University Of Wisconsin-Madison

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT Yellow fever (YF) is a deadly disease transmitted by the bite of infected Aedes aegypti mosquitoes. Although an effective vaccine for YF was invented in the 1930s, it is not amenable to large-scale production and has a high rate of adverse events (including death). As a result, YF continues to kill approximately 30,000 people each year. Moreover, due to the re-emergence of Aedes mosquito vectors, substantial portions of Southeast Asia, Oceania, and even Europe and North America are at increasing risk for introduction of the YF virus (YFV), which would be a public health disaster. In humans, YF presents as a classic viral hemorrhagic fever (VHF), causing severe coagulation abnormalities (i.e. coagulopathy) and end-organ damage. This contrasts with what happens in mice, where YFV infection is largely benign and characterized by a distinct lack of coagulopathy/hemorrhagic features. The host factors that determine the development of VHF in YF in humans are unknown. This project aims to identify these factors at both cellular and molecular levels. Currently, VHF in YF is thought to be driven by the infection of hepatocytes. However, unpublished data collected from Brazilian YF patients suggests that hepatocyte destruction alone is insufficient to explain the coagulopathy observed in YF. Aim 1 will test the hypothesis that the infection of immune cells plays a major (and previously underappreciated) role in the development of VHF in YF. This Aim will utilize YFV infection of transgenic mice that have been engrafted with human hepatocytes or human immune cells – two cell populations that are thought to play a major role in the VHF disease process – allowing the study YFV infection of these cell populations in isolation in this otherwise YF-resistant host. A range of coagulation tests will be performed on infected mice to determine the mechanism by which coagulopathy develops during YFV infection. Blood samples drawn daily from YFV-infected macaques – the gold-standard animal model for studying VHF in YF – also will be analyzed, as this will be essential for determining the timing and causal relationships between key events such as clot formation, clotting factor depletion, and liver damage. In Aim 2, a CRISPR-Cas9 genome wide knock-out screen will be performed utilizing human and mouse, hepatocyte and Kupffer cell lines, to identify the factors that render humans (and primates) uniquely susceptible to YFV. The factors that restrict YFV replication in the murine host – hypothesized to be an interferon-stimulated gene (ISG) based on preliminary data – also will be identified. ‘Hits’ from this screen will be validated using targeted gene knock-out and trans-complementation in vitro. This will identify host factors that are essential in the YFV life cycle, and may lead to the generation of new YF-susceptible mouse models. This research will generate information and biological tools that can be used to combat YF specifically, and VHF more generally. This award will also help the principal investigator, Dr. Adam Bailey, MD, PhD, establish himself as an independent researcher and enable him to pursue high-reward projects that would otherwise not be feasible.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT The need to provide safe and cost-effective measures to prevent Cancer is self-explanatory. The University of Wisconsin (UWISC) as the Lead Academic Organization (LAO) together with our collaborating institutions (Affiliated Organizations/AOs) propose to continue performing early phase cancer prevention trials, for the National Cancer Institute (NCI), Division of Cancer Prevention (DCP), as a Cancer Prevention Clinical Trials Network (CP-CTNET) site funded as a UG1 Consortium as described in RFA-CA-18-031. Our overarching goal is to evaluate the effects of novel preventive agents/interventions on pertinent biological endpoints in order to efficiently determine their appropriateness for further testing and potential viability toward becoming recommended societal interventions to lessen the burden of cancer. We will pursue this overarching goal via the following specific aims: (1) Efficiently design, conduct and perform Phase 0, 1, 2 Chemoprevention Clinical Trials of novel and/or re-purposed immunologic-based or molecular targeted agents, assessing their effects upon relevant biological (biomarkers/clinical effects) endpoints. (2) Build upon existing consortium infrastructure to complete Aim 1 via intra- or inter-consortium (CP-CTNET) multi-institutional collaborations in concert with NCI/DCP contributing three to four new Phase 0, 1 or 2 clinical trials and accrual of ≥ 40-50 subjects per year. We are well positioned to successfully achieve the above aims due to our combined experience with prevention agent development (consortium members currently hold >90 DCP sponsored R, U or PREVENT awards) and our consortium's staffing, organization, and management which results in the timely development, performance and completion of impactful, multi-institutional early phase cancer prevention trials. Our enhanced consortium consists of >10 NCI-designated Cancer Centers, institutions providing access to robust numbers of at-risk patients (e.g. Mayo Clinics, Seattle Cancer Care Alliance) and expanded access to at-risk under-represented minorities and special populations (e.g. University of Puerto Rico, Alaska Native Medical Center). Supported by the Carbone Cancer Center, the University of Wisconsin CP-CTNet site is determined to contribute to advancing the field of Cancer prevention through performance of early phase prevention trials.

NIH Research Projects · FY 2025 · 2020-09

ABSTRACT The CISNET Breast Working Group (BWG) conducts innovative modeling research focused on new precision oncology paradigms that are expected to re-define breast cancer control best practices. In the parent award, we selected significant topics where modeling is suited to fill evidence gaps and facilitate clinical and policy translation. The activities of this award have encompassed multiple lines of activity including analyses for the US Preventive Services Task Force, publishing a cross-CISNET monograph, dedicating Rapid Response funds to address priority areas for new investigation, and fostering the career of early-stage investigators. The models share common inputs and provide a standard set of outcomes for benefits (e.g., distant recurrences and deaths avoided, mortality reductions, life years and quality-adjusted life years), harms (e.g., false positives and benign biopsies, advanced stage diagnoses, overdiagnosis and treatment impact on quality of life), and costs. Unique components of our approach include modeling of absolute risk of disease accounting for multiple risk factors, evaluating emerging screening modalities, addressing important comorbidities—specifically type 2 diabetes—that affect both disease risk and survival, and providing guidance regarding new precision systemic treatments and their impact on outcomes among survivors. The specific aims of this extension are to: 1) complete manuscripts in progress; 2) fully implement common inputs that consider important recent trends in screening and treatment; 3) update documentation of program code and manuscript specifications; and 4) retain essential programming staff to complete coding, finalize documentation, and facilitate future use of breast cancer modeling resources. This scope of work would not be feasible without the availability of five distinctive BWG models: Dana Farber (D), Georgetown-Einstein (GE), MD Anderson (M), Stanford (S), and Wisconsin (W). This extension period will ensure that the BWG models are supported to incorporate the latest input data and record key decisions as a foundation for future use. Retention of key programming staff will guarantee that modeling resources will be updated, documented, and accessible for utilization in new studies. Continuously funded for the past 25 years, the modeling teams have published >230 research papers informing public health and trained >20 junior investigators. An experienced Coordinating Center provides the infrastructure to support the project goals including resource sharing and model accessibility. The exceptional environment provides unprecedented synergy and leveraging of resources to address new research questions and support career development that would not otherwise be possible. Overall, infrastructure support for the Breast Working Group will advance modeling research and guide breast cancer control decision making.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT At least 90% of individuals with Parkinson disease (PD) experience significant vocal communication deficits that appear preclinically, negatively impact quality of life, and are refractory to current therapeutic approaches. Management of PD voice deficits is restricted by a limited scientific understanding of the preclinical disease framework resulting in a critical gap in knowledge. This research proposal directly aims to improve the understanding of preclinical PD biology to ultimately reduce disease progression and vocal symptom burden. Neuroinflammation plays an important role in the initial insult, spread, and severity of neurodegenerative disease and epidemiological studies suggest that anti-inflammatory drugs may be therapeutic and reduce the incidence of PD. Results of our current award, using the Pink1-/- rat model of preclinical PD, so far demonstrate that NF-κB and MAPK signaling result in upregulated inflammation in the brainstem vocal pathway. This continuing proposal is grounded in the new central hypothesis that inflammation within vocal motor brainstem nuclei, particularly the reciprocal modulation between microglia and astrocytes, contributes to neuronal dysfunction and death. Further, we hypothesize that this mechanistic action within vocal nuclei results in the preclinical onset and progression of PD voice deficits, and that anti-inflammatory drugs targeting these mechanisms will prevent progression PD vocal deficits. To accomplish this, Specific Aim 1 will use Pink1-/- and wildtype control co-cultures (astrocyte, microglia, and neurons) and a series of cross-talk experiments to quantify gene and protein changes in the NF-κB and MAPK inflammation pathways as well as evaluate their responses to anti-inflammatory drugs. We expect that, compared to wildtype controls, Pink1-/- co-cultures and media will demonstrate high levels of cytotoxic and apoptotic markers that will be decreased with anti- inflammatory drugs. Specific Aim 2 will use spatial transcriptomics within the brainstem descending vocal motor pathway to evaluate cell type and gene expression changes with age and sex between Pink1-/- and wildtype rats. We expect to see significant upregulation and clustering of immune cell numbers with age in the Pink1-/- rat compared to controls. Then, Pink1-/- rats will be given site-specific administration of anti- inflammatory drugs into the vocal pathway, tested for vocalization behavior, and inflammatory gene expression will be re-analyzed. We expect that vocalization acoustic parameters will increase with anti-inflammatory drugs (to control-like levels) and correlate with reductions in the production of proinflammatory cytokines. This proposal is the first to adopt an innovative approach combining both in vivo and in vitro methods for studying preclinical CNS inflammation in PD and vocalization. This work is significant because these data will initiate long-term advancements to the field by identifying the early inflammatory pathophysiology of voice deficits as well as anti-inflammatory treatments that target these mechanisms.

