University Of Washington

NIH Research Projects · FY 2025 · 2024-08

SUMMARY/ABSTRACT The burden of noncommunicable diseases (NCDs), including cardiometabolic disease, is increasing globally, particularly in low- and middle-income countries (LMICs). NCDs are responsible for an estimated 35% of all deaths in sub-Saharan Africa, and in Mozambique nearly 60% of all DALYs are attributable to NCDs and injury. The prevalence of hypertension (HTN) and type II diabetes (T2D) are on the rise, from 33.1% in 2005 to 38.9% in 2015 for HTN and 2.9% to 7.4% for T2D across the same period. Effective strategies to meet the growing disease burden that can be integrated into – and scaled through – health systems and disease-specific platforms, like those for HIV, are needed. Similarly, little information exists on effective, population-based primordial prevention of cardiometabolic in LMIC community-based settings, including efforts to intervene in the relationship between an individual's environment, health behaviors, and risk factors for HTN and T2D. In recent years, several implementation research studies have applied the Systems Analysis and Improvement Approach (SAIA) to optimize care cascades for preventing mother-to-child HIV transmission (R01HD0757; PI: Sherr), addressing HTN among people with HIV (SAIA-HTN, R01HL142412, PI: Gimbel; SCALE SAIA-HTN, 1UG3HL156390- 01/UH3HL156390,PI: Mocumbi/Gimbel), and to pilot T2D care optimization within facilities where SAIA-HTN has been implemented, coupled with food environment assessments and Citizen Science-based nutritional education modules in three institutional cafeterias. This F31 proposal will leverage the data gathered under these studies to conduct formative research to characterize determinants for effective implementation of integrated care for T2D, HTN, and HIV embedded within ongoing health systems strengthening efforts in public sector facilities in Mozambique. We will also conduct pilot research to determine possible associations between community-based participatory education modules and changes in diet. We will evaluate barriers, facilitators, and organizational readiness to deliver medical services for T2D alongside integrated HTN and HIV services (Aim 1), leveraging qualitative data gathered through interviews and focus group discussions among stakeholders in two Mozambican health facilities and organizational readiness for implementing change (ORIC) assessments. In Aim 2, we will leverage data collected in food intake questionnaires administered under the parent study to assess the presence of longitudinal or dose-response relationships between exposure to community education modules and food intake. This formative research will contribute to scarce evidence for effective systems approaches to treat and prevent cardiometabolic disease in LMICs. This research plan will provide the F31 candidate rigorous predoctoral training including 1) application of implementation science frameworks for qualitative research, 2) development of participatory research methods, and 3) advanced statistical analyses using quantitative data for implementation science.

NIH Research Projects · FY 2024 · 2024-08

The Ras family of proteins contain four major isoforms, and altogether these proteins are constitutively activated in a third of cancers. In the past decade, inhibitors to mutant Ras (RasG12C) have been developed, but most patients administered RasG12C inhibitors (RasG12Ci) relapse. Interestingly, these Ras inhibitor resistant tumors have Ras signaling reactivated and the signaling mechanisms underlying this drug resistance are unknown. To uncover these drug resistance mechanisms, I developed Ras activity sensors and Ras activity dependent proximity labelers, applied them to RasG12C-addicted cancer cells treated with RasG12Ci, and observed that RasG12Ci blocked mutant Ras signaling at the plasma membrane while wildtype (WT) Ras is activated at endomembranes to fuel oncogenic signaling and cell growth. While these results are preliminary as these studies were done in 2D cell culture and do not delineate which particular Ras isoforms enable RasG12Ci resistance, these exciting findings beg the question of whether cancer cells can evade other recently developed Ras inhibitors targeting RasG12C, G12D, G12R, or G12S by also reactivating Ras signaling. Therefore, the objective of this K99/R00 proposal is to expand the molecular toolkit for Ras and utilize these tools to profile and uncover the molecular mechanisms driving Ras inhibitor resistance. The central hypothesis driving this work is that WT Ras compensation for mutant Ras inhibition is a general feature cancer cells employ to evade Ras inhibitors. Profiling the subcellular Ras activities during Ras inhibitor treatment and uncovering the molecular components driving this reorganization of Ras signaling will allow better understanding of Ras inhibitor resistance and illumination of new therapeutic targets. To investigate this hypothesis, the following specific aims will be addressed: (1) Developing and applying Ras sensors in complex cancer cell models (K99); (2) De novo design of Ras isoform selective tools (K99/R00); and (3) Profiling and dissecting the mechanisms underpinning Ras inhibitor resistance (R00). In the proposed research, I will protein engineer current and new Ras tools (sensors, proximity labelers, perturbators) along with microscopy and proteomic techniques to determine how Ras inhibitors impact compartmentalized Ras signaling. The expected outcomes are (1) an expansion of tools that can be applied to in vivo models and probe specific Ras isoforms and (2) a better understanding of how Ras inhibitors operate and how drug resistance can occur. Of note, I believe these new Ras tools will be of great interest to the cancer community (e.g. NCI’s Ras initiative) and can be useful for other applications beyond the scope of this proposal such as diagnostics and therapeutics. Towards completion of the proposed work, I will be trained in protein design methods and complex cancer models and guided by an advisory committee composed of experts in cancer, Ras signaling, and cell culture. The long-term goal of this project is to develop an independent research program that bridges protein design with cancer cell biology to understand how oncogenic signaling pathways rewire themselves during oncogenesis and drug resistance.

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT Community-based surveillance studies of respiratory viruses provide the opportunity for early viral detection and situational awareness, and may inform early public health efforts to mitigate the transmission of respiratory viruses, as was evidenced by the SARS-CoV-2 pandemic. Case-ascertained household transmission studies are an efficient and epidemiologically rich method to study respiratory virus transmission. Our team of investigators has deep expertise in community surveillance and case-ascertainment trials of respiratory viruses. Our group of collaborators include specialists in infectious diseases, epidemiology, biostatistics, virology, immunology, and infectious disease modeling. In 2018, we developed the infrastructure for a pandemic preparedness platform in Seattle, Washington, leading to the first identification of SARS-CoV-2 community transmission in the United States. We have developed innovative methods for fully remote observational studies of respiratory viruses in households, and have built a logistics and laboratory insfrastructure for rapid delivery and pickup of biospecimens, multiplex testing for a panel of respiratory pathogens, in-depth immunologic characterization, and whole genome sequencing and visualization. In this study, we propose to build on these platforms to conduct the following studies. First, we propose a prospective longitudinal cohort study of 2000 children and adults in the Seattle area, with weekly symptom screening and collection of nasal swabs for respiratory illness. We will conduct multi-pathogen testing for respiratory viruses and whole genome sequencing for RSV, SARS-CoV-2, and other respiratory viruses. We will calculate incidence, assess epidemiology and burden of respiratory viruses, measure participant knowledge, attitudes and perceptions, and measure the effectiveness of interventions in prevention of disease. We additionally propose to perform in-depth immunologic assessments by collection of serum samples longitudinally in a subset of individuals to understand antibody kinetics over the course of multiple seasons, and the effects of vaccines and infections on antibody titers across age groups. For our second study, we propose to conduct a case-ascertained household transmission study to understand transmission dynamics of SARS-CoV-2, RSV, and other viruses. Index cases will be enrolled remotely along with their household members, for serial self-swab collection for a period of two weeks after enrollment. We propose collection of environmental samples within a subset of households. Molecular testing and sequencing will be performed for SARS-CoV-2, RSV, and other viruses, as indicated, allowing for calculation of household attack rates, serial intervals, and risk factors for within household transmission for known and novel pathogens. These proposed studies will provide real-time situational awareness of community respiratory epidemiology, and our team has the experience and expertise to pivot for identification of novel pathogens and pandemic response.

NIH Research Projects · FY 2025 · 2024-08

Alcohol use can accelerate the detrimental processes of aging in terms of cognitive, physical, and emotional outcomes and has been identified as the strongest modifiable risk factor for the development of Alzheimer’s Disease and Related Dementias (ADRD). Common biological mechanisms such as inflammation, amyloid-β deposition, and cell death have been observed in patients with ADRD and those who consume high amounts of alcohol in a chronic fashion. Despite evidence of this causal link, minimal focus has been placed on understanding synergistic interactions of alcohol, aging, and tau-driven neurodegeneration; which is a hallmark of multiple neurodegenerative disorders. Preclinical models of pathological tau expression, such as the PS19 tau mouse model, have been well characterized in the study of ADRD but have rarely been utilized to study consequences of chronic alcohol consumption. The prevalence of alcohol consumption in older adults is increasing and our society is aging, leaving our population even more vulnerable to the neurodegenerative effects of alcohol. Thus, the study of the intersection of aging and alcohol is more urgent than ever. The current literature on this topic is limited, so it will be critical to determine foundational knowledge about the chronic effects of alcohol in a model of neurodegeneration. The current proposal therefore aims to investigate the progression of behavioral impairment, the time course of aging-related biomarkers, and development of neurodegeneration as a result of alcohol intake and development of tau pathology. Behavioral and cognitive impairment is a critical standard that we will measure as an outcome, as it is a common feature present in pathological aging and neurodegenerative diseases. Peripheral biomarkers represent a method of quantifying the level of biological impairment and assessing the overall state of aging and degeneration in multiple systems across the body. All of the biomarkers we have selected to measure in this proposal have a parallel clinical assessment that can be measured noninvasively, which will be beneficial for translational purposes. SPECIFIC AIMS: (1a) Determine how voluntary chronic alcohol intake in the presence of pathological tau expression impacts behavioral and aging-related outcomes across the lifespan, (1b) Delineate the synergistic effects of specific low or high alcohol intake and pathological tau expression, (1c) Utilize machine learning techniques to predict behavioral and neurological outcomes from acute biomarker measures, and (2) Investigate the acute effects of alcohol in a mouse model of tauopathy. The successful completion of this proposal will provide a holistic characterization of how aging is affected by alcohol over the lifespan, highlight new biomarkers to predict chronic adverse outcomes, and ultimately provide potential clinical targets to combat physical, emotional, and cognitive impairments in the aging population.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Chromosomes must be highly packaged to fit with cells. Modern tools like 3D chromosome capture allow for visualization of chromosome organization but are limited in describing gene-level packaging and cannot reveal the mechanisms that underlie packaging. Understanding these mechanisms is critical to understand genome function and to identify chromosome vulnerabilities as targets for future therapeutics. We propose to attack two major challenges limiting our understanding of chromosome organization in bacteria: understanding supercoiling and structuring protein function. First, supercoiling represents the DNA winding about itself, with “positive” supercoiling (+SC) describing an overwound structure, which is refractory to DNA duplex melting. +SC is generated as a byproduct of replication and must be removed by topoisomerase enzymes or replication will cease, leading to cell death. Prior to my work, a lack of tools to map +SC and a lack of knowledge of topoisomerase regulators meant that our cellular understanding of +SC was limited. Second, bacterial chromosomes are structured by nucleoid-associated proteins (NAPs). NAPs are a group of sequence-diverse, functionally heterogeneous, and poorly understood proteins. Our discoveries in Caulobacter crescentus of an essential NAP and +SC regulator called GapR has empowered us to begin untangling chromosome packaging. We initially showed that GapR binds +SC in a sequence-independent manner to stimulate topoisomerase activity and promote replication. We then developed GapR into technology that allows us to “see” where +SC occurs. While GapR homologs are ubiquitous in ⍺-proteobacteria, analogous NAPs that regulate topoisomerase activity in other bacteria have not been identified. Our preliminary data suggests that bacterial viruses (phages) encode GapR homologs that hijack bacterial GapR to promote infection, uncovering a beneficial role for phage hijacking of bacterial NAPs during infection. We have also developed technology to capture topoisomerase regulators in bacteria. In this proposal, we will 1) elucidate GapR mechanism as a model for understanding how bacteria and phage control +SC to proliferate, 2) mine bacteria and phage genomes to characterize additional topoisomerase regulators, and 3) examine NAP hijacking during phage infection. These projects will describe fundamental paradigms of bacterial chromosome organization and improve our understanding of phage-bacterial warfare. Further, topoisomerases are important antibacterial targets while phages are potential antibiotic alternatives, thus the findings of this proposal will identify vulnerabilities in bacterial chromosome regulation as future antibiotic targets and improve our understanding of phage infection for phage therapy.

NSF Awards · FY 2024 · 2024-08

This award supports a CAREER project that engages in research and teaching activities related to the implications of neuroscience research.The project provides guidelines to stimulate greater collaboration between the neurosciences, academic communities, and the public. Ongoing results from this project will be made available to the public through open forum engagements and to university and high school students through classroom-based lectures to educate a wide audience about neuroscience’s benefits and challenges. This project uses a qualitative approach to map the relationships between overlapping neuroscientific epistemic communities—funding bodies, publication mechanisms, professional organizations, agencies, research labs, and bioethical domains. The project provides recommendations and educational resources for neuroscience educators and researchers. The project contributes to existing literature in Bioethics, Sociology, Science of Science, and Science and Technology Studies (STS) on the impacts of neuroscience research. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-08

Sea level rise remains one of the most pressing and widespread risks of climate change. The West Antarctic Ice Sheet (WAIS), with an ice volume equivalent to 4.3 meters of sea-level rise, provides substantial uncertainty and may dominate sea-level changes in the near future. As climate changes, WAIS surface mass (the balance of snow added and snow lost) changes, which then drives a change in the flow of ice into the ocean. Both the surface mass balance and the shifts in ice flow into the ocean, alter sea level. To determine how climate change will impact WAIS contributions to sea-level changes, it is therefore critical to understand the atmospheric processes driving WAIS surface mass balance and ice flow. In particular, the ice sheet's response to surface mass balance is nonlinear, meaning small changes in weather patterns triggered by variations over the tropics may produce large changes in ice flow. It is therefore critical to assess how changes in climate at both low (e.g., tropics) and high (e.g., polar regions) latitudes combine to impact WAIS surface mass balance and ice flow, and the potential to cross tipping points that would result in rapid changes in sea level. This project will also leverage the existing University of Utah Masters of Science in Secondary School Teaching (MSSST) program, which supports motivated middle and high school teachers to earn an M.S. while still actively engaging in classroom teaching. This project addresses two primary research questions motivated by the need to improve our understanding of atmospheric forcing of WAIS surface mass balance and dynamic response: (1) What climate mechanisms generate interannual-to-multidecadal surface mass balance variability and trends in different sectors of WAIS? (2) How do climate-driven variations at multiple frequencies and spatially divergent trends in surface mass balance project onto ice sheet dynamics? We use a combination of multi-platform, spatially dense surface mass balance records, two century-length climate reanalyses, and climate and glacier numerical modeling to address these two questions. The observational work will use multivariate statistical methods to identify internal versus tropically-forced modes of climate variability relevant to spatiotemporal variations in SMB across WAIS, leveraging spatially dense surface mass balance records from cores and radar in conjunction with multiple century-length climate and sea surface temperature data sets. The resulting hypotheses of climate driver causality will then be tested by conducting boundary-forcing experiments with a global atmospheric model. Finally, the response of ice sheet dynamics to the spectrum of climate forcing from the observational and atmospheric model results will be investigated using a hierarchy of numerical ice sheet models. Ultimately, this work will allow us to deconvolve the role of tropical and intrinsic climate variability and trends on the dynamic response of WAIS to SMB variance and trends and how that varies across different regions of WAIS. The results will point directly to the climate mechanisms most likely to significantly influence ice sheet SMB and dynamics in the coming decades to centuries. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

Project Summary/Abstract The overarching objective of this project is to develop an advanced computational super-resolution model to enhance the throughput and resolution of chemical imaging. Chemical imaging, which includes advanced techniques such as stimulated Raman scattering (SRS) microscopy, holds immense potential in intraoperative cancer detection. This is due to its ability to generate intrinsic molecular contrasts without tissue processing or labeling, which can improve the accuracy and speed of intraoperative diagnosis. Such improvement is crucial to better patient outcomes by providing near real-time feedback to the surgeon and reducing the risk of leftover cancerous tissues. However, existing chemical imaging techniques grapple with an inherent limitation – the tradeoff between spatial resolution and imaging field of view, resulting in low imaging throughput and prolonged imaging durations for larger tissue samples. For other applications involving live cell imaging, the limited resolution of SRS (> 300nm) hinders visualization of subcellular organelles and fine structures. Computational super-resolution, bolstered by advancements in artificial intelligence, can address these challenges by transforming low-resolution images into high-resolution versions. This has been achieved with the Convolutional Neural Network and the Generative Adversarial Network. However, super-resolution chemical imaging is scarce. There are no datasets available for super-resolution training. It is also unclear whether existing super-resolution microscopy techniques could work for chemical images due to vast differences in imaging contrasts. Here we propose to develop a new super-resolution technique ChemDiffuse that is based on the diffusion-based deep generative network. Diffusion-based models are widely used in popular image-generation tools such as Midjourney and DALL-E. While superior in stability and image quality to CNN and GAN models, they need extensive training data. Leveraging on our recent progress in image augmentation and a new diffusion model for 2D and 3D data, we will develop the ChemDiffuse model to significantly improve SRS imaging throughput and resolution. We aim to test the application of the ChemDiffuse super-resolution model in two different areas: 1. Fast gigapixel SRS imaging of tissue at submicron resolution for pathology application. We will use mouse brain tissue as our test system to train the ChemDiffuse model to enable fast 3D SRS imaging at 10 million pixels/sec, a 40-fold improvement in lateral dimension, and another 10-fold improvement in axial dimensional. Such improvement is crucial for intraoperative stimulated Raman histology of large tissues. 2. Label-free SRS imaging of live cell organelles at 150 nm resolution. We will train the super-resolution model to enable 2-fold resolution enhancement with regular SRS imaging, an improvement that will allow unprecedented label-free tracking of multiple organelles and single cell analysis for a wide range of drug discovery applications. Though our focus is on SRS imaging, this methodology can benefit various other chemical imaging techniques, including Raman microscopy, IR imaging, transient absorption microscopy, and photothermal microscopy, etc.

NSF Awards · FY 2024 · 2024-08

The National Nuclear Physics Summer School, held annually since 1988, is an effort by the nuclear physics community to acquaint students with the forefront issues of the field and with the experimental and theoretical methods available to nuclear physics researchers. The intent is to broaden student appreciation for and interest in the field, while also strengthening them technically. Typically most of the field's major directions are represented in the lectures: hot and cold nuclear matter, electromagnetic physics, weak interactions, astrophysics, and nuclear structure. Advanced graduate students and beginning postdoctoral researchers are the target audience. School organizers are charged with identifying outstanding lecturers from theory and experiment, conducting the school in a manner that encourages discussion and interaction, and developing supporting materials, including an online archive of lectures that can serve as a long-term reference for participating students and for others who are interested in the current status of nuclear physics. School organizers are assisted by the Principal Investigators and by a Steering Committee appointed by the Division of Nuclear Physics of the American Physical Society. The school is often the first opportunity for students to recognize the breadth of nuclear physics and to interact across subfields. As such, the National Nuclear Physics Summer School represents a community-building activity that provides students with excellent opportunities to interact with leading researchers in the field and to network with their peers. Students leave the school with stronger backgrounds in nuclear physics, and with new knowledge of their chosen specialties. As the lectures are preserved in an on-line archive, the students can return to this material when a need arises during their subsequent training and research. Students are given a chance to present talks and thus to gain experience in making presentations to a diverse audience. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-08

Environmental change has contributed to our planet currently undergoing a “6th mass extinction”, in which 40% of vertebrate species have experienced severe population declines. Identifying the mechanisms underlying species responses to environmental change is a pressing challenge and essential to mitigating global biodiversity loss. Global environmental change remains a leading threat to biodiversity, and altered species interactions are a primary driver through which climate variability impacts populations. In particular, changes in predation and intraguild competition (i.e., competition between species that share the same food resources) are among the proximate mechanisms most widely implicated in climate-driven population declines. Animal behavior can strongly mediate how individuals and species respond to environmental variability, and thus should mediate how climate variability impacts interspecific interactions like predation and competition. However, little research has examined the behavioral mechanisms driving climate impacts on species interactions, particularly at top trophic levels. This research will advance our understanding of how animal behavior mediates changes in upper-level trophic interactions by assessing how climate variation affects the behavior and interactions of four African large carnivore species in Botswana. In addition, this work will build local capacity for wildlife research by providing training for African field assistants, as well as providing training for undergraduate, graduate, and postdoctoral researchers. Finally, by partnering with a local environmental education program, we will integrate our research into a new learning module on local wildlife. Because altered patterns of predation and competition are primary mechanisms by which global environmental change leads to population declines, it is critical to understand how and why climate variability modifies species interactions. Animal behavior is likely an underappreciated pathway through which climate changes alter these interactions, providing a key missing link between environmental change and ecological patterns. The goal of this research is to understand the patterns, drivers, and consequences for predation and competition of individual- and species-level variation in the behavioral responses of top predators to climate variability. This work builds upon a unique behavioral dataset from high-resolution GPS and accelerometer data from four African large carnivore species, spanning an 8-year period that included three drought events and the hottest local temperature on record. We will couple behavioral observations and the collection of new collar-derived data to parameterize a machine learning model to classify behaviors (stationary, mobile, hunting, feeding). We will apply our classification model to retroactively identify behaviors in the historical data. We will then integrate these classifications with high-resolution environmental data to assess how climatic variation influences individual behavior and subsequently predation and intraguild competition. In Aim 1, we examine intra- and interspecific variation in behavioral responses to temperature and drought severity within the large predator guild, and evaluate the mediating role of two key predator functional traits – body size and hunting mode. In Aim 2, we examine how variation in climate-driven behavioral responses scales up to affect patterns of predation and intraguild competition. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-08

Removing carbon dioxide (CO2) from the atmosphere benefits society. One promising approach to CO2 removal is enhanced mineral weathering (EMW). In EMW, specific rocks are crushed and spread on agricultural fields. The rocks then react with CO2 and water to generate a dissolved form of inorganic carbon called bicarbonate. Successful CO2 removal requires the generated bicarbonate to travel from agricultural fields through rivers into the ocean. However, during the journey through rivers, bicarbonate can get converted back to CO2 and re-enter the atmosphere. The amount of bicarbonate re-emitted to the atmosphere is unknown. This project will address this gap in our knowledge by quantifying how much bicarbonate gets converted to CO2 and released to the atmosphere. The measurements will be made in an agricultural river system in Illinois. Successful completion of this project will benefit society by ensuring that EMW removes more CO2 from the atmosphere than it contributes, and creating a community quantification standard for EMW that aligns with the forefront of scientific discovery. Additional benefits to society will result from the development of a university-level course that is publicly available through ‘Teach the Earth’, aiming to enhance public access to STEM education. The goal of this project is to quantify the fate of weathered carbon within an agricultural river system in Illinois where lime (CaCO3) is applied to manage soil pH. Lime dissolution is a form of EMW with currently unknown CO2 removal potential that is now practiced at large enough magnitude to be detectable at stream network scale. Previous efforts to estimate the amount of carbon lost in rivers have all been model based. These efforts have only focused on CO2 degassing and carbonate precipitation as the dominant loss processes. In this project, the research team will exploit the fact that lime carbon is radiocarbon dead to allow differentiation between modern carbon and lime carbon in the stream. In addition, the research team will measure how submerged plants and algae uptake bicarbonate and convert it back to CO2. Finally, the team will assess how seasonal changes in temperature and stream flow control the balance between sequestration and re-emission of EMW-produced bicarbonate. Together, these experimental measurements will be used to generate a comprehensive dataset that supports the development and validation of future models. Successful completion of this project will lead to new insights into how temporal variation in biological processes and other stream processes affects the amount of carbon sequestered by EMW. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-08

When speaking, the brain checks the accuracy of the sounds that are produced. This monitoring of auditory feedback is used to try and adjust speech movements in case the output does not sound correct. Thus, the brain tries to adapt its motor behavior based on the auditory input. This is known as auditory-motor adaptation. Situations where speech movements may not yield the correct sounds include wearing dentures, oral surgery, and developmental or acquired neurological disorders that affect mouth movements. Little is known about which brain parts are involved in speech auditory-motor adaptation or how the underlying mechanisms can be enhanced to optimize this learning. Such information is critical to understand basic brain function and to help millions of children and adults with temporary or permanent speech difficulties. Studies on limb movements suggest that connections between the brain's cortex and deeper (subcortical) structures play an important role. It is unclear, however, if those findings also apply to speech. This doctoral dissertation project studies the role of two subcortical structures by testing patients who, as part of their clinical treatment, have had electrodes implanted inside those structures (deep brain stimulation, DBS). Parkinson's disease (PD) and essential tremor (ET) patients have DBS electrodes implanted in the basal ganglia or thalamus, respectively. The project assesses differences in how the two groups of patients use auditory feedback to adjust their speech with the DBS device on or off. In some cases, the DBS device also allows brain activity to be directly recorded during the speech task. This research provides new insights into the role of the subcortical structures in speech auditory-motor adaptation. Overall, it improves the understanding of how the brain controls speech movements. It also has implications for understanding speech development during childhood and the decline of speech with aging. Lastly, it informs the development of novel treatments for individuals with speech impairments. To test how the brain adjusts speech based on auditory feedback, this doctoral dissertation project uses a behavioral adaptation paradigm that involves consistent perturbations of one or more resonance frequencies (formants) in the auditory feedback. For example, when the perturbation shifts the first formant (F1) upward, a speaker who produces the word "pet" hears "pat" in the altered auditory feedback. The brain then adjusts the involved speech movements to lower F1 in subsequent productions of "pet" (moving it closer to "pit") in order to partially compensate for the perturbation—a phenomenon known as speech auditory-motor adaptation. This project investigates the distinct roles of two subcortical structures, the subthalamic nucleus (STN) of the basal ganglia and the ventral intermediate nucleus (Vim) of the thalamus, in speech auditory-motor adaptation. STN and Vim are critical components of two important circuits that connect the cortex and different subcortical structures: one circuit between the cortex and the basal ganglia, and the other between the cortex and the cerebellum. STN and Vim are common Deep Brain Stimulation (DBS) implantation sites for patients with Parkinson’s Disease (PD) and essential tremor (ET), respectively. To elucidate the role of these nuclei in the adjustment of speech movements during auditory-motor adaptation, this project tests two forms of adaptation and contrasts the results with a non-adaptation form of speech motor learning. Specifically, patients with DBS electrodes at either STN or Vim complete (a) auditory-motor adaptation with a gradually introduced perturbation, (b) auditory-motor adaptation with a suddenly introduced perturbation, and (c) syllable sequence learning. The results provide direct evidence for the contribution of these nuclei in speech auditory-motor adaptation that has remained limited due to methodological barriers. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

The goals of the Northwest Genomics Center (NWGC) for All of Us (AoU) are to provide high-quality, cost-effective genotyping and whole genome sequence (WGS) data; to return genomic results for medically actionable and pharmacogenomic genetic variants; to enhance the scientific value of AoU by adding innovative -omic technologies including long-read sequence data. The NWGC brings together three internationally recognized Principal Investigators–Gail Jarvik, Chia-Lin Wei, and Evan Eichler–with decades of expertise in high-throughput clinical genomics. Together with their Co-Investigators—Christina Lockwood, Colin Pritchard, and Phil Empey—they have returned tens of thousands of variant interpretations to patients including AoU participants. Since August 2020, the NWGC has delivered genotype array data for 169,414 samples and genome data for 61,606 samples to the AoU Data Research Center (DRC). We have completed variant review and classification resulting in genomic return of results reports for more than 15,000 AoU participants including 411 medically actionable reports (i.e., American College of Medical Genetics pathogenic or likely pathogenic classifications). To advance the core business goals and objectives of AoU, the NWGC will generate high-quality genotyping array and whole genome sequence data for up to 50,000 AoU participants per year. In addition, we will interpret and classify variants for individuals who request genomic return of results (gRoR). We will continue to lead and support expanding gRoR content to participants (i.e., additional genes and medications) and leverage long-read -omics projects for investigators in the Researcher Work Bench (RWB). We will contribute our in-depth experience in sequencing technologies to support the FDA IDE supplemental validation plans for the NovaSeq X Plus platform and construct long-read resources in the discovery of pathogenic variants in medically- relevant genes and PGx targets. We will participate in analysis working groups and work with the DRC to improve genotype imputation and phasing tools, when funding is available, we will deploy innovative multiomics technologies to expand variant interpretation and functional annotation. The NWGC is ideally positioned to support and maximize the potential of AoU and its central mission to understand human genetic variation that impacts disease prevalence in the United States population.

NSF Awards · FY 2024 · 2024-08

Faculty and staff at the University of Washington (UW) will organize and lead GeoFutures, a conference-based mentoring program housed within a professional scientific conference in 2025. The Geo-Futures program will provide student attendees with mentoring before, during and after their conference experience to help them maintain the intellectual momentum acquired during their time at the conference. The program will help students develop a sense of community within the geosciences and assist them as they begin to define their academic and professional pathways through the discipline. GeoFutures will use previously developed engagement strategies for recruiting and retaining students and perspectives that will be required to address the emerging workforce needs of 21st century geoscience. GEOFutures offers students pre and post-conference training that will prepare them to participate in a national scientific research conference. Pre-conference activities will provide students with an introduction on navigating a scientific conference and networking with geoscientists in attendance. Conference activities will provide students with an introduction to original research being conducted in the geosciences and examples of career pathways that can be engaged in as a professional geoscientist. Beyond providing students with guidance on how to pursue academic and professional careers in geoscience, the GeoFutures program can help them understand how their talents and perspectives can contribute to a scientific workforce that will be called upon to address the emerging needs of 21st century geoscience research. The GeoFutures program forms an alliance among geoscience professionals to help recruit, engage and retain students in the geosciences through a conference based mentoring program. The GeoFutures program aims to 1) recruit up to forty student participants and those with prior experience in an NSF Geoscience Research Experience for Undergraduates (REU) program; 2) engage students in innovative professional development activities; 3) provide students with high-caliber mentoring; and 4) provide rigorous support and training for students to ensure continued interest and involvement in the geosciences. A vigorous recruitment and outreach effort leverages connections with early career research programs from across the United States and the experience of the leaders in engaging with students. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

Abstract The serine/threonine Raf Kinases function as downstream modulators of RAS GTPase-driven signaling and are key drivers of many oncogenic signaling networks. Despite extensive efforts to characterize the molecular functions of Raf kinases and to develop pharmacological modulators as cancer therapies, many aspects of Raf kinase regulation and intracellular function are not well understood. Recent work suggests that the interplay of different Raf isoforms (A/B/C-R Raf) can have a profound effect on downstream signaling and may be a major determinant in the ability to pharmacologically target cancers that are driven by mutant RAS and Raf. Here, we propose a systematic biochemical analysis of Raf regulation, intracellular function, and pharmacology. Leveraging a suite of molecular tools for dissecting the “druggability” of RAF isoforms, we will define pathways that make Raf kinases sensitive or resistant to specific classes of ATP-competitive inhibitors and co-inhibition strategies. In addition, by applying a new RNA barcoding methodology that we’ve developed, we will perform deep mutational scans of the structure, function, and pharmacology of Raf isoforms. Furthermore, we will systematically analyze how upstream and downstream modulators of Raf kinase signaling affect the activity and intracellular interactions of Raf kinases. Together, these studies will provide a more complete picture of Raf kinase regulation, intracellular function, and may facilitate more effective therapeutic targeting of RAS- and Raf- driven cancers.

NSF Awards · FY 2024 · 2024-08

This project investigates how to more effectively enable the success and advancement of neurodivergent employees in the technology workplace. We do so by investigating specific success enablers grounded in an asset model of disability and considerate of the unique needs, interests, and strengths of neurodivergent individuals in the context of their work. Success enablers are a broader set of practices and work designs that include accommodations but are not limited to them. Success enablers include both support and, more importantly, changes to the workplace environment. The goal of SEEN Tech Professionals is to 1) empower neurodivergent professionals in the technology sector to determine, identify, and use what they deem as appropriate success enablers based on their intersecting identities and 2) build the capacity of organizations to systemize and normalize the use of success enablers more readily. The SEEN Tech Professionals project will improve technology workplace environments to advance equity for neurodivergent professionals by creating The Neuroinclusive Success Enablers Toolkit. The Toolkit compiles interventions designed to empower neurodivergent employees and build the capacity of their managers, HR professionals, and providers to leverage and normalize success enablers. The Toolkit will be freely available in accessible formats on the project website and distributed widely through the University of Washington and the project Advisory Group. The SEEN Tech Professionals research project investigates three central questions: 1) What are the most common success enablers neurodivergent individuals identify as critical in the workplace? 2) What role do managers play in identifying, normalizing, and systemizing success enablers to support their ND employees? 3) What are the organizational characteristics that support or limit the identification, implementation, and systemization of success enablers? Our work is based on Annabi and Locke’s Organizational Interventions Mitigating Individual Barriers (OIMIB) framework, grounded in the neurodiversity paradigm and critical disability studies and emphasizing intersectionality. Findings from this research will advance our theoretical and practical understanding of how neurodivergent technology professionals leverage various success enablers to address barriers they face and the role organizational interventions (e.g., Neurodiversity Hiring Programs) and managers play in facilitating the use of success enablers. Our project will be carried out in three phases over three years. The primary outcome of this study is to create high-impact interventions organized in a comprehensive Neuroinclusive Success Enablers Toolkit designed for technology employers and neurodivergent professionals. This award has been made in response to the NSF solicitation “Workplace Equity for Persons with Disabilities in STEM and STEM Education” (NSF 23-593). The project is funded by the Division of Equity for Excellence in STEM’s EDU Core Research program (ECR). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY / ABSTRACT Zika virus (ZIKV) re-emerged over the last several decades to cause large outbreaks in the Asian Pacific and Americas. ZIKV disease, though usually mild, can occasionally cause severe neurological complications, including developmental defects in babies born to a ZIKV-infected person. Much remains to be learned about ZIKV host-pathogen interactions, including how the innate immune system fights the early stages of infection in human cells. Given their role in inhibiting viral replication, innate immune factors could help explain the heterogeneity in ZIKV clinical outcome and could lead to the development of treatments for ZIKV. The type I interferon (IFN) response plays a particularly critical role in the innate immune response to viruses, including ZIKV. IFN is secreted from virus-infected cells, binds its receptor on nearby cells, and sets off a signaling pathway that culminates in a transcriptional program turning on hundreds of interferon-stimulated genes (ISGs), some of which encode antiviral proteins against a given virus. While the IFN response is clearly important in restricting ZIKV, systematic studies to define which host genes are responsible for this potent effect are lacking. While several specific antiviral ISGs have been identified, there have not been broader attempts to define all host genes that contribute to IFN restriction of ZIKV, including non-ISGs that may play a regulatory role in the pathway. Our lab performed a CRISPR knockout screen to identify genes that contribute to IFN restriction of ZIKV. The screen approach successfully identified genes involved in IFN signaling (positive controls). IFI6, a previously described ISG and flavivirus restriction factor, was also identified, as was AMOTL2, a non-ISG in our cell type and a gene with no described role in innate immunity. Despite the fact that AMOTL2 is not itself IFN-induced, AMOTL2 knockout increased ZIKV replication in the presence, but not absence, of IFN. These findings confirmed the IFN-specific antiviral phenotype of AMOTL2 and led to the hypothesis that AMOTL2 acts upstream of ISG transcription in the IFN response. My preliminary results support this model and are the basis for the proposed studies. In Aim 1, the mechanism of AMOTL2’s viral restriction will be better elucidated. This will be achieved by first validating the specificity of the observed phenotype through rescue experiments, then assessing the involvement of AMOTL2’s binding partners YAP and TAZ in IFN restriction of ZIKV through double knockouts and co-IP assays. In Aim 2, I will test the breadth of AMOTL2’s antiviral phenotype across diverse viruses (Aim 2a) and with ZIKV infections in a biologically-relevant primary cell type (Aim 2b). The complementary results of my two aims will enhance our understanding of the IFN response against ZIKV and possibly other viruses.

NIH Research Projects · FY 2025 · 2024-08

The discovery of BRCA1 and BRCA2 and other genes of homologous recombination repair, and the characterization of the roles of these genes in inherited predisposition to breast and ovarian cancer, have had a tremendous impact on cancer prevention and treatment. Yet family history remains one of the most important risk factors for these cancers, and a great deal of that risk remains unexplained. We propose two hypotheses.: (1) In some families, breast cancer is due to previously cryptic non-coding variation altering transcription and/or expression of known breast cancer genes (BRCA1, BRCA2, PALB2, ATM, CHEK2, BARD1, BRIP1, RAD51C, RAD51D, and TP53); and (2) In some families, breast cancer is due to rare or private non-coding variants regulating expression of critical genes with no cancer-predisposing alleles in coding sequence. To test these hypotheses, we will deploy new technologies: long-read sequencing of genomic and cDNA; Fiber- seq identification of regions of open chromatin in cell types of choice; massively parallel reporter assays (MPRA) to compare effects of variant versus reference alleles for many sites simultaneously; and CRISPR inhibition (CRISPRi) of candidate regulatory regions. We propose to integrate these approaches to discover and characterize classes of non-coding pathogenic variation in 1253 extended families, severely affected with breast and/or ovarian cancer, but for whom no causal variants have been found by any current genomic technology. In Aim 1, we will use adaptive-sampling long-read genomic sequencing to determine the spectrum of all classes of rare non-coding variants in the extended TADs of the known breast cancer genes. In Aim 2. we will use long- read cDNA sequencing to characterize transcriptional consequences of the complex SVs and rare deep intronic SNVs co-segregating with breast cancer (from Aim 1). In Aim 3, we will use Fiber-seq analyses (already in hand) of fallopian tube epithelial cells and MCF10A mammary cells to identify candidate regulatory regions of breast cancer genes. Then, for rare variants in these peaks that are co-inherited with breast cancer in unsolved families, we will use MPRA to test allele-specific differences in reporter activity; and in parallel silence the regions with CRISPRi to evaluate their effects on gene expression. In Aim 4, we will extend Aim 3 genome-wide, to identify variants near other breast cancer-relevant genes that lie in open chromatin regions, that co-segregate with breast cancer in unsolved families, and that reveal allele-specific differences in regulatory activity. We expect to discover clinically meaningful genetic variation in non-coding regulatory regions, including in repetitive genomic neighborhoods particularly subject to complex rearrangement. We expect that this approach will be directly applicable to any complex diseases with an inherited genetic influence.

NIH Research Projects · FY 2025 · 2024-08

ABSTRACT Cannabis is a widely used psychoactive substance among young adults (YAs), with ~44% using in the past year. Daily use by YAs not attending college (14.5%) is 3x the prevalence of daily use by college attending peers (4.7%). Cannabis is associated with acute and longer-term consequences. A growing concern among researchers and clinicians is of frequent and/or high-intensity use (i.e., daily/near daily use, use multiple times a day, and use of higher potency THC products). Although published research has grown exponentially in the last decade regarding correlates and consequences of cannabis, development and evaluation of efficacious brief interventions to reduce cannabis use is still needed. Mobile health (mHealth) technology may help address gaps between clinical need and formal clinical services, particularly for non-treatment seeking YAs and those typically underserved and underrepresented in research such as non-college YAs. The proposed R34 application uses a multi-method user-informed approach to develop a novel brief motivational mHealth intervention that incorporates cutting-edge cannabis intervention research. The proposed intervention is an integrated mobile-enhanced web-based program with subsequent text messages for 5 weeks that provides reflection on YAs’ cannabis use in 5 domains: (1) patterns/potency of use, (2) motivations for use, (3) social connections and use, (4) personal use goals and use and (5) protective behavioral strategies. The intervention prioritizes autonomy and will allow YAs to direct their journey through the program and allowing for self- identification of personal reduction goals. YAs are at different places with respect to readiness to change, thus by allowing the YA to direct their journey, the program will present appropriate content based on their interests and tailored to their self-efficacy and readiness to change. YAs will have opportunity to reflect on motivations for using cannabis, and when appropriate, cognitive behavioral skills-training for substance-free strategies for coping with negative mood and emotions, potential for engaging in substance-free activities, social skill building, exploration of the relationships among values, future goals, and cannabis use, and opportunities to explore lower-risk use. Aim 1: Develop a YA cannabis intervention (Take 5) to reduce cannabis use and negative consequences among non-collegiate YAs engaging in frequent/high-intensity cannabis use. Focus groups and user experience interviews will inform the development of the intervention. Aim 2: Conduct a 6- month pilot study with non-collegiate YAs in WA (who use cannabis 15+ days/month, use 2+ times per day when use, or use high-potency THC products [60%+]) to determine feasibility and acceptability. 120 YAs will be randomized to Take 5 (n=60) or attention control condition (n=60). The present application addresses several priorities in NIDA’s Strategic Plan, including using technology (e.g., smartphones) to deliver interventions and inclusion of high-risk vulnerable populations (i.e., non-college attending YAs).

NSF Awards · FY 2024 · 2024-08

Some of the greatest earthquake hazard on the planet is located along the Alaska-Aleutian subduction zone, where the Pacific Plate is diving underneath North America. Understanding seismic hazard in this region is complicated by a phenomenon known as slow slip, in which tectonic plates slide past each other without causing damaging earthquakes. Changes in water content and in rock type along the subduction zone are thought to control whether slow slip or fast earthquakes happen, but these properties are not well understood. In addition, there are gaps in seismic data coverage that make it difficult to understand how water and rock type vary along the plate boundary. This study focuses on one of these gaps, in the Cook Inlet region of Alaska. The research team has deployed two types of seismic instruments to record local seismic events and use data from earthquakes around the world to understand the deep structure of the subduction zone. One is a traditional experiment using broadband instruments deployed on land in the Kenai Peninsula. These instruments are placed on the property of volunteers, including two K-12 schools, enabling community participation and outreach. The other experiment uses Distributed Acoustic Sensing (DAS) technology – using fiber optic cables as seismic sensors – along and across the Cook Inlet. The goal of this project is to combine both types of data to model the structure and speed of seismic waves in the Pacific plate and its interface with the North American plate. From this water content and rock type, and how these properties vary between regions where slow slip does and does not happen, can be understood. Ultimately, this work will help scientists understand the processes that control seismic hazard where multiple modes and rates of slip coexist. Broader Impacts from this work include showcasing project field practices, and facilitating K-12 school outreach and community participation in broadband data collection. Slow slip events and tectonic tremor have been linked to eclogitization in the subduction interface, but the mechanisms of how these properties vary along the subduction zone and how they govern seismicity style are not well understood. This project harnesses novel and cutting-edge seismic data to investigate the processes that govern these phenomena in the Cook Inlet region of the Alaska-Aleutian subduction zone. The study area encompasses two locations where the subduction interface undergoes recurrent, prolonged, Slow Slip Events (SSEs) as well as damaging megathrust earthquakes. Crucially, only one of the SSE-prone regions is associated with tectonic tremor, providing a natural laboratory for isolating tremor and slip mechanisms. In this project, seismic data from a traditional land-based broadband seismometer deployment will be combined with data from a Distributed Acoustic Sensing (DAS) experiment within the Cook Inlet to fill in a crucial resolution gap over a patch of the subduction zone that experiences slow slip but not tremor. A suite of seismic imaging products, including receiver functions, P-wave autocorrelations, and surface wave dispersion from ambient noise correlations, will be generated and jointly inverted to constrain P and S wavespeed within both plates in order to infer variations in fluid properties and eclogite content across slipping and locked sections of the subduction zone, and evaluate the relative importance of these processes. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

Vestibular deficits are highly prevalent and cause debilitating symptoms. Degeneration and ototoxin- induced injury of vestibular hair cells (HCs) are underlying causes of some vestibular deficits. Remarkably, adult mammals, including humans, can replace some vestibular HCs once they are lost. However, in rodents and likely in humans, only one type of vestibular HC – type II (HCII) – is naturally replaced, and this degree of regeneration does not restore vestibulo-motor functions. Functional testing by our group and others’ provides strong evidence that the other type of vestibular HC – type I (HCI) – must be replaced to reverse vestibular deficits. HCI are very different from HCII; for instance, they have longer and thicker stereocilia (the mechanosensitive organelle of HCs), and they synapse on a single large calyx-shaped afferent terminal of a vestibular ganglion neuron (VGN) rather than on the VGN’s bouton terminals, as HCII do. These features are thought to endow HCI with functional specializations such as the ability to detect fast-onset, high-frequency stimuli. However, we understand very little about how these specializations develop or which molecular mechanisms control their genesis, differentiation, and maintenance in adult mammals. This lack of knowledge hampers scientists’ efforts to determine how to drive functional regeneration in mammals after HC loss. Recently, we found that conditional knockout (cKO) of the transcription factor Sox2 from normal HCII and from naturally regenerated HCII in adult mice drives many of them to partially convert toward the HCI fate, including establishing the long, thick stereocilia unique to HCI. Sox2 cKO from adult HCII also triggers VGNs to remove bouton terminals and extend a full or partial HCI-specific calyx terminal on the converting HCII. However, reprogramming and synaptic remodeling of regenerated HCII after Sox2 deletion are insufficient to recover vestibulo-motor functions. The objective of the proposed studies is to exploit these new findings to investigate molecular mechanisms by which HCIs establish and maintain their type-specific hair bundle and innervation phenotypes, which are critical for function. Aim 1 will use mouse multiomics and CUT&RUN to define gene regulatory networks by which SOX2 in HCII blocks acquisition of HCI features, and it will begin to define SOX2-independent mechanisms that promote the HCI fate. Aim 2 will use targeted gene deletion in mice to identify novel SOX2-regulated genes that are required to develop and maintain the stereocilia morphology of HCI. Aim 3 will use targeted loss and gain of gene function in mice to define novel SOX2- regulated genes that are necessary in HCI to establish and maintain the calyx-type terminal and will employ snRNAseq to identify new ligand-receptor pairs that may regulate VGN innervation of HCI and HCII. This project will engage experts from four laboratories to generate new fundamental knowledge about how vestibular HCs develop and maintain structures that are critical for sensory transduction and synaptic transmission, providing new insights for strategies to regenerate vestibular HCs in adult mammals.

NIH Research Projects · FY 2023 · 2024-08

ABSTRACT The University of Washington Injury Control Research Center at the Harborview Injury Prevention and Research Center aims to prevent injury and violence across the lifespan. We are committed to collaborative community engagement as a key factor in “Achieving Injury-related Health Equity across the Lifespan.” We describe a scientifically rigorous 5-year plan to prevent injury and violence in our communities that are most affected by injury and violence, to advance the scientific base for the prevention and control of injury and violence, to translate research efforts for community and public health practitioner use, and to train and educate the next generation of interdisciplinary injury control scientists and public health practitioners, all with a health equity lens. Our proposed Cores and Research Projects all focus on CDC/NCIPC priorities. Our Administrative Core, including Research, provides vision, direction, oversight, and management for ICRC’s research, training and education, outreach, and communication efforts. Our Outreach Core develops the transdisciplinary community academic network and translates results into public policy and best practice implementation. Our Training and Education Core emphasizes interdisciplinary and active community collaboration in the identification and setting of training and education priorities with a commitment to equitable access to opportunities. We propose four translational Research Projects, and an Exploratory Research Program, all of which support early-stage investigators. Two of the four projects address CDC/NCIPC Director’s Priorities—Combatting Overdose and Preventing Adverse Childhood Experiences and two address other key CDC/NCIPC priorities—Youth Violence Prevention and Older Adult Falls. Each Research Project addresses health equity and leverages interdisciplinary expertise to change the national paradigm for injury and violence prevention and control research.

NIH Research Projects · FY 2025 · 2024-08

Abstract/Project Summary Opioid use disorder is defined as chronic, maladaptive opioid use despite negative consequences. Prolonged opioid exposure can induce long-lasting changes to neural circuitry involved with reward and motivation, leading to drug cravings and tolerance to the drug’s rewarding effects over time. One brain region known to play a key role in opioid reward processing is the nucleus accumbens, which contains two subpopulations of GABAergic medium spiny neurons that express either the D1 or D2 dopamine receptor, as well as a variety of interneuron cell types and glia. Medium spiny neurons project to multiple brain regions, and µ-opioid receptors are also expressed on many neurons throughout the brain. Thus, it is reasonable to predict that chronic opioid exposure will be associated with global alterations in neuronal activity patterns. Improving our understanding of both the molecular and circuit-level mechanisms underlying opioid use disorder is crucial to facilitating the development of more effective treatments. To address this gap in knowledge, I plan to leverage a rodent model of chronic, voluntary oral fentanyl intake that I have developed and implemented. My goal for this training proposal is to use this paradigm to specifically assess what changes occur to gene expression and neural circuitry over periods of sustained opioid self-administration. In Aim 1, we plan to conduct single nuclei RNAseq in the nucleus accumbens of mice after chronic fentanyl intake and withdrawal. Aim 2 will utilize transgenic mice to visualize expression of the early immediate gene cFOS, a marker of neuron activity. Whole-brain clearing and light-sheet microscopy will be used to quantify the neural activation patterns associated with long-term fentanyl escalation and withdrawal. Together, these aims will lead to a more complete understanding of the impact of chronic opioid exposure, which may inform the development of novel therapeutic interventions for opioid use disorder.

NSF Awards · FY 2024 · 2024-08

With support from the Chemical Mechanism, Function and Properties Program, Professor Frantisek Turecek at University of Washington will develop new reactions for chemical crosslinking in biomolecular ions representing components of proteins and nucleic acids, establish product structures, and study reaction mechanisms. Crosslinking is a process that has been used in industry to modify the properties of various commodity polymer materials such as tire rubber, polystyrene, and epoxy adhesives. Its implementation in biomolecular structure studies relies on highly reactive intermediates that are produced by laser radiation and react rapidly by forming chemical bonds with other parts of the molecule or another molecule in a complex. The Frantisek research group has been developing chemical reactions to be used in crosslinking in peptide conjugates that are charged and isolated in the gas phase. Under these conditions, reaction products and mechanisms can be studied free of interferences from the surrounding environment. Exploration of new types of organic reactions is important as it may have a broader impact in biochemistry and biology to provide new tools for molecular structure determination and advance scientific knowledge to foster science literacy. The features of the proposed research, combining advanced experimental methods with quantum chemistry calculations, will contribute to the education of undergraduate and graduate students and a post-doctoral associate, including those from underrepresented groups, while also providing outreach to high-school students for research internships in the PI’s laboratories. Development of a diverse and globally competitive STEM workforce is a major goal. In addition to the standard research training, the students and postdoctoral associates will continue having frequent opportunities to present their results at scientific conferences nationally and internationally. International collaborations and short exchange stays will foster culture immersion and professional growth of the participating students and trainees. The research to be developed concerns four aims which are focused on the generation and properties of peptide and oligonucleotide conjugates of nitrile imines, exploring newly discovered crosslinking reactions of the involved functional groups. In the first aim, the PI and his team will explore methods for the synthesis of conjugates in which peptides and nucleotides are tagged with 2,5-diaryltetrazoles as nitrile imine precursors. This research will provide access to stable precursors of reactive intermediates for nitrile-imine based covalent crosslinking. In the second aim, the PI and his team will carry out conformational analysis of the photo-tagged peptide ions and photodissociation products. The PI and collaborators will use cyclic ion mobility mass spectrometry to separate precursor ion conformers and product isomers and correlate their collision cross sections with those of low Gibbs energy isomers generated by Born-Oppenheimer molecular dynamics (BOMD) and density functional theory (DFT) calculations. Progress in this project will provide the PI with a comprehensive view and understanding of the precursor conjugate stereochemistry. The third aim is focused on the combination of ultraviolet-visible (UV-VIS) action spectroscopy, time-dependent-DFT calculations, cyclic ion mobility mass spectrometry (c-IMS), and calculations of theoretical collision cross sections to aid identification of the photoproducts. The results will help answer the critical question of transferring the efficient gas-phase crosslinking to the condensed phase for explorations of biomolecular structures with applications in structural biology. Finally, conjugates will be constructed in which 2,5-diaryltetrazoles and peptides are mounted on cyclic scaffolds to control steric access of the reactive nitrile-imine intermediate to the target moiety. These synthetic peptide scaffolds will provide fine steric control of nitrile-imine-amide and nitrile-imine-nucleobase interactions leading to covalent bond formation and crosslinking. The study is aimed at elucidating the electronic and structural factors that play a role in nitrile-imine crosslinking to complex biomolecules. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2025 · 2024-08

Project Summary / Abstract Corneal epithelium is maintained by stem cells residing in the limbus, the area between the cornea and conjunctiva. These stem cells are known as limbal stem cells (LSCs) and give rise to progenitor cells, which migrate toward central and apical cornea to form the stratified corneal epithelial cell layers. Loss of LSCs result in limbal stem cell deficiency (LSCD), one of the most common causes of vision loss in the world. While unilateral LSCD can be treated by transplantation of autologous limbal tissue, patients with bilateral LSCD have to rely on allogeneic transplantation and the clinical outcome is relatively poor. In addition, there are multiple post-operative complications reported including recurrent/persistent epithelial erosion, intraocular pressure elevation, and rejection. Moreover, the shortage of corneal donors reduces the opportunity for allogeneic transplantation. There is, therefore, a critical need to develop a less invasive therapy to treat LSCD that also addresses the donor shortage. Besides the graft condition, it is important to control recipients’ limbal niche microenvironment for a successful transplantation, since there has been growing awareness of the fact that limbal niche cells play a critical role for the maintenance of LSCs. Of the niche cells, limbal fibroblasts are thought to contribute to the maintenance of corneal epithelial phenotype by secreting fibroblast growth factor 7 (FGF7), whose secretion is promoted by platelet-derived growth factor (PDGF)-BB and interleukin 1 beta (IL1β). However, it remains largely unknown how limbal fibroblasts are affected in the LSCD and how the recovery of limbal fibroblasts contributes to the success of the treatment of LSCD. Our long-term goal is to develop a novel treatment for LSCD by recovering the function of limbal niche. Our overall objective in this proposal is to reveal the contribution of limbal fibroblasts in normal and LSCD conditions, and to establish a limbal fibroblast transplantation technique. Our central hypothesis is that limbal fibroblasts secrete FGF7 in response to PDGF and IL1β secreted by limbal dendritic cells to maintain the limbal stem/progenitor cells. Moreover, we hypothesize that limbal niche including limbal fibroblasts are disrupted in LSCD and transplantation of limbal fibroblasts regenerate the corneal phenotype in LSCD. In addition to the human donor corneas, we will utilize a novel technique of eye-like organoid formation from induced pluripotent stem cells (iPSCs) to create limbal fibroblasts as a cell source of transplantation to resolve the corneal donor shortage. Proposed study will reveal the characteristics of limbal fibroblasts and limbal dendritic cells (Aim 1), evaluate the interaction of limbal niche cells and limbal epithelial cells (Aim 2), and establish limbal fibroblast transplantation for the treatment of LSCD (Aim 3). Successful completion of this project will improve our understanding of limbal niche cells in normal and LSCD conditions, and provide us with another strategic option to treat LSCD, namely limbal fibroblast transplantation. This will allow us