity, with the focus on genome/epigenome stability and Alzheimer’s disease (AD) and related dementias (ADRD), in long-lived mammalian species. Mammals differ over 100-fold in their maximum lifespans, from 2 years in a shrew to over 200 years in the bowhead whale. Characterization of the processes responsible for this disparity in lifespan will enable the development of interventions to prevent or cure age-related diseases including AD and ADRD. The central hypothesis of this PPG is that long-lived species have evolved more efficient mechanisms to maintain genome/epigenome stability and prevent Alzheimer’s and ADRD, which can be adapted to extend human healthspan. In the previous phase of the PPG, we generated exciting data that support our hypothesis. Specifically, we identified DNA double strand break repair as a mechanism that strongly correlates with longevity; improved DNA repair in mouse cells by specific amino acid changes; showed that the naked mole rat hyaluronan synthase 2 gene improves mouse health and lifespan and delays ADRD pathology; showed that mutation rates are lower in long-lived species; developed epigenetic clocks for naked mole rats; discovered mechanisms responsible for longevity and genome stability in the longest-lived mammal, the bowhead whale; identified omics profiles of long-lived species; identified a potential mechanism for sporadic AD in degu; and generated new mouse models with genes from long-lived mammals. In the next cycle we will expand our studies to developing interventions and applying novel omics techniques. This PPG is comprised of four integrated projects and three cores. Project 1 (Gorbunova; Garcia) is focused on mechanisms responsible for more efficient genome/epigenome stability and developing anti-aging and AD and ADRD interventions. Project 2 (Seluanov; O’Banion) develops novel animal models of AD resilience (naked mole rat) and sporadic AD (degu). Project 3 (Vijg; Fang) investigates mutations and epimutations in animals with diverse lifespans and in AD models using novel, high throughput single-molecule approaches. Project 4 (Gladyshev; Tyshkovskii) uses multi-omics to identify genes and pathways involved in genome/epigenome stability across species and developing aging, longevity and AD biomarkers. The research team consists of investigators dedicated to longevity research who are experts in comparative biology and DNA repair (Gorbunova), long-lived rodents (Seluanov), AD and ADRD (O’Banion), mutagenesis and single-cell approaches (Vijg), comparative genomics and biomarkers (Gladyshev; Tyshkovskiy); cutting-edge omics techniques, proteomics, and mass spectrometry (Garcia); long-read sequencing (Fang) and bioinformatics (Zhang, Core C). Moreover, we developed a collection of primary cells and tissues, and animal colonies, to facilitate comparative studies of longevity and AD (Seluanov, Core B). This assembly of expertise uniquely positions our team to achieve unprecedented insight into the biology of longevity.

NIH Research Projects · FY 2026 · 2014-04

The long-term goal of this project is to define the molecular mechanisms which regulate epigenome stability during aging and the role of the mammalian sirtuin, SIRT6, in this process. Growing evidence indicates that epigenome structure becomes compromised with age which may be the root cause of age-related decline in cell and organ function. SIRT6 emerged as a critical regulator of multiple pathways related to aging such as genome and epigenome stability, tumorigenesis, inflammation and glycolysis. Additionally, SIRT6 overexpression extends the lifespan of mice. Our laboratory has demonstrated that SIRT6 is an upstream regulator of DNA double strand break (DSB) repair. We demonstrated that SIRT6 is phosphorylated by JNK1/2 in response to oxidative stress on amino acid S10 and this phosphorylation is required for the stimulation of DSB repair. We have shown that, in addition to controlling DSB repair, SIRT6 maintains genome stability by repressing transposable elements, and that oxidative stress causes re-localization of SIRT6 from the promoters of transposable elements to the DNA breaks. SIRT6 has two biochemical activities deacetylase (deacetylase) and mono-ADP ribosylase. The function of SIRT6 deacetylase activity is best characterized. In contrast, mono-ADP ribosylation activity of SIRT6 is much less studied. Our work has implicated this activity in DNA repair and epigenome stability. Our recent unpublished data analyzing biochemical functions of a SIRT6 variant found in centenarians, showed that centenarian SIRT6 has reduced deacetylation activity and enhanced mono-ADP ribosylation activity. This suggests that SIRT6 mono-ADP ribosylation activity is important for longevity. Therefore, we set out to identify the function and new targets of SIRT6 mono-ADP ribosylation. Using mass spectrometry we identified novel targets for SIRT6 mono- ADP ribosylation including histone H1, H2A, H2A.J, and chromatin remodelers BRG1 and SMARCC2. Furthermore, we showed that SIRT6-mediated ribosylation of SMARCC2 is required for activation of Nrf2 target genes in response to oxidative stress. Thus, we are ideally positioned to conduct further mechanistic studies of the role of SIRT6 mono-ADP ribosylation activity in epigenome stability. We will pursue the following specific aims: (1) examine the role of SIRT6 in maintaining epigenome stability in the context of cellular senescence and aging; (2) examine the mechanisms of SIRT6 effect on epigenome; specifically, the biological function of H1, H2A, H2A.J, BRG1 and SMARCC2 mono-ADP ribosylation; and (3) determine the role of SIRT6 mono-ADP ribosylation activity in epigenome stability and longevity by analyzing the knock-in mouse model which expresses ribosylation deficient SIRT6 mutation. The proposed research will delineate new pathways regulated by SIRT6, which are relevant to epigenome stability and aging. As such, we expect that these experiments will reveal critical, new information about the aging process, and will help to develop new strategies for treating age-related diseases.

NIH Research Projects · FY 2025 · 2013-04

Project summary Lipid droplets are the intracellular sites for fat storage, with essential functions in lipid metabolism and energy storage. They also have a second major role in controlling the fate of specific proteins, namely mediating their maturation, transport, refolding, storage, and turnover. While there has been great progress in unraveling how droplets control lipid metabolism, studies of their protein-handling roles remain limited. The goal of this project is to dissect the mechanism, regulation, and physiological relevance of this protein handling function, by taking advantage of the best characterized example of this phenomenon, the sequestration of histone H2Av to lipid droplets in Drosophila ovaries and embryos. H2Av is dynamically stored, exchanging constantly between lipid droplets via the cytoplasm. Lipid droplet sequestration prevents H2Av degradation in oocytes and its premature import into nuclei in embryos. It is also developmentally regulated, being switched off in embryos at the mid- blastula transition. Progress in the last granting period identified the importins Impa2 and Ipo9 as critical factors mediating H2Av exchange, uncovered a correlation between the activity of the cell cycle kinase Cdk1, the phosphorylation state of Impa2, and the rate of H2Av exchange, and discovered that excess nuclear H2Av dramatically alters the transcriptome. These insights led to new models about the mechanism of H2Av exchange, its regulation, and its biological role, models that the current application proposes to test. It is hypothesized that Impa2 promotes H2Av exchange by physically interacting with Jabba and reducing its affinity to H2Av and that Ipo9 acts as cytoplasmic chaperone, accompanying H2Av on its journey between LDs. This model will be tested by determining intracellular distribution and dynamics of the two importins in ovaries and embryos and by analyzing how mutants in key functional domains affect H2Av exchange. To understand the developmental regulation, point mutants in Impa2 will be generated to prevent or mimic phosphorylation and Cdk1 activity will be inhibited. The consequences of these manipulations for H2Av exchange and the physical interactions between importins, the H2Av anchor Jabba, and H2Av will be determined. In nurse cells lacking Jabba, absence of sequestration results in increased levels of nuclear H2Av. Using RNA seq analysis, manipulation of H2Av dosage, and Jabba mutants unable to bind H2Av, it will be determined whether the altered H2Av levels result in changes to the transcriptome. If successful, these studies will test key predictions about the mechanism, regulation, and function of H2Av sequestration by lipid droplets and develop general paradigms for how protein handling by lipid droplets can control processes elsewhere in the cell. These studies will thus provide the groundwork for determining if and how protein sequestration by lipid droplets contributes to their prominent roles in health and disease.

NIH Research Projects · FY 2025 · 2010-04

The goal of this proposal is to establish a new model for masked detection and frequency resolution, applicable to listeners with normal hearing and hearing loss, based on realistic physiological response properties. We are developing a new, fundamental framework for neural representations of acoustic stimuli that can predict a wide range of psychoacoustic phenomena. This framework is focused on neural fluctuations of auditory-nerve (AN) fibers, rather than on energy, average rates, or phase-locking to temporal fine structure. Neural fluctuations (NFs) refer to the relatively slow changes over time in AN responses (i.e., changes with rates ranging from 10s to a few 100 Hz). Neural fluctuations in this frequency range are of interest because they strongly excite, or suppress, neurons in the auditory central nervous system. The NF model is based on known nonlinear properties of inner-hair-cell and AN responses, and thus has important implications for interpreting masking results in listeners with sensorineural hearing loss. A representation of masked sounds based on the NF model is an alternative to the commonly accepted excitation-pattern representation provided by the power spectrum model of masking. The NF model successfully describes basic masking thresholds, as well as many experimental paradigms for which the power-spectrum (or energy) model fails. The NF model is not limited to low frequencies, as are models based on phase-locking to temporal fine structure. Here, the NF framework will be applied not only to masking paradigms, but also to stimulus paradigms that focus on frequency resolution, such as discrimination of the fundamental frequency of harmonic complex tones, or detection of increments in profile-analysis stimuli. Current models for the representation of these stimuli rely on a conceptual peripheral filter bank with critical bandwidths, estimated from human masking results using the power spectrum model of masking. Critical bandwidths, assumed to limit the frequency resolution of the auditory representations of complex sounds, are not consistent with known physiology. In contrast, frequency resolution according to the NF model is grounded on physiologically realistic response properties of AN fibers and sensitivity to neural fluctuations observed in the midbrain. Finally, to explain perception based on NF cues across the entire range of audible sound levels, we will extend our AN model to include NF-driven feedback gain control, guided by the known physiology and anatomy of the medial olivocochlear efferent system. The studies proposed here include: i) computational modeling to predict human thresholds, including re-examination of classical datasets that can, and those that cannot, be explained by the power-spectrum model, ii) related physiological studies in the midbrain, where cells are strongly sensitive to fluctuating inputs, and iii) new psychophysical studies designed to challenge the NF model, in listeners with normal hearing and those with sensorineural hearing loss.

NIH Research Projects · FY 2025 · 2009-08

Abstract Rheumatoid arthritis (RA) is a destructive inflammatory joint disease associated with increased morbidity and mortality. While biologic therapies have improved treatment response, unmet clinical needs remain due to the refractory nature of RA, and recurrent disease flares despite aggressive treatment. In the prior funding periods, we developed a multidisciplinary translational research program that combined longitudinal near infrared (NIR) imaging of indocyanine green (ICG) with targeted therapies to elucidate how TNF, B cells and lymphatics converge to trigger arthritic flare in murine models and RA patients. Specifically, we found that prior to RA signs and symptoms, there is an increase in lymphatic vessel (LV) contraction frequency to enhance the efflux of CD11b+ myeloid cells from the affected joints, which ultimately gets overwhelmed at early onset. RA disease proceeds with expansion of joint draining lymph nodes from an influx of unactivated-polyclonal CD23+/CD21hi/CD1d hi B cells in inflamed nodes (Bin) and lymph, until a sudden loss of LV contractions is observed along the ipsilateral axis, which results in lymph node collapse and Bin clogging of the sinuses. Interestingly, knee synovitis following popliteal lymph node (PLN) collapse in TNF-Tg mice is ameliorated by anti- CD20 B cell depletion therapy (BCDT), which restores passive but not active lymph flow. Most recently, we demonstrated that loss of LV contractions in TNF-Tg mice is secondary to activated macrophage adherence to the lymphatic endothelial cells (LEC), and subsequent LEC and lymphatic muscle cell (LMC) damage from chronic inflammation. Remarkably, this major defect can be corrected with anti- TNF therapy that ameliorates the inflammatory-erosive arthritis, and restores LEC-LMC integrity. To further understand the role of lymphatics in arthritic flare, we propose three Specific Aims. In Aim 1 we will demonstrate perivascular LMC progenitor incorporation into PLVs during growth and flare in WT growing mice (homeostasis), and TNF-Tg mice with Expanding vs. Collapsed PLN treated with anti- TNF or placebo. We will also confirm their LMC progenitor potential in adoptive transfer studies in vitro and in vivo. In Aim 2 we will demonstrate the role of PDGF signaling in LMC during the Expanding and Collapsed phases of arthritic progression via functional genomic studies, and genetic loss of function in the setting of acute collagen antibody-induced arthritis and chronic TNF-induced arthritis in mice. To correlate these animal studies with human disease, in Aim 3 we will complete a clinical pilot of RA patients receiving anti-TNF therapy for hand flare, to formally demonstrate the utility of NIR-ICG imaging as a biomarker of LV recovery, and its correlation with response to therapy. Completion of these Specific Aims will substantiate our paradigm-shifting hypothesis of RA flare, and may provide novel insights into refractory disease that can be diagnosed by assessing efferent lymphatics.

NIH Research Projects · FY 2024 · 2008-09

Advancements in computational psychiatry allow us to isolate multiple, specific cognitive mechanisms that determine human behavior. This formal modeling framework generates quantitative parameter estimates that can serve as bridges between pathophysiology and psychopathology. A major goal of computational psychiatry is to translate these laboratory tools so that they can be used in the clinic. Two critical hurdles need to be overcome. First, the enhanced validity and sensitivity of computational metrics needs to be established relative to standard behavioral performance metrics in key psychiatric and nonpsychiatric populations. We propose to do that by addressing a range of cognitive and motivational domains that have been strongly implicated in psychopathology, including working and episodic memory, visual perception, reinforcement learning, and effort based decision making. Second, we need to establish and optimize the psychometrics of these computational metrics so that they can be used as tools in treatment development, treatment evaluation, longitudinal, and genetic studies. These powerful metrics must have adequate test-retest reliability, and not be limited by ceiling and floor effects. We propose to develop these methods using an open, flexible, and scalable framework and demonstrate that they provide valid data both in the laboratory and in large-scale Internet-based data collection, facilitating “big data” studies of cognitive processes. To this end, the current project will leverage the expertise of Cognitive Neuroscience Task Reliability and Clinical applications in Serious mental illness (CNTRACS) consortium, a multi-site research group with an established record of rapid cognitive tool development and dissemination. Aim 1 is to establish that model based parameters for the measurement of cognitive function are more sensitive than standard behavioral methods in assessing deficits across a range of common mental disorders, and have an enhanced capacity to predict clinical symptoms and real-world functioning, with a sample of 180 patients with psychotic and affective disorders (both medicated and unmedicated) and 100 healthy controls. Aim 2 is to measure and optimize the psychometric properties (test re-test reliability, internal validity, floor and absence of ceiling and practice effects) of computational parameters described in Aim 1, in a new sample of 180 psychiatric patients and 100 healthy controls. Aim 3 is to establish the feasibility and replicability of model-based analytic approaches outside the laboratory for assessing RDoC dimensions of interest, and to assess their relationships to variation in psychotic-like experience, depression and anhedonia, as well as real- world functioning in a community sample of 10,000 recruited over the Internet. Aim 4 is to validate key model based parameters against well-characterized neurophysiological measures acquired using EEG recordings during task performance. Successful completion of these Aims will significantly advance the field by providing easily administered and scalable web-based tools for estimating the integrity of key neural systems that underlie normal cognition and motivation and form the basis of common forms of cognitive and affective psychopathology.

NIH Research Projects · FY 2026 · 2008-08

Abstract Saliva hydrates and lubricates the oral cavity and contains proteins that begin to digest food. Saliva also contains numerous substances that protect the oral cavity and upper gastrointestinal tract from microbial infection. The importance of salivary secretion is generally underappreciated; however, a reduction of salivary fluid flow (xerostomia) leads to a severe deterioration in the quality of life. Patients report difficulty swallowing and chewing food, with a marked increase in dental carries and susceptibility to oral candidiasis. Xerostomia is associated with collateral damage following g-irradiation used as a therapy for head and neck cancers and in Sjogren’s disease, a relatively common autoimmune disease. In both maladies, profound xerostomia is reported before any evidence of glandular destruction indicating that a defect in stimulus-secretion coupling underlies reduced secretion early in disease. To develop therapy for xerostomia it is fundamentally important to understand the processes that lead to saliva secretion physiologically to appreciate how these mechanisms are altered in pathological states. The overarching principle driving this long-term project is that a synergistic combination of experimental investigation and quantitative theoretical modeling can be used to further our understanding of both salivary gland physiology and pathology in a manner that neither single approach can accomplish in isolation. In the current proposal, we will build on preliminary data that demonstrates that fluid secretion is dependent on Ca2+ signaling that occurs in an apical microdomain in acinar cells, and not the bulk cytoplasm. This microdomain, which we term the “secretory synapse”, consists of the endoplasmic reticulum expressing inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channels within ~50 nm of the apical plasma membrane harboring the Ca2+ activated Cl channel, TMEM16a. TMEM16a activity is dependent on sustained local Ca2+ release in the secretory synapse which is in turn reliant on efficient refilling of the ER in the microdomain and influenced by Ca2+ flux from endolysosomal Two Pore Channels (TPCs). Notably, Ca2+ signaling and the structure of the secretory synapse are altered in a mouse model of Sjogren’s disease and following g-irradiation of salivary glands. We will construct a new computational model of fluid secretion intimately based on experimental data, that now reflects the specialized subcellular architecture and local signaling events occurring in the apical secretory synapse microdomain. This new acinar cell model will be integrated into our current, anatomically realistic 3D model of the secretory unit, developed in the previous funding period. The model will be used to both qualitatively explain the data and to predict how changes on a micro-spatial scale can influence the secretion of the whole gland. We will subsequently utilize the models to understand how the apical microdomain is disrupted in disease states to suggest and subsequently test how function may be restored. It is envisioned that the model may ultimately suggest novel therapies to restore salivary gland function, which would not be readily evident from a traditional purely experimental methodology.

NIH Research Projects · FY 2025 · 2007-09

PROJECT SUMMARY Humans are not aware that their eyes are always in motion. Even when attending to a single point, fixational eye movements (FEM) continually shift the stimulus on the retina in ways that would be immediately visible had the motion originated from objects in the scene rather than oculomotor activity. It is now clear that FEM are vital for visual sensitivity, fine pattern vision, and acuity. Furthermore, a considerable body of evidence, in part from our NIH-funded research, indicates that this behavior embodies a sensorimotor strategy by which the visual system processes spatial information in the temporal domain. While much has been learned about the monoc- ular functions of FEM, little is known about their consequences for binocular vision. Ocular drifts—the incessant inter-saccadic movements—differ considerably in the two eyes. How does the visual system combine continually changing inputs from independently jittering eyes? Here we focus on FEM consequences for 3D spatial repre- sentations, specifically their role in stereopsis (Aim 1), their decoding mechanisms (Aim 2), and their binocular control (Aim 3). The research strategy consists of assessing FEM effects on the binocular visual input and ex- amining the resulting implications for neural coding, perception, and control. Our driving hypothesis is that the active space-time encoding strategy that emerged in the monocular processing of luminance also applies to pro- cessing binocularly-derived features at later stages of the visual stream. Since this theory yields counter-intuitive hypotheses, each aim builds on a supporting preparatory study that sets the stage for the proposed experiments. Aim 1 builds on the surprising observation that stereopsis is impaired when fixational disparity modulations are selectively eliminated from the visual flow, even in the presence of otherwise normal luminance modulations on the retina. We will explore the causes for this impairment and elucidate FEM contributions. Aim 2 focuses on the mechanisms by which the fixational visual flow is interpreted. Contrary to traditional assumptions, our prelimi- nary evidence indicates that the visual system has access to extraretinal knowledge of ocular drift and uses it to infer spatial relations at high spatiotemporal resolution. Aim 3 examines these ideas in the context of oculomotor control. We provide the first comprehensive high-resolution measurements of head-free binocular FEM in natural real-world tasks and test the hypothesis that eye drifts are actively controlled to encode task-relevant features (e.g., disparity, spatial contrast, etc.). The experiments rely on the combination of (a) binocular measurements of human eye movements with unprecedented accuracy; and (b) highly flexible, binocularly synchronized, gaze- contingent control of retinal stimulation, an approach made possible by our recent instrumentation developments. All experiments are theoretically grounded and all hypotheses supported by new preliminary data. They are, to our knowledge, entirely novel, and confirmation of any of them will have broad implications for understanding the functional principles of the visual system, the computational mechanisms of perception, possible oculomotor contributions to neuro-ophthalmologic disorders, and the development of rehabilitative strategies and prostheses.

NIH Research Projects · FY 2025 · 2007-08

PROJECT SUMMARY The goal of the project is to understand the genetic basis and evolution of thermotolerance in the Saccharomyces species. Whereas much has been learned about the causes of phenotypic variation within species, reproductive barriers have limited genetic analysis to closely related, inter-fertile species. Consequently, we know little about the genetic basis of substantial phenotypic differences that arise over longer time periods and the importance of evolutionary hotspots and historical contingency. To overcome this reproductive barrier we will use hybrid, proteomic and transgenic analysis of yeast species. In Aim 1 we will map thermotolerance in interspecific hybrids using reciprocal chromosome loss and CRISPR induced mitotic recombination. This will enable us to measure the combined effects of many genes, but also identify thermotolerance genes and determine: whether they evolved through the cumulative effects of multiple changes, and whether their effects depend on changes in other genes. In Aim 2 we will investigate divergence in protein thermal stability and whether it results in temperature dependent fitness effects not detected by mapping. In Aim 3 we will examine the reuse of thermotolerance genes during thermal divergence along independent lineages. Doing so will determine whether thermal divergence requires certain genes or can be achieved in different ways. Together, these aims will push the limits of interspecific genetic analysis in order to provide insight into the acquisition of a complex and broadly relevant phenotype that has taken millions of years to evolve.

NIH Research Projects · FY 2026 · 2007-02

The University of Rochester (UR) Infectious Diseases Division (IDD) has meaningfully contributed to the NIAID HIV/AIDS Clinical Research Networks (CRNs) for over 30 years. The enduring mission of the UR Clinical Trials Unit (CTU) is to support and conduct clinical research on HIV/AIDS that contributes to the discovery of a preventive vaccine for HIV, as well as to the discovery of a cure for HIV, and of treatments that reduce the burden of disease due to HIV infection and its complications. It will acheive this mission by providing exceptional scientific and administrative leadership, and outstanding clinical trials infrastructure, and by engaging with all relevant stakeholders and communities, to ensure that the research it conducts is responsive to community needs. Five aims are proposed. The first is to strengthen our well-established Clinical Research Sites (CRSs). To do this, we will support 2 domestic CRSs that will participate in the following NIAID CRNs: (i) the `AIDS Clinical Trials Group' (ACTG); and (ii) the `HIV Vaccine Trials Network' (HVTN). We will also develop a plan to diversify UR CTU leadership, and to enhance integration and coordination of these CRSs. The second aim is to provide exceptional administrative leadership that accelerates and enhances NIAID clinical research. We will do this by continuing and enhancing effective financial management procedures and a robust quality management program, and by continuously assessing well-defined metrics of operational performance and capacity; these measures will be used to determine improvement opportunities that will be addressed by targeted actions and follow-up evaluation to assess the efficacy of such interventions. Performance data will be coupled to resource allocation, and poorly performing CTU components will be terminated, if necessary. The third aim is to provide exceptional scientific leadership that promotes important contributions to the advancement of HIV/AIDS science, and mentors the next generation of CTU researchers. The CTU will oversee and coordinate groundbreaking research activities at the participating CRSs, develop and execute a strong research agenda for the ACTG and HVTN, mentor and train the next generation of CTU researchers, and draw upon campus-wide intellectual capital. Aim four will provide outstanding clinical trials infrastructure, including data and quality management, regulatory support, laboratory, pharmacy and other resources. Innovations will include procedures that facilitate flexibility and surge capacity, allowing a rapid response to emerging Network needs, as well as shared resources that promote cost efficiency. Finally, our fifth aim is to communicate and engage effectively with all UR CTU stakeholders. The Rochester Community Advisory Board (CAB) and our diverse Community Education and Recruitment (CER) Team will engage with local communities, to increase community awareness and education, and facilitate recruitment and retention of research participants. Finally, we will also continue highly responsive communication and engagement with NIAID, its CRNs, and all other key constituencies.

NIH Research Projects · FY 2026 · 2006-07

SUMMARY: The long-term goal of the proposed project is to identify strategies to safely restore genome and epigenome stability in aged cells and tissues. Sirtuin 6 (SIRT6) is a chromatin regulator; SIRT6 KO mice exhibit premature aging and genomic instability, while SIRT6 overexpressing mice show extended lifespan. In the previous funding cycle, we identified SIRT6 as the regulator of several DNA repair pathways, most notably, DNA double strand break repair. We showed that SIRT6 is more active in long-lived species and identified a variant of SIRT6 with a missense mutation in the C-terminus enriched in human centenarians. The centenarian SIRT6 has higher mono-ADP ribosylation activity and stronger interaction with Lamin A. Our unpublished data shows that overexpression of SIRT6 reduces epigenetic age of human cells from aged donors and represses expression of transposable elements. Furthermore, we found that delivering SIRT6 to aged mice using AAV vectors reduces frailty. We demonstrate that SIRT6 activator, fucoidan, extends lifespan and healthspan of aged mice. Following these findings, we would like to understand the mechanisms by which SIRT6 “rejuvenates” the epigenome. SIRT6 contains an intrinsically disordered region (IDR) in its C-terminus. Our unpublished data shows that SIRT6 forms biomolecular condensates in vitro with RNA, DNA and nucleosome arrays. Formation of condensates is dependent on the SIRT6 C-terminal IDR. Interestingly, the mutations in the centenarian variant of SIRT6 are located in the IDR. Four Lys to Ala substitutions in the SIRT6 IDR abolished condensate formation in vitro, and disrupted DNA repair function, but not LINE1 suppression function of SIRT6 in vivo. Using stochastic emission depletion super-resolution fluorescence microscopy (STED), we observed that in vivo SIRT6 forms condensates that are evenly distributed throughout the nucleus, while the phase separation mutant had a more diffuse distribution. Our preliminary RIP-seq analysis identified lncRNAs interacting with SIRT6. Our unpublished data show that SIRT6 interactome is enriched in RNA binding proteins and chromatin modifiers. We hypothesize that SIRT6 via its IDR interacts with lncRNAs and chromatin helping maintain epigenetic blueprint of the cell. SIRT6 overexpression in aged cells restores the epigenome to a more youthful state by using the epigenetic blueprint. In this application, we propose to understand the function of SIRT6 IDR in maintaining chromatin organization, SIRT6-RNA interactions, and test the role of SIRT6 IDR in aging and epigenetic rejuvenation. We will pursue the following aims: (1) Characterize SIRT6-RNA interactions, SIRT6 biomolecular condensates, and the structure of SIRT6 IDR. (2) Examine the role of SIRT6 IDR domain and condensate formation in SIRT6 canonical functions: DNA repair, telomere maintenance and LINE1 suppression. (3) Examine the role of SIRT6 IDR and SIRT6-interacting RNAs in rejuvenation and aging. The proposed experiments will outline a novel facet of SIRT6 function in epigenome maintenance and uncover the RNA-mediated epigenetic blueprint which may be used for cellular rejuvenation and safe epigenetic reprogramming.

NIH Research Projects · FY 2025 · 2005-08

When an observer moves through the environment, self-motion creates large-field patterns of visual motion known as optic flow. Cortical processing of optic flow is important for perception of self-motion, as demonstrated in animal models. In most navigation behaviors, such as driving a vehicle, optic-flow processing is part of an active sensing loop in which self-motion produces optic flow which is, in turn, used to change direction at the next instant of time. While this is a natural mode of optic flow processing, almost all previous studies of neural processing of optic flow involve passive self-motion without an active control component. Indeed, the patterns of optic flow that are experienced when steering a vehicle along a curved path are quite different than those typically used to study cortical neurons. Thus, what we know about cortical processing of optic flow is generally not applicable to control of steering. Furthermore, to effectively steer, control theory indicates that optic flow signals should be combined with efference copy of motor commands and working memory signals, but essentially nothing is known about these critical interactions. We propose a tightly integrated program of research, involving behavior, neurophysiology and theory, that will provide the first systematic study of the neural mechanisms of visually guided steering control. In Aim #1, we train monkeys to steer through a virtual environment to align their heading with the remembered direction of a briefly flashed target. We develop optimal stochastic control theory for our task, and we fit the model to behavioral data to extract estimates of key latent variables that govern steering control. In Aim #2, we record from populations of neurons in areas MSTd and 7a during performance of the steering task. Using both model-free and model-based analyses, we test specific hypotheses about how and where the critical variables for steering control are represented in the brain. Our strong preliminary data suggest that we will make important advances in understanding cortical processing of optic flow in a more natural active-sensing context. The proposed research is directly relevant to the research priorities of the Strabismus, Amblyopia, and Visual Processing program at the National Eye Institute.

NIH Research Projects · FY 2025 · 2004-07

SUMMARY The goal of our project is to understand the structural features guiding the initial stages of pre-mRNA splice site recognition, which are critical for accurate pre-mRNA splicing and frequently dysregulated in human diseases. We focus on the U2AF, SF1 and SF3B1 splicing factors directing the U2 small nuclear ribonucleoprotein (U2 snRNP) to the 3´ splice sites of pre-mRNAs. The U2AF subunits, U2AF2 and U2AF1, recognize the polypyrimidine and AG-dinucleotide splice site signals. A third subunit, SF1 initially associates with the branch point sequence of the pre-mRNA then is displaced by the SF3B1 subunit of the U2 snRNP. Dynamic phosphorylation and dephosphorylation of SF3B1 is required for formation of the active spliceosome. Previously, we made progress towards understanding the molecular underpinnings of the initial steps of 3´ splice site selection. Using X-ray crystallography, biophysical techniques, and functional assays for pre-mRNA splicing in human cells, we have shown that representative cancer-associated mutations at the U2AF2 – RNA interface disrupt RNA binding and splicing. We have deciphered structural details showing how U2AF2 accommodates diverse nucleotides of splice site signals. By complementary single molecule Förster resonance energy transfer approaches, we further revealed that the U2AF2 conformation changes in response to different splice site sequences as well as the U2AF1 subunit and its recurrent cancer-associated mutation. We have established important, functional interfaces of U2AF2 with SF1 and SF3B1 during pre-mRNA splicing in human cells and discovered that phosphorylation strongly reduces SF3B1 binding to U2AF2. However, these provocative results raise new questions. First, what are the effects of recurrent U2AF2 mutations in neurodevelopmental disorders compared to those in cancers? Second, how is dynamic SF3B1 phosphorylation and dephosphorylation temporally regulated with U2AF2 dissociation prior to splicing? Third, how are the U2AF2, U2AF1, and SF1 subunits arranged to accurately recognize the splice site signals and ensure the fidelity of splicing? We address these questions in the aims of this proposal by leveraging structural approaches (including X-ray crystallography, cryoelectron microscopy, calorimetry, and fluorescence) and complementary functional assays (including co- immunoprecipitations, pre-mRNA splicing assays, and transcriptome-wide sequencing). Altogether, the results of these aims contribute to understanding the structural and functional underpinnings of 3´ splice site recognition and its dysregulation in human disease.

NIH Research Projects · FY 2025 · 2004-06

The goal of this renewal application is to maintain and further develop the world's most comprehensive research resource specializing in the use of the amphibian Xenopus laevis as a multi-faceted experimental platform for biomedical and immunological research and for the benefit of the whole scientific community. Interests and medical relevance of X. laevis are due to the remarkable similarity of its immune system with that of human, the accessibility to experimentation at all developmental stages, as well as the availability of large genetic and genomic resources, invaluable MHC-defined inbred strains, clones and transgenic (Tg) lines of frogs, as well as tools such as cell lines, monoclonal antibodies, MHC tetramers, tagged recombinant reagents and batteries of validated PCR primers for immune-relevant genes. These animals and reagents that are not commercially available need to be preserved, enriched, and made available to the scientific community. As in previous proposals, two major main aims are proposed: (1) Preserving and promoting the X. laevis research resource for immunobiology by keeping on producing, managing and distributing animal stocks and reagents to laboratories in the US and abroad. We will maintain the quality, productivity and welfare of our inbred and Tg animals. We will assist, train and inform scientists, students and educators interested in using X. laevis for research. We will foster the information, accessibility, networking and public awareness of the resource using our web site, and by interacting with other Xenopus resources nationally and internationally. (2) Developing new methodologies and generating new experimental animals and reagents by leveraging X. laevis to advance research in 3 main areas: (1) Genetically modified X. laevis lines of frogs: to improve genome annotation, generate new CRISPR/Cas9-mediated inbred Tg knockout (KO) lines and lines with traceable immune cell types by knocking-in long single stranded donor templates (lssDNA). (2) X. laevis cell culture systems and reagents: to produce transfected and deficient cell lines, recombinant tagged immune reagents and MHC tetramers as well as to develop VSV-lentiviral transducing vectors for in vitro and in vivo studies in Xenopus. (3) X. laevis experimental platforms for infectious disease and developmental immunotoxicology: to develop cost-effective and reliable experimental platforms to study infection and pathogenicity of emerging human non-tuberculosis mycobacterium (NTM) pathogens (e.g., M. abscessus); and assess overlooked risk for immunity and human health of water pollutants with endocrine disruption activity. In addition to maintaining a research resource that is crucial for the Xenopus scientific community, this project will develop new approaches and technologies broadly applicable for gathering innovative insights into tissue and organ physiology, immunology and developmental biology. This will contribute to the efforts of the scientific community assisted by the NIH to establish Xenopus as a relevant model for biomedical research.

NIH Research Projects · FY 2025 · 2003-07

ABSTRACT In the US there are >700,000 new heart attacks (acute myocardial infarctions) a year, and >300,000 patients undergo scheduled ischemia during cardiac surgery. Beyond tissue reperfusion (angioplasty, thrombolysis) there are no FDA-approved interventions to limit acute cardiac injury due to ischemia and reperfusion (IR). This renewal proposal supports our ongoing research program in cardiac metabolism, and is focused on novel cardioprotective metabolic signaling events downstream of glycolysis. It is built on the following premise: (i) Acidic pH during IR is cardioprotective, and many therapies that boost glycolysis work in-part by enhancing metabolic acidosis. (ii) Succinate accumulation is a key event in ischemia, and its oxidation at reperfusion drives reactive oxygen species generation. We propose acid can regulate succinate dynamics (accumulation, oxidation, transport). (iii) The glycolytic byproduct methylglyoxal (MGO) causes glycative stress in diabetes, but type-I diabetic hearts are acutely protected against IR injury, and MGO inhibits the mitochondrial permeability transition (PT) pore, a key event in IR injury. Some cardioprotective interventions also elevate MGO. (iv) The mitochondrial enzyme ALKBH7 is necessary for necrosis. Inhibition or ablation of ALKBH7 is cardioprotective, and this protection requires the MGO metabolizing enzyme GLO-1. Based on these published and preliminary findings, our central hypothesis is that the cardioprotective effects of elevated glycolysis are mediated by pH, succinate, and MGO. This hypothesis will be tested by addressing 3 related aims… Aim 1 will investigate succinate dynamics in IR injury and the impact of pH. Aim 2 will investigate the role of MGO as an acute cardioprotective signal, including identifying its targets in mitochondria. Aim 3 will study ALKBH7, identify its substrates, and develop ALKBH7 inhibitors as cardioprotective drugs. The aims will use established experimental systems (adult cardiomyocytes, perfused hearts, LC-MS based metabolomics, in-vivo IR injury, and a high-fat diet model of diabetes) and engineered mice including Alkbh7-/-, Glo1-/- and hGlo1TG. Innovation is embedded in the idea that MGO can serve an acute protective signaling role separate from its well-known chronic pathologic effects (i.e. hormesis). This work will advance basic knowledge on ischemic cardiac metabolism, will develop small molecule therapeutics, and will offer mechanistic insights to other pathologies beyond IR.

NIH Research Projects · FY 2025 · 2002-08

Abstract. Secretion of saliva, a watery fluid containing ions and a host of protein constituents, is the primary function of the major salivary glands. The importance of salivary fluid secretion is most palpable when it is reduced. Commonly, this occurs in the autoimmune disease, Sjogren’s syndrome (SS). Notably in SS, a decrease in fluid flow occurs prior to any deleterious morphological changes in the gland that occurs because of immune cell infiltration. Importantly, this indicates that early in disease, defects in the stimulus-secretion coupling machinery occur prior to any gland destruction. A fundamental understanding of the earliest alterations in signaling in SS are however lacking. To model early events in SS, we have used a mouse model where the Stimulator of Interferon Genes (STING) pathway is activated. This ubiquitous pathway, known to be activated in SS, is initiated by sensing cytoplasmic cyclic dinucleotides derived from DNA arising from virus, mitochondria and dying cells to result in a type -1 interferon response and thus mirrors key features of SS disease. Following parasympathetic nervous input, the appropriate stimulation of fluid secretion is absolutely dependent on the proper localization and activation of the elements of the stimulus-secretion coupling machinery to distinct domains of the polarized acinar cell. Our preliminary data show that fluid secretion is reduced ~50% following STING activation, but paradoxically the peak acinar cell Ca2+ signal measured in vivo in an animal expressing a genetically encoded Ca2+ indicator in acinar cells is markedly augmented. In addition, while stimulated Ca2+ signals are apically confined in physiological situations, the spatial characteristics of the Ca2+ signal are disrupted in the model. We posit that disruption of the Ca2+ signal contributes to both the initial hypofunction and ultimately the progression of disease. The proposed studies will investigate the mechanisms occurring at the onset of SS which result in hypofunction together with the pathways associated with progression of disease. We will use complimentary, but independent approaches to address these goals. In Specific aim 1, single cell RNA sequencing will be used to identify genes and target pathways in salivary gland cell populations involved in the disruption of fluid flow and progression of disease and will inform all subsequent studies. We will validate findings at the protein level and by functional assays. In tandem, we will explore promising candidates involved in fluid secretion suggested by our preliminary functional data whose abundance, localization or activity is disrupted in the SS disease models. Where possible targets and pathways will be validated in human tissues provided by our collaborators at NIDCR. In specific aim 2, we will use in vivo imaging to define mechanisms which are involved in the disruption of the Ca2+ signal in the disease models. We hypothesize that changes in Ca2+ sequestration together with Ca2+ release/influx mechanisms will be revealed. We hypothesize that the altered Ca2+ signals are a compensatory mechanism in response to reduced secretion that are ultimately detrimental. Consistent with this idea, mitochondrial morphology is severely disrupted in the disease model. In Specific aim 3, we will investigate how mitochondrial Ca2+ handling, bioenergetics and reactive oxygen species production are altered in disease. In total our studies are designed to provide insight in events occurring at the onset of SS which ultimately should provide targets for novel therapeutic intervention.

NIH Research Projects · FY 2025 · 2001-09

As the only NIMH-funded postdoctoral training program dedicated solely to suicide prevention research, the CSPS T32 continues to shape the field. This application is for competitive renewal (Years 21 through 25) of an NIMH Institutional National Research Service Award (T32 MH20061) titled, “Postdoctoral Training Program in Suicide Prevention Research.” We seek support for six postdoctoral training slots. A component of the University of Rochester’s (UR) Center for the Study of the Prevention Suicide (CSPS), the “CSPS T32” has as its long-term objective to develop a cadre of early career scientists with the knowledge and experience necessary to establish careers as independent investigators and members of interdisciplinary teams dedicated to the study of suicide and its prevention. Training emphasizes suicide prevention research consistent with the revised NIMH Strategic Plan for Research – 2020, which calls suicide “an urgent priority for NIMH”. Fellows in psychology, public health, medicine, and related disciplines undergo a two-year training sequence guided by an “individual development plan” designed by each fellow and her/his mentor at the outset of training and annually. The curriculum includes a coordinated series of courses, seminars, and workshops designed to provide the fellow with a firm foundation in the theories and methods of suicide prevention research. Core areas of knowledge and skill development include (a) suicidology, (b) research methods, (c) academic career development and “survival skills”, and (d) professional integrity and the ethical conduct of research. Each fellow works with one or more Mentors selected from the UR faculty on development and implementation of an individually tailored program of applied suicide prevention research. A third year of mentored research training is available on a selective basis. An evaluation plan closely tracks the performance of each trainee and, over the longer term, of the program itself in preparing its graduates for careers in intervention, outcomes, and health services research in general and suicide prevention studies in particular. Suicide is a complex, multi-determined behavior. The CSPS T32 has been successful in recruiting fellows who are from cultural and academic backgrounds that reflect the diverse perspectives needed to understand suicide and its prevention. Retention through the program and into careers in suicide prevention research has been excellent. Program innovations with this application include increased emphases on training in (a) the use of digital technologies, artificial intelligence and health analytics in suicide prevention research; (b) the translational neuroscience of suicide; (c) the role that sensory (hearing and vision) deficits play in suicide risk, and the recruitment of fellows from those communities; and (d) the lived experience of individuals and families affected by suicide. The fellowship is further enriched by the addition to its already robust network of collaborators of linkages with (i) the UR Health Lab for digital technology innovation, (ii) the UR Del Monte Institute for Neuroscience, (iii) UR Center for Eye and Brain Health.

NIH Research Projects · FY 2024 · 2001-04

Project Summary RNA plays a central role in many gene-regulatory processes and is viewed as an important drug target. Riboswitches are natural RNA sensors typically found in bacterial mRNAs where they bind cognate metabolites. This process elicits riboswitch conformational changes that control expression of downstream genes. Although most riboswitches bind only a single ligand, we discovered that the Type I preQ1-I (class I) riboswitch recognizes two metabolites, leading to positive cooperativity that extends the riboswitch’s preQ1-sensing range in bacterial cells. The discovery that a small, single-domain RNA can bind two metabolites is unprecedented in the field. The importance of this discovery is heightened by the fact that the Type I sub-class is the most prominent preQ1- sensing riboswitch in the biosphere and exists in many human pathogens. Surprisingly, cooperative binding by the Type I preQ1-I riboswitch has gone undetected for more than a decade — until now. The overarching goal of this proposal is to define the underlying molecular attributes that confer cooperativity and its interplay with preQ1- sensing during bacterial gene regulation. To address this challenge, we developed innovative tools including: (i) adaptation of a bacterial reporter assay in which the Type I preQ1 riboswitch controls GFPuv expression, which revealed two EC50 values for preQ1 binding; (ii) development of software for analysis of cooperative isothermal titration calorimetry (ITC) data, which yields microscopic binding constants whose ratio quantifies cooperativity; and (iii) refinement of our GFPuv reporter coupled in cell (ReCo-ic)SHAPE (selective 2´-hydroxyl acylation analyzed by primer extension) assay to pinpoint functionally-relevant, preQ1-dependent riboswitch conformations in live bacteria. These approaches form a rigorous foundation to define the chemical determinants of cooperative ligand binding and to evaluate their effects on biological function. We will address our central goal in three aims: (1) Determine crystal structures of Type I preQ1 riboswitches in apo and bound states; (2) Define riboswitch chemical attributes that confer cooperative binding and evaluate their role in bacterial gene regulation; and (3) Define gene-regulatory conformational changes using ReCo-icSHAPE in concert with all-atom computational approaches to delineate cooperative binding pathways. We are a team of experts with strong records in: RNA crystallography, effector binding, RNA chemical modification and bacterial reporter assays (Wedekind, P.I.); RNA dynamics and computational prediction of RNA structure using experimental restraints (Mathews, co-I); next- generation sequencing (Pritchett, collaborator); and biophysical approaches (Jenkins, collaborator). Given our novel premise, expertise and team synergy, we are uniquely qualified to perform this work. High-value outcomes include a new structural and chemical understanding of cooperative binding by the smallest natural riboswitch aptamer, which we hypothesize is a hallmark of the entire Type I sub-class. Overall, the proposed analysis will broaden our understanding of RNA-mediated gene regulation, which has implications for antibacterial targeting.

NIH Research Projects · FY 2026 · 2001-04

Social communication depends on the integration of sensory, emotional and cognitive information by a large network of brain regions. While many brain regions integrate auditory and visual information, the inferior frontal lobes (IFG) receive a wealth of sensory afferents from multiple modalities, has influence over many brain regions involved in motor and cognitive processes, and plays a major role in our speech and language processes. Previous investigations in primate ventrolateral prefrontal cortex (VLPFC), a proposed homologue of human IFG, revealed that neurons are selectively responsive to species-specific faces and to vocalizations and perform complex integration of these communication stimuli. Furthermore, VLPFC is essential during crossmodal working memory. These varied functions of VLPFC occur over several cytoarchitectonic regions, each of which receives different anatomical afferents and demonstrates diverse sensory and cognitive responses. Inability to integrate face and vocal information impairs speech processing, language learning, recognition and semantic processing, all essential elements of social communication that VLPFC participates in. Hence, identifying the integrative and cellular functions of VLPFC subdivisions will clarify the organization of the primate frontal lobe and its role in disorders of communication. In this proposal, we will determine what components of face and vocal information are integrated within VLPFC subregions and what neural mechanism underlie integration in these subregions. In Aim 1, we will investigate which features of face and vocal information are integrated and whether subregions of VLPFC process and integrate these stimuli differently (i.e. What is integrated Where). We hypothesize that modality specific subregions of VLFPC will show differential responses to manipulations of spectrotemporal features of face and vocal stimuli. In Aim 2, we will interrogate how face and vocal stimuli are integrated by single neurons and across simultaneously recorded ensembles during the presentation of dynamic naturalistic audiovisual stimuli. In particular we will determine if VLPFC neurons perform time division multiplexing, where the neural response to face-vocalization movie shows evidence of “switching” back and forth between neural representations of the face or the vocal stimulus during integration. Furthermore we will degrade the face and vocal stimuli in an audiovisual pair in order to selectively diminish the contribution of the face or the vocal stimuli during integration and to determine how this affects our temporal model of these responses. Merging ensemble recordings of responses to naturalistic stimuli in the prefrontal cortex of Old World Primates with novel computational methods, will provide new and valuable data on how we combine social communication information. This is of particular relevance to understanding changes in sensory integration in intellectual disabilities such as autism spectrum disorders.

NIH Research Projects · FY 2025 · 1997-04

The overarching goals of the Rochester Environmental Health Sciences Center (EHSC) are to prevent disease and improve health by advancing innovative and impactful translational environmental health research, engaging communities to address environmental health issues, and enhancing career development of talented environmental health investigators. More specifically, the goals of the Center are to: (1) enrich the ability to hypothesize and implement cutting-edge translational environmental health research using modern approaches that also move knowledge into action; (2) advance science by stimulating the prediction and prevention of detrimental exposures on health across the lifespan by developing and using the best available tools and approaches, and integrating new information to understand cumulative risks; (3) promote career development of the next generation of environmental health investigators in a collaborative environment; (4) foster interactions of Center members and community partners to cultivate new ideas and respond to issues of concern at the local, national, and international level; and (5) support existing and build new institutional and inter-institutional partnerships. The Rochester EHSC achieves these goals by providing a framework that is anchored in our overall mission to improve public health through the generation of fundamental knowledge and elaboration of mechanisms by which chemical exposures, alone or through interaction with other modifying factors, contribute to cumulative health risk across the life span. A key strength of the Center is that activities are not siloed within the study of a specific organ system or a single disease. As such, the central theme that integrates EHSC research and community engagement programs is the desire to understand how exposures and interactions of environmental factors affect health and disease across the lifespan. This theme weaves together and synergizes Center member efforts, such that the totality of our impact is greater than each part would achieve on its own. Further supporting the Center is a strong tradition of emphasizing and integrating basic mechanistic research in model systems with clinical and epidemiological approaches, which catalyzes multidirectional transformation of new information into actions. The Center also sustains strong community partnerships that develop, advise, and also learn from new methods and engage with communities in our region and across the nation. Unique and expanded strengths of our Center combined with emerging new areas underlie proposed Center activities, programs, and use of resources, and provide the foundation for future success. Further supporting our success is the broad-based scientific expertise of our faculty, which ideally positions us to apply integrative and innovative strategies to address critical questions in environmental health sciences.