NIH Research Projects · FY 2024 · 2020-09

Radiochemistry: Analytical Track Food Defense 2020 Wisconsin State Laboratory of Hygiene FDA Laboratory Flexible Funding Model PAR-20-105 Project of Radiological Discipline Abstract/Summary Establishing a radiological baseline for the dairy products of Wisconsin The Wisconsin State Laboratory of Hygiene (WSLH) is Wisconsin’s public and environmental health laboratory. As part of the University of Wisconsin-Madison, the WSLH is committed to exploring new ideas and developing new programs to benefit the state and nation. The WSLH’s Radiochemistry unit has been a member of the Food and Drug Administration’s Food Emergency Response Network (FERN) for over twelve years. This agreement has enabled the WSLH radiochemistry department to maintain and enhance its testing capability and capacity for rapid and accurate response in the event of radiological food emergencies. The department has experience in gamma and alpha spectrometry, gas proportional counting, and liquid scintillation counting. The Radiochemistry unit uses only approved Environmental Protection Agency (EPA) and FDA FERN Cooperative Agreement Program methodologies. The unit is certified and inspected by the EPA, a complete quality assurance and quality control program is maintained. This laboratory is not a regulatory laboratory for human or animal food, nor does it perform animal diagnostic laboratory services. According to the Department of Agriculture, Trade and Consumer Protection, Wisconsin produced over 30 billion pounds of milk last year and is the country’s largest producer of dairy products. With the cessation of the EPA RadNet milk sampling program in November 2014, WSLH’s Radiochemistry unit is seeking to fill this deficit in the nation’s food monitoring by testing Wisconsin’s raw milk to further the FDA’s efforts to prevent foodborne exposures to contaminants from a radiological event. The program will focus on analysis of raw unpasteurized milk samples from the state’s dairy plants for gamma emitting isotopes, including 137Cs and 131I. All results obtained for this project would be reported to the nationally integrated science system FERN. Future goals include increasing the number of dairy plants beyond the initial scope of this project and expanding to incorporate alpha and beta emitters, such as americium, plutonium, and 90Sr.

NIH Research Projects · FY 2024 · 2020-09

Presynaptic inhibitory synapses positioned across axon terminals of sensory neurons critically regulate information flow across sensory circuits, allowing meaningful interactions of an organism with its external environment. Whereas much is known about the functional role of presynaptic inhibitory synapses across sensory circuits; little is known about the mechanisms that regulate the development, maturation and maintenance of these inhibitory synapses. Using the well-characterized dim-light (rod) visual circuit of the mammalian retina we uncovered a synaptic reorganization during assembly of GABAergic presynaptic inhibitory synapses that regulate dim-light retinal output. The current proposal aims to determine the cell-autonomous and non-cell autonomous mechanisms that regulate this developmental plasticity during assembly of inhibitory feedback circuits that regulate the gain of sensory (retinal) signal transfer. Our research will yield fundamental information about: (i) retinal circuit assembly (ii) organization of sensory circuits and mechanisms that regulate sensory feedback, and (iii) principles that regulate receptor plasticity during establishment of inhibitory circuits across the CNS. We will combine murine transgenic approaches with high resolution light microscopy, 3D electron microscopy and electrophysiology to address the following three Aims. In Aim 1 we will determine if cell-autonomous alterations in chloride transporter expression across developing retinal rod bipolar neurons drive and regulate the timing and/or occurrence of the developmental GABAA receptor reorganization. Aim 2 will determine the contribution(s) of excitatory and inhibitory neurotransmission onto the retinal rod bipolar neuron in regulating the developmental GABAA receptor plasticity. Aim 3 will determine the role of early visual experience in regulating GABAA receptor reorganizations and assembly of feedback inhibitory synapses of the dim-light retinal circuit. Our research will reveal the interplay between cell-autonomous mechanisms, synaptic input, network activity and environmental cues during establishment and maturation of feedback inhibitory circuits that regulate sensory output. Our study will also reveal circuit plasticity motifs that can be recruited to ameliorate dysfunction during retinal diseases. Furthermore, our findings will determine the developmental sequence of maturation during assembly of invivo presynaptic inhibitory circuits to compare with exvivo retinal assembly such as when pluripotent stem cells are used for retinogenesis.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY/ABSTRACT This career development proposal will provide Dr. Michelle Kelly, a Pediatric Hospitalist Physician at the University of Wisconsin School of Medicine and Public Health, with the training and mentorship required for success as an independent, physician-scientist leveraging tools and technologies supporting family engagement to improve the quality and safety of care of hospitalized children. Dr. Kelly has championed efforts to promote family engagement, including redesigning hospital rounds to include families and implementing a bedside portal – an online application giving families’ access to parts of their child’s inpatient medical record. She has attained an MS in Clinical Investigation to develop skills in conducting literature reviews, analyzing quantitative data, and designing clinical and translational studies. Building on this foundation, her proposed K08 career development plan focuses on three knowledge gaps: qualitative methods, human-centered design of interventions to support health communication and literacy, and systematic intervention implementation and evaluation. With the protected time afforded by this award for coursework, participation in national meetings and mentored research, Dr. Kelly will attain the critical skills necessary to develop and disseminate healthcare interventions. In her proposed research plan, she will develop BedsideNotes as a model intervention and implementation bundle. This bundle will consist of expanding the bedside portal to share inpatient doctors’ admission and progress notes with families and complementary implementation strategies to optimally support family engagement while mitigating unintended negative consequences. She will develop this bundle using these aims: 1) identify family and clinician perspectives of barriers, facilitators and strategies to share inpatient doctors’ notes; 2) develop BedsideNotes design requirements; and 3) implement and evaluate the feasibility and preliminary efficacy of BedsideNotes in a pilot study with families of hospitalized children. This proposed study responds directly to NOT-HS-13-011 and addresses multiple research areas, including health IT design and implementation, and will provide preliminary data describing the use and impact of health IT on family engagement and patient safety outcomes. As faculty at an institution with extensive research infrastructure, Dr. Kelly is in an ideal environment to complete this proposed research and pursue advanced training. Her career development plan includes protected time for coursework and mentorship from a committed team of experts in human factors and systems engineering, patient safety, qualitative methods, health communication/literacy, pediatric ethics, and health IT intervention design, implementation, evaluation and dissemination. At the end of this project, Dr. Kelly will have the preliminary data necessary to support a competitive R01 proposal in which she will test whether this intervention improves family engagement and safety concern reporting. This K08 award will also allow her to achieve her goal of independently leading a program leveraging tools and technologies to improve the safety of care of hospitalized children, an AHRQ priority population.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY Optimal brain health requires effective cerebrovascular function, adequate perfusion, and highly responsive blood flow regulation. If any of these, or a combination of these, are compromised, there are implications for brain health. Previous research demonstrated that cerebral hypoperfusion and inadequate cerebrovascular responses to vasoactive stimuli may precede the onset of cognitive impairment. Indeed, adults with cognitive impairment, including vascular dementia and Alzheimer’s disease, exhibit inadequate cerebral perfusion. Yet, the majority of evidence linking hypoperfusion to cognition comes from preclinical models, and there is minimal research on how chronic cerebral hypoperfusion may impact cerebrovascular control in humans. Accordingly, there is a critical need for more research on the pathophysiology of cognitive decline in humans. Our preliminary data indicate that adults with cerebral anatomical variations demonstrate cerebral hypoperfusion and reduced cerebrovascular reactivity. This finding is important as it presents our investigative team with a group of individuals with a cerebral anatomical variant that may naturally model a state of chronic cerebral hypoperfusion. Our overarching hypothesis is that chronic hypoperfusion, resulting from a specific variation in cerebrovascular architecture, impacts cerebral blood flow regulation which increases the risk of cognitive impairment. Thus, the objectives of this application are to investigate chronic models of hypoperfusion in humans, examine compensatory mechanisms to maintain perfusion, and determine the potential impact on cognitive health. For each aim, we will recruit participants from a unique, risk-enriched cohort of middle-aged and older adults from the University of Wisconsin-Madison Alzheimer’s Disease Research Center. This cohort has extensive longitudinal data on medical health, genetics, and cognitive biomarkers. We will use state-of-the-art imaging modalities to identify differences in cerebrovascular architecture and quantify cerebral blood flow regulation in the following specific aims: In Specific Aim 1, we will examine the compensatory responses to a model of acute hypoperfusion and determine the impact of chronic cerebral hypoperfusion observed in adults with specific cerebral anatomical variations. In Specific Aim 2, we will utilize aerobic exercise to characterize the cerebrovascular responses to acute hyperperfusion and determine the impact of cerebral anatomical variations. In Specific Aim 3 we will determine the impact of cerebral anatomical variations on cerebrovascular control and establish whether adults with cerebral anatomical variations are at a higher risk of cognitive decline. This project will be the first systematic investigation of cerebrovascular control mechanisms in acute and chronic cerebral hypoperfusion in humans, and will address the potential implications of long-term hypoperfusion for cognitive health. Upon completion, we will understand the impact of cerebral anatomical variations on cerebrovascular health and the risk of Alzheimer’s disease and related dementias.

NIH Research Projects · FY 2025 · 2020-08

Patients with high-grade serous ovarian cancer (HGSOC) are frequently diagnosed with extensive metastatic disease, resulting in a poor prognosis. In HGSOC, metastasis occurs primarily by transcoelomic spread, where tumor cells detach from the primary tumor, float through the peritoneal fluid, and attach to the mesothelial layer to form new metastases. Tumor cells in patient ascites exist as single cells or in multi-cellular aggregates similar in size to experimental spheroids. We hypothesize that single cell and aggregate-based metastasis are distinct processes in HGSOC transcoelomic spread. To test this hypothesis, we will utilize a combination of engineering-based approaches (in vitro culture systems, multivariate modeling, computational fluid dynamics) and biological methods (molecular and cellular assays, analysis of patient samples, xenograft models). This proposal leverages a diverse, collaborative team that includes experts in engineering, biology, and the clinical presentation of HGSOC. Completion of the proposed studies will result in an improved understanding of mechanisms regulating transcoelomic spread and identification of potential targets for future work to control metastatic spread.

NIH Research Projects · FY 2025 · 2020-08

SUMMARY SARS-CoV-2, the causative agent of COVID-19, has emerged as a global human pathogen sweeping through our communities. The development of strategies to prevent or treat COVID-19 is essential for mitigating disease and limiting further viral spread. A key target of antiviral therapeutics is the virus RNA replication complex. The SARS-CoV-2 replication complex is a large multi-subunit machine with multiple co-factors, enzymes and host modulating proteins. How these protein subunits assemble and cooperate to carryout viral RNA replication and transcription remains unclear. The overarching theme of this work is to examine understudied aspects of the coronavirus replication complex with an emphasis on characterizing the effects of existing antiviral therapeutics as well as the discovery of novel targets and compounds. We are interested in how the virus nsp14 exonuclease contributes to viral RNA proofreading, reducing nucleotide misincorporations and providing natural resistance to nucleoside analogue drugs. We will also explore the function of the enigmatic nsp12 nucleotidyltransferase (NiRAN) and will piece together the network of viral protein interactions responsible for the assembly of the RNA synthesis complex. To this we will use diverse methods including biochemistry, biophysics, cell biology, chemical biology and cryo-electron microscopy. These studies will provide new insight into the workings of this complicated machine, provide new mechanisms of action for existing therapeutics and discover novel antiviral compounds. In addition, the high conservation of components of the replication machinery across the coronavirus family allows these studies of SARS-CoV-2 to be applicable to future emerging coronaviruses to head off future viral pandemics before they become global crises.

NIH Research Projects · FY 2024 · 2020-08

Summary Demand for the common marmoset (Callithrix jacchus) in biomedical research has increased tremendously over the past five years, as they have emerged as a critical biomedical model system in a variety of study disciplines. The increased use of marmosets has been most acute in neuroscience, where the need to study cognition, behavior, and mental illness in primate models has grown. Among non-human primate (NHP) models, the marmoset provides unique practical advantages for neuroscientific studies, including small size, easy handling, rapid reproductive maturation, high fecundity, compressed life cycle, and social behaviors and communication that more closely resemble those observed in humans. These advantages have also contributed to the recent surge in the demand for marmosets in studies using embryonic new genomic editing techniques. This recent upsurge in demand has resulted in shortages in availability of marmosets estimated to be in the range of 2000 marmosets needed by the overall research community each year, with our own estimate in the range of a 200- animal shortfall for the neuroscience community alone. The Southwest National Primate Research Center (SNPRC) and the Wisconsin National Primate Research Center (WNPRC) propose a major collaborative effort to dramatically increase the availability of healthy common marmosets for national neuroscience research projects. In response to the upsurge in demand for marmosets in neuroscience-related projects, the SNPRC and WNPRC propose a comprehensive plan to 1) double their output of marmosets for provision to national institutions in need of these experimental animals for neuroscience research, 2) continue to ensure the highest standards of husbandry and veterinary care and thus, the excellent health of these colonies, 3) maintain pedigree records and develop genomic databases to document and maximize genetic diversity among the expanded marmoset colonies, 4) continue collaboration between the two Centers in new studies focused on improving husbandry, breeding, and veterinary practices to maximize the health and lifespan of these animals, and 5) communicate on a regular basis with the U24 Marmoset Colony Coordination Center, and with the NIH Marmosets for Neuroscience Steering Committee. The collaborative approach proposed by the two NPRCs is both innovative and appropriate given the resources and expertise available and ensures successful completion of these aims resulting in a dramatic increase in the number of marmosets available to the neuroscience community.

NIH Research Projects · FY 2024 · 2020-08

PROJECT SUMMARY AHRQ previously funded the development of Elder Tree (ET), a laptop-based eHealth system shown in a randomized trial to improve quality of life and health factors among older adults with multiple chronic conditions (MCCs) such as diabetes and hypertension. MCCs are pervasive and costly among older adults, reducing quality of life and accounting for 90% of Medicare spending. Self-management can help patients control their conditions, but these skills are rarely taught in primary care. ET provides tools and support for such self- management and addresses the frustration, discouragement, and loneliness shared by all patients with MCCs, regardless of specific disease. Despite positive results, though, many study participants did not use ET extensively, a problem for all health apps. In a separate survey, we learned that more than 60% of non-users abandoned ET because the computer was too hard to use. Voice-controlled "smart" technology may solve this problem. Smart speakers, used by speaking and listening rather than typing and reading, avoid barriers to use such as poor vision, tremors, and interface complexity; they have been adopted at a rate faster than the Internet or TV, especially among older adults. Smart displays enhance the system with a visual element, helping users remember content and creating access to media meant to be viewed. This application proposes to test the ability of voice-activated technology to expand the implementation and sustain the use of a proven electronic health system. Specifically, we propose to test whether older adults with MCCs will use and benefit more from ET delivered by a smart system (speaker plus display), due to ease of use, than from a laptop version of ET. The proposal has 2 aims: 1) develop the smart system platform (ET-SS) and 2) test it against laptop ET (ET-LT) in a 2-arm RCT. The trial will randomize 220 patients age 65+ with 4 or more chronic conditions to receive for 8 months either ET-LT or ET-SS. We hypothesize that: · patients with ET-SS (vs. ET-LT) will have greater ET use and better quality of life in month 8. · these clinical outcomes will improve: a) 30-day hospital readmissions), b) medication adherence, and c) composite score of HbA1c, mg/dL, mmHg, FEV-1, BMI, PHQ-8, FAS-GFR, and Brief Pain Inventory. · the amount of ET use at 4 months will mediate the effects of ET at 8 months (i.e. greater use at 4 months=greater effects at 8 months), and negative affect and Self-Determination Theory constructs of competence, social relatedness, and intrinsic motivation will mediate the effects of ET use. · ET-SS will have better outcomes for women and for patients with 6+ (vs. 4-5) chronic conditions. We, the investigators who developed ET, have conducted numerous RCTs of eHealth systems and published extensively. To our knowledge, this will be the first large RCT of a smart system as a platform to implement and disseminate a health intervention. The result could make ET more accessible and effective for older patients, improve their quality of life, and point the way to more impactful delivery of future eHealth innovations.

NIH Research Projects · FY 2024 · 2020-08

The premise of this RO1 is to test a R21-derived hypothesis that inappropriate intrusion of a mitochondrial anchoring protein, Syntaphilin (SNPH), into neuronal dendrites is harmful in Progressive Multiple Sclerosis (MS). Progressive MS refers to the late-phase of MS and currently this disease phase has no treatments. SNPH is normally expressed only in axons. Surprisingly, under support from a R21, we discovered that SNPH intrudes into dendrites of Purkinje cells in the cerebellum and causes excitotoxicity in a rodent model (Shiverer) for Progressive MS (Joshi et al., 2019, Cell Reports, Article In Press 15th October). This discovery suggests that targeting SNPH to block intrusion into dendrites is a novel treatment for Progressive MS. In this follow-up RO1, we will address three important questions raised by our R21 discovery highly relevant to the basic science and clinical aspect of MS. In Aim #1, we will test the hypothesis that the pathology of dendritic SNPH intrusion in the grey matter is de-coupled from the pathology of white matter. We will test this hypothesis by showing that curing white matter pathology in the Shiverer model (by genetically suppressing axonal degeneration and by remyelination therapy) will not prevent the pathology of dendritic SNPH intrusion. In Aim #2, we will test the hypothesis that dendritic SNPH intrusion causes excitotoxicity by biasing the activation of NMDA receptors towards the pro-death, extra-synaptic NMDA receptors. In Aim #3, we will test the hypothesis that the glutamate released by synaptic activity, when it spills over to the extra-synaptic region as exacerbated by dysfunctional glutamate uptake, constitutes an early glutamate signaling cascade that triggers dendritic SNPH intrusion. Conclusion – SNPH is a key protein that controls mitochondrial movement with multi-faceted effects on neuronal behaviors in health and disease. Since the cloning of SNPH in 2000, the studies of SNPH in neurons have been exclusively in axons. The Novelty of this RO1 is a paradigm shift to pioneer the study of SNPH in dendrites. The Translational Significance is the surprising discovery that dendritic SNPH mediates excitotoxicity in Progressive MS, thereby opening up new insights to treat MS in this incurable late-phase.

NIH Research Projects · FY 2025 · 2020-08

Abstract Voltage-gated potassium ion channels (Kv) are responsible for reestablishing the resting potential of cells upon membrane depolarization. They consist of two domains, a voltage sensing domain (VSD) and a pore domain. The voltage-dependence of the VSD is sensed by the positively charged TM4 helix, causing a translation and/or secondary structure change that tugs on the pore domain to open the channel. There exist many high-resolution atomic structures of Kv channels in the depolarized state, but since it is experimentally difficult to obtain structural information under an electric field, most of the information about the polarized state comes from mutated proteins that approximate the hyperpolarized structure and from molecular dynamics simulations. During the last round of funding, we developed a method for measuring two-dimensional infrared (2D IR) spectra of membrane- embedded proteins under an electric field. We did so by using surface enhanced plasmons to increase the sensitivity of 2D IR spectroscopy by many orders of magnitude, making possible measurements on a single bilayer of membrane-embedded proteins across which a potential can be applied. Working with the Valiyaveetil lab, we have now measured voltage-dependent structural changes in the VSD of KvAP using cyano-labeled amino acids incorporated into the TM4 helix with in vivo nonsense suppression. In collaboration with the Kananenka group, we simulated the local electric field on the cyano group from molecular dynamics simulations, thereby relating the structural features of the hyperpolarized VSD to the experimental measurements. We also developed a sample cell that can create a fast voltage jump, enabling successive 2D IR spectra to measure the structural kinetics of the proteins as they switch from the depolarized to hyperpolarized state (or vice versa). In Aims 1 and 2 of this proposal, we will apply this methodology to study the voltage-dependent structural changes of the VSD from KvAP and Shaker, respectively, as well as in the full ion channels. In Aim 3, we will further develop and apply voltage-triggered 2D IR spectroscopy to time-resolve their structural motions. This combination of 2D IR spectroscopy, cyano tags or isotope labeling, and molecular dynamics simulations is a new means of investigating the structural dynamics of the hyperpolarized states of potassium ion channels and other membrane proteins.

NIH Research Projects · FY 2024 · 2020-07

SUMMARY The standard view of how we make sense of the world around us focuses on reconstructing our environment from the information received by our sensory organs. In this view, low-level brain areas (e.g., primary sensory cortex) represent basic features of objects, which are elaborated on in successive processing stages, until representations become increasingly complex in high-level areas (e.g., frontal cortex). An alternative view is predictive coding (PC), in which we model our environment to generate sensory predictions. In PC, high-level brain areas generate predictions of sensory activity and transmit them to low-level areas. A prediction that does not match the sensory information gives rise to a prediction error. This error signal is sent from low- to high-level brain areas to update the model of our environment, thereby improving future predictions to minimize errors. Modeling studies show PC is a fast and efficient way to process sensory information, and PC provides innovative hypotheses for understanding sleep and anesthesia, particularly when disconnected consciousness occurs (consciousness without awareness of the environment), like dreaming. PC also holds great promise for conceptualizing and treating brain disorders, including schizophrenia and depression. But key central features of PC have not been empirically tested and little is known about the underlying neural mechanisms. The goal of the proposed project is to characterize the neural dynamics, circuits and receptors enabling PC. There are two principle hypotheses. First, predictions depend on N-methyl-D-aspartate receptors (NMDAR) because NMDAR influence the activity of high-level brain areas where predictions are generated, and NMDAR are enriched on neurons in lower-level areas receiving predictions. Second, in disconnected consciousness, a breakdown of information transmission from low-level to high-level brain areas, as well as a breakdown of computations within each area, explains why models of our environment are not updated by external sensory information. These breakdowns prevent the comparison of predictions and sensory information, as well as the transmission of prediction errors to high-level brain areas. To test these hypotheses, we use a cross-species experimental design connecting cellular, circuit and systems levels to behavior. We will perform electroencephalography, machine learning and computational modeling to define the neural basis of PC in humans performing prediction tasks. Then we will manipulate PC using different anesthetic agents with diverse mechanisms, establishing causal relationships between receptors, large-scale brain networks and PC. In parallel, we will simultaneously record activity from sensory and high-level brain areas of non-human primates (NHPs) using the same PC tasks and pharmacological interventions to measure cellular and circuit level contributions to PC. Investigating PC will illuminate the fundamental mechanisms of perception, providing critical insights to guide therapeutic development for multiple health conditions.

NIH Research Projects · FY 2025 · 2020-07

This proposal requests continued support for the NIGMS T32 Biotechnology Training Program at UW-Madison. Our plan addresses the evolving landscape of science and career development. Trainees will become highly educated, technically adept individuals with operational and professional skills capable of addressing the needs of society across all realms of academic, commercial, governmental, and non-profit enterprises. In this renewal application, the BTP embraces the need to train students ready to contribute to the Regional Innovation and Technology Hubs (Tech Hubs) authorized by the 2022 CHIPS and Science Act. The 31 Tech Hubs announced in October 2023 will catalyze investment in technologies critical to economic growth, national security, and job creation. This investment will help communities across the country become centers of innovation critical to American competitiveness. The Wisconsin Biohealth Tech Hub (WI) led by BioForward Wisconsin – a strong partner with the BTP – seeks to advance critical genomic technologies and accelerate domestic biotech manufacturing to support advances in personalized medicine. Our deep commitment to industrial internships supports this initiative by creating new awareness of these career options and prepares students for the multifaceted roles and diverse career paths available in the modern scientific workforce. Our plan for mentorship training at the individual and leadership levels is a vital component of this proposal, recognizing its importance for career development and innovation. We have assembled a BTP faculty that is advancing biotechnology at the forefront of scientific and societal challenges. The ~300 TGE students working with BTP faculty will acquire unique and specific expertise in areas such as emerging diseases, personalized medicine, metabolic engineering, food security, bioenergy, and technology development. Each year, BTP receives many applications from these TGE students interested in joining our program. This enthusiasm stems from our known track record with a highly successful internship program, promotion of excellence and innovation in the BTP Seminar, BTP Foundations of Biotechnology and BTP Responsible Conduct of Research courses, and our increasingly visible support of many other opportunities for internships, networking, and career development across all STEM disciplines. With this large pool of eligible students and increasing enthusiasm of both students and faculty for BTP training opportunities, we request that NIGMS provide support for 12 new trainees per year (24 total), which represents only a small fraction of the clearly interested, motivated, and highly qualified students seeking to participate in our training program each year.

NIH Research Projects · FY 2025 · 2020-07

The Cellular and Molecular Pathology Graduate Training Program T32 (CMP) is a joint venture of the University of Wisconsin-Madison Graduate School, the Department of Pathology & Laboratory Medicine, and the School of Medicine and Public Health (SMPH). CMP provides rigorous didactic training in fundamental basic biomedical sciences and interdisciplinary and innovative training in the pathogenesis of human diseases. The CMP program creates stimulating, and robust intellectual environment for predoctoral training embedded in a dynamic basic and clinical translational research atmosphere to facilitate successful transitions of trainees into careers in the biomedical workforce. Biomedical Graduate Training for the future demands that first-rate academic scientists graduate with appropriate operational, technical, and professional skills to position them for diverse bioscience careers in academia, industry, or government. The primary mission of the CMP program is to answer this demand and prepare our graduates for productive careers in the rapidly evolving biomedical field. The Program provides operational (in-depth knowledge, rigorous and reproducible experimental design, critical thinking), technical (teaching state-of-the-art methods, rigor, and reproducibility), and professional (career development, individual development plans, networking opportunities, and grant writing) skills to ensure student success. The overall objective of CMP is to educate trainees to have a fundamental knowledge of pathology, molecular medicine, and translational clinical research. CMP’s specific objective is to ensure optimal Ph.D. completion rates and time-to-degree. CMP objectives integrate measurable outcomes, such as completion rates, career placement outcomes, and student retention rates. The Program draws 83 Ph.D., MD/Ph.D., or MD. funded faculty trainers. We request six non-dissertator, and two dissertator slots under the Molecular Medicine Program. Non-dissertator trainees will be supported in Y 1 or 2, and dissertators will be supported in Y 3 or 4 of training. Candidates will be selected based on commitment to research, letters of recommendation, and the potential to make significant contributions toward the health-related research needs of our nation. Our curriculum provides interdisciplinary and integrated training in fundamental concepts in modern pathobiology, emphasizing biochemical, cellular, and molecular approaches to studying human disease, including training in statistics, rigor, and reproducibility, and responsible conduct in research. Intended trainee outcomes include 100% success in trainee transition into biomedical research positions that align with the health-related needs of our nation. Modified CMP T32 Specific Aims Section The primary mission of the CMP T32 program is to prepare graduates for productive careers in biomedical or clinical research, education, or service and to position them to make significant contributions toward the health-related research needs of our nation. The CMP program will provide graduate students with operational, technical, and professional skills in interdisciplinary and integrated training in the pathogenesis of human diseases with emphasis on molecular, cellular, and biochemical approaches. Since pathology is a broad discipline, there are four focus areas for disease pathogenesis in our T32 program: Cancer Biology, Immunopathology, Neuropathology, and Signal Transduction in the pathogenesis of human diseases. To provide operational skills and in-depth understanding of common mechanisms in cellular and molecular changes that underlie diseases, we offer courses where clinicians and basic researchers provide side-by-side training. Pre-doctoral graduate training is conducted in an intellectual interdisciplinary environment embedded in an exciting and challenging basic and clinical translational research context. Training in Responsible Conduct of Research (RCR) is part of the operational/technical and professional training in the Program. Trainees will receive informal training from faculty and mentors, and formal training through the CMP RCR introductory and advance courses, and workshops. The program will enhance the training environment, and not simply provide financial support to graduate trainees, by sponsoring numerous student gatherings over the course of the year to ensure that all our trainees form a cohesive group. Specific Aim 1. Provide Operational Skills to trainees • Focus on rigorous fundamental knowledge, significant training in statistics, ethics, rigor and state-of-the art methods for innovative research design and critical thinking. • Experience for conceptualizing scientific problems, hypotheses and developing appropriate experimental approaches to test these. • Empower students with rigorous knowledge combined with analytics that will enable them to think critically challenge existing paradigms of disease treatments and pathogenic mechanisms. • Provide collaborative team-based interdisciplinary educational environment. • Require participation in innovative courses, including the Pathology 802 Histopathology for Transitional Scientists. This course is unique among the graduate curricula, introducing students to the pathogenesis of disease via integration of actual autopsy patient cases. In addition to attending twice-weekly lectures, students will participate in weekly autopsy gross organ conferences as well as microscopic review sessions. In this way, the concepts covered in lectures will be applied and reinforced in the interactive autopsy sessions. Students will also observe at least one full autopsy, gaining a three-dimensional understanding of human structure and disease. Specific Aim 2. Provide Technical Skills for trainees • Training in state-of-the-art methods and technology. Innovative hands-on cutting-edge technology training provided by the Translational Research in Pathology (TRIP) laboratory. The TRIP offers yearly training in spatial and molecular profiling methods. New technologies, including spatial gene expression methodologies are introduced to our trainees. • State-of-the-art quantitative and computational training. • Integrate responsible training in research with all courses and teaching experiences. Aim 3. Provide Professional Skills for trainees. • Provide skills needed for transition into careers in the rapidly evolving field of biomedical research and emphasize trainee development. Organize professional development panels and career workshops. • Provide platforms for presenting research findings. • Provide oversight to all trainees through the completion of their training by offering individual development plans (IDPs) integrated with careful mentoring. • Provide teaching experience that will prepare CMP trainees for all careers in the rapidly evolving biomedical research. • Provide leadership training and opportunities ensuring student participation on all program committees including the Steering Committee. Listening to our students is a crucial component of CMP training and it is important for program improvement.

NIH Research Projects · FY 2026 · 2020-07

ABSTRACT Regenerative capacity is widespread across almost all animal phyla, but the distributing pattern appears inexplicable. Teleost fishes and urodele amphibians can regenerate amputated appendages, such as fins and limbs, whereas this ability is largely restricted to digit tips in adult mammals. This diverse regenerative distribution raises questions about how animals evolve toward loss or gain of regenerative capacity. This proposal will use zebrafish fins as a model to elucidate cellular and molecular mechanisms controlling regenerative ability. A critical aspect of appendage regeneration is nerve dependence, as denervated appendages display impaired regeneration phenotypes. Previous studies showed that upon injury nerves and their associated Schwann cells provide trophic factors to drive appendage regeneration. However, the accurate reconstruction of nerves during appendage regeneration and their target cells remain unknown. Moreover, the cellular and molecular mechanisms governing production of neurotrophic factors and interaction between nerves and Schwann cells remain elusive. Recent studies also revealed that sensory cells interplay with immune cells and nerves to influence tissue homeostasis and regeneration. Yet, the cross-talks among neuro-immune-sensory cells for appendage regeneration are unexplored. Our research programs will address these key questions in the field of appendage regeneration by employing zebrafish genetic models combined with cellular, molecular, spatial transcriptomic methodologies. First, we will elucidate cellular and molecular mechanisms underlying peripheral nervous system reconstruction and neural modulation of fin regeneration. Second, we will dissect cellular and molecular dynamics that enable special epithelial cells and their interacting partners to engage in neuro-immune- epithelial interactions for fin regeneration. Third, we will utilize MERSCOPE to generate spatial maps detailing the cellular and spatial remodeling processes underlying fin regeneration. The results of these research programs will offer a comprehensive insight into the basic principles of appendage regeneration, providing new avenues to study how innervation, immune cells, and local tissues drive appendage regeneration.

NIH Research Projects · FY 2024 · 2020-07

Project Summary/Abstract Up to 65% of adults aged 65 and over will lose their ability to independently ambulate during hospitalization primarily because they were not engaged in walking during their stay. Loss of independent ambulation is now identified as a hospital acquired disability and a significant patient safety concern. Older adults spend greater than 80% of the time in bed during hospitalization and only engage in ambulation 4% of the time. Costs for new onset disability in the United States are estimated to be 26 billion dollars annually to cover increased medical and long term care needs. Having a recent hospitalization and restricted activity were strongly associated with development of a new functional impairment in older persons. Although patient ambulation may fall within the domain of multiple healthcare providers (nurses, physical and occupational therapy, and physicians), nurses have traditionally been responsible for promoting and maintaining patients’ functional mobility. But nurses infrequently ambulate patients due to multiple personal and organizational barriers that prevent them from getting patients up to walk. Asking nurses to do more will not fix the harm that is being caused to hospitalized older adults or mounting costs due to increased need for health care resources post discharge. Innovative models of care are needed that address and overcome barriers that prevent nurses from walking older patients. Pilot study results of our model of care, Mobilizing Older adults Via a systems-based INtervention (MOVIN) have demonstrated statistically significant increases in patient ambulation and change in nursing practice and unit culture, which have been sustained on the study unit for over 3 years. Our next step is to conduct an RCT using an incomplete stepped wedge cluster randomization design across four adult medical inpatient units in two hospitals. The overarching hypothesis of this project is that MOVIN will improve functional outcomes for older adult patients by producing a change in nursing practice and culture of ambulation on inpatient units. Specific aims are to: 1) test the effectiveness of MOVIN to improve functional ability of older adult patients at discharge, and 1, 3, and 6 months post discharge; 2) test the effectiveness of MOVIN to reduce healthcare utilization of older adults at discharge, and 1, 3, and 6 months post discharge; 2a) analyze a return on investment of MOVIN based on program costs and health utilization measures across different hospitals; and 3) measure change in nurse behaviors and unit culture and identify ongoing systems barriers that impact translation of MOVIN across inpatient units and different hospitals. We propose to accomplish these aims with the overarching goal of eliminating loss of independent ambulation in hospitalized older adults. This proposed project has the potential to prevent development of physical disability (loss of independent ambulation) and functional decline, improve overall quality of life for older adults during and after their hospital stay, and decrease healthcare utilization. The proposed project has the capacity to be immediately translated to other hospitals to improve care quality to older adults across the United States.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Tetherin, also known as BST-2 or CD317, is an interferon-inducible transmembrane protein that inhibits the detachment of enveloped viruses from infected cells. Under conditions of interferon-induction, tetherin is upregulated on virus-infected cells and captures nascent virions as they attempt to bud from the cell surface. Whereas most simian immunodeficiency viruses (SIVs) use Nef to oppose the tetherin proteins of their nonhuman primate hosts, HIV-1 Vpu and HIV-2 Env have acquired the ability to counteract human tetherin because of the absence of a five amino acid sequence in human tetherin that confers susceptibility to Nef. We previously demonstrated that tetherin antagonism by Vpu protects HIV-infected cells from antibody-dependent cellular cytotoxicity (ADCC). We now show that the anti-tetherin activity of Vpu also protects HIV-infected cells from antibody-dependent cellular phagocytosis (ADCP). These findings imply that by trapping virions on the cell surface, tetherin increases the sensitivity of HIV-infected cells to Fc-mediated antibody responses, and that the antiviral activity of tetherin may be much greater in vivo than previously appreciated. The current proposal builds on these studies to address the overarching hypothesis that tetherin serves as link between innate and adaptive immunity to enhance the susceptibility of virus-infected cells to antibodies. In Aim 1, we will determine the immunological mechanisms by which tetherin enhances antibody-mediated phagocytosis of HIV-infected cells. These studies will focus on the factors that influence the extent to which tetherin can promote ADCP, which will provide a better understanding of how to use these interactions to improve antibody-based treatments for HIV-1 infection. In Aim 2, we will take advantage of the power of SIV infection of the rhesus macaque as an animal model to assess the contribution of viral countermeasures to tetherin and SERINC5 to lentiviral replication and pathogenesis. These studies will reveal the impact of tetherin and SERINC5 on lentiviral infection and the therapeutic benefit that may be derived from antiretroviral drugs designed to increase the sensitivity of HIV-1 to these restriction factors. In Aim 3, we will test the hypothesis that tetherin enhances antibody-mediated control of virus replication in SIV-infected macaques. These studies are fundamental to our basic understanding of the synergy between tetherin and antibodies and the potential to exploit these interactions for the treatment and prevention of HIV-1 infection.

NIH Research Projects · FY 2024 · 2020-06

ABSTRACT Alzheimer's disease (AD) is increasing in prevalence in the United States and despite efforts to date an effective treatment remains elusive. AD presents clinically as amyloid plaque load, neurofibrillary tangles comprised of hyper phosphorylated tau, and abnormal vasculature, but the mechanistic basis for cognitive decline is not known. We have shown that the anti-aging intervention of caloric restriction (CR) preserves brain volume and neuronal synaptic density, and lowers age-related astrogliosis. Importantly, age-related shifts in redox metabolism and mitochondrial energy metabolism in brain are abrogated by CR. Our hypothesis is that neuroprotection by CR will slow AD pathology development specifically through its impact on brain metabolism. We will implement CR in APP PS1 (amyloid plaques) and hTauP301 (neurofibrillary tangles) mouse models of AD to determine the impact of CR-induced changes in brain metabolism on pathology development and the consequence for cellular networks of neurons, glia, and the vasculature. Experiments include behavioral testing, ex vivo electrophysiology, and in vivo imaging technology. Brain metabolism will be tracked using histochemistry and 2-photon metabolic imaging. Additional mechanistic studies using pharmacological and genetic approaches in primary neurons and astrocytes will determine the impact of metabolism on brain cell-cell networks. There are three specific aims: Specific Aim 1: To determine the impact of CR on AD pathology advance, documenting hippocampal dependent memory and behaviors, ex vivo measures of synaptic transmission and hippocampal neuronal networks, and brain metabolism. Specific Aim 2: To determine the impact of metabolism and AD pathology on neuron-glial crosstalk using co-cultured primary neurons and primary astrocytes. Live imaging studies will investigate how neurons with amyloidopathy and tauopathy respond to changes in astrocyte metabolism in real time. Specific Aim 3: To determine the in vivo impact of CR-induced changes in brain metabolism and AD pathology on vascular responsivity and adaptation using implanted transparent electrodes and opto-genetics coupled with coherence tomography. These studies focus on the interaction between disease pathology and the local brain metabolic environment, acknowledging the importance of layers of communication among neuronal, neuron-glia, and vascular networks, and establishing mechanisms behind the neuroprotective effects of CR. The proposed research will advance our understanding of the role metabolism plays in AD progression, and will determine if strategies to preserve brain metabolism as a function of age might have therapeutic potential as a means to ameliorate outcomes of AD, translating basic biology to clinical promise.

NIH Research Projects · FY 2025 · 2020-06

PROJECT SUMMARY/ABSTRACT Interdisciplinary women’s health research thrives at the University of Wisconsin-Madison (UW) because of the environment of excellence, the culture of teamwork, the tradition of stellar research career development programs, and the expectation of collaboration between varied disciplines. This outstanding environment will foster the next generation of leaders in women’s health research. The UW BIRCWH will provide novel, competency-based training and career development opportunities for these future leaders. The UW BIRCWH vision is to improve women’s health by developing a scientific workforce capable of leading research programs in biomedical, behavioral, and clinical research using the most advanced techniques in team science, community partnerships, and implementation science. The Multiple PIs bring extensive knowledge, experience, and skills in women’s health research, didactic training, mentoring, and research leadership. The program assembles 40 NIH-funded faculty mentors from 21 Departments in 14 Schools/Centers across UW and spanning four strategic priority areas aligned with the 2024-2028 ORWH draft Trans-NIH Strategic Plan for Women’s Health Research. The UW BIRCWH program aims to: a) identify/prepare committed and capable scholars; b) ensure scholars achieve rigorous and reproducible women’s health and sex differences research; c) enhance fundamental research skills; and d) foster fulfilling, funded, interdisciplinary research careers, increase productivity, and maximize impact. The UW BIRCWH will support four early career scholars for two to three years with the goal of attaining scientific independence and sustainable extramural support. The program will provide scholars with a competency-based curriculum, courses on leadership and women’s health, individualized experiential opportunities, and novel training in team science, the responsible conduct of research, community engagement, and implementation science. The program will align each scholar with at least three established faculty mentors, each with designated and complementary roles, to provide interdisciplinary team mentoring. The program will systematically gather actionable feedback from scholars, faculty mentors, and senior advisors to evaluate both process and outcome measures designed for continuous improvement, for programmatic evolution, and to ultimately meet program goals. UW BIRCWH leadership will complete the transition to a conceptual evaluation model called the Translational Sciences Benefits Model that ensures scholars’ research is translated to health outcomes and policy change to advance women’s health. The UW BIRCWH program will leverage a highly interdisciplinary environment, committed leadership, and ambitious early career faculty to fuel discoveries and accelerate translation to drive toward a future in which women’s health and sex differences research positively impacts all women, families, and communities.

NIH Research Projects · FY 2024 · 2020-05

Early detection of ovarian cancer using screening algorithms is ineffective, even in high-risk populations. Patients who carry germline mutations, such as BRCA, have limited options to lower their ovarian cancer risk, short of removing their ovaries and fallopian tubes. There is a critical need for novel methods to prevent ovarian cancer without the negative consequences of surgical menopause. Drugs that inhibit OXPHOS, such as atovaquone, have potential as effective chemoprevention agents. Atovaquone is a mitochondrial complex III inhibitor. Preliminary data from our laboratory support atovaquone's ability to effectively block OXPHOS by interfering with mitochondrial electron transport. Atovaquone is currently FDA approved for the treatment of malaria, and is a well-tolerated, orally available medication. It slows ovarian cancer growth in vitro and in vivo and increases p53-related apoptosis. Hypothesis: We hypothesize that atovaquone will block oxidative phosphorylation, increase oxidative stress, and potentially activate p53-mediated apoptosis, preventing precursor lesions from progressing to ovarian cancer in a genetically engineered mouse model. Aim 1. Examine the role of atovaquone in delaying the onset of ovarian cancer in an OVGP1 mouse model. The OVGP1 BPRN genetically engineered mouse model is based on fallopian tube transformation and mimics human high-grade serous carcinoma development. This mouse model will be used to determine if atovaquone delays the onset of ovarian cancer in mice predisposed to develop this disease. Additional studies will investigate short-term transcriptome changes seen in the ovary and fallopian tube that could serve as additional exploratory biomarkers in our proposed window-of-opportunity clinical trial. Aim 2. Complete a window of opportunity clinical trial examining the effects of atovaquone on normal fallopian tube and ovarian epithelium in patients undergoing planned gynecologic surgery. Eligible patients will be women scheduled to undergo removal of at least one fallopian tube for benign indications. Baseline cytology sampling of the fallopian tube will be performed using office hysteroscopy. Cells collected can be used for transcriptome analysis. The subjects will be exposed to atovaquone for 25-35 days pre- operatively. MDA expression, a marker of inhibition to OXPHOS, will be measured after atovaquone exposure to confirm its proposed mechanism of action. IHC expression for p53 and p53 phosphorylation will be performed. Additional biomarkers from our mouse work may be added. Aim 3. Investigate potential barriers to atovaquone therapy. The Nrf-2 chemoresistance mechanisms pertinent to oxidative phosphorylation will be explored. It is critical to develop strategies to overcome the antioxidant mechanisms induced by Nrf-2 regulated genes, including superoxide dismutase (MnSOD), catalase, and hemoxygenase-1 (HO-1).

NIH Research Projects · FY 2026 · 2020-05

PROJECT SUMMARY/ABSTRACT The eukaryotic transcriptome is defined by both genomic sequence and pre-mRNA processing steps that define the composition of mature mRNAs. Nearly every human mRNA must be processed by splicing to remove introns and cleavage and polyadenylation to be released from the transcription machinery and prepared for translation. These processes are catalyzed by large, megadalton-sized macromolecular machines [the spliceosome and cleavage and polyadenylation factor (CPF)] that assemble on RNAs and must precisely recognize which regions of the RNA to keep and which to discard. Failure to recognize the proper splice or 3' end cleavage sites can destroy the protein coding potential of a message; alter the amino acid composition of the encoded protein; or dramatically change when, where, or how much protein is produced from a given message. The Hoskins Laboratory is focused on understanding spliceosome and CPF function in biochemical depth to illuminate how they shape the transcriptome. To study these machines, we integrate single molecule fluorescence microscopy with genetic, transcriptomic, and biochemical assays. In the next funding period, we will focus on how splice and cleavage sites are recognized by the spliceosome and CPF. We will pioneer single molecule methods to follow assembly of CPF and 3' end cleavage in real-time to answer fundamental questions about CPF function that have proved elusive for decades. By studying how 5' splice site recognition occurs co-transcriptionally, we will elucidate how splicing factors influence the transcriptional machinery and vice versa. We will use genetics and transcriptomics to probe how 3' splice site recognition is coupled with proofreading to maintain the integrity of genetic information flow. Finally, we will develop new high-throughput assays and microscopy hardware that will provide quantitative biochemical data for splice and cleavage site recognition at the transcriptome-scale. Together, these research directions will reveal fundamental mechanisms of the pre-mRNA processing machineries and insights into how these machines malfunction in human diseases including cancers and neuromuscular disorders.

NIH Research Projects · FY 2026 · 2020-05

PROJECT SUMMARY Our long-term goal is to elucidate the molecular mechanisms that restructure the genome to allow for developmental transitions. This process is orchestrated by sequence-specific, DNA-binding, transcription factors that occupy discrete regulatory regions and drive gene expression. However, the packaging of the genome into chromatin limits the ability of many transcription factors to bind the underlying DNA. A specialized class of transcription factors, called pioneer factors, are uniquely capable of binding histone-wrapped DNA, establishing accessible chromatin domains, and facilitating the binding of additional transcription factors. These distinctive properties enable pioneer factors to act at the top of gene-regulatory networks to drive changes in cell state. Indeed, pioneer factors are required for reprogramming the early embryonic genome in all species studied. We identified two pioneer factors in Drosophila, Zelda and GAGA factor, that are each essential for embryonic development and together partition the embryonic genome into transcriptionally active and inactive states. Nonetheless, the mechanisms by which these two factors function to drive this conserved developmental transition remains unclear. Despite the powerful ability of pioneer factors to restructure the genome, there are barriers to both their binding and activity. By combining our development of novel tools to interrogate transcription-factor function in the early embryo with the strengths of the well-studied fly system, we are uniquely positioned to determine essential features of pioneer-factor mediated genomic reprogramming. We will use genetic, genomic, biochemical, and imaging strategies to determine how pioneer factors partition the early embryonic genome into the transcriptionally active and inactive compartments and identify pioneer- specific features that promote their ability to restructure the genome. Our proposed research is significant because we will identify unifying principles by which pioneer factors drive the changes in gene-regulatory networks that promote developmental transitions and, when dysregulated, disease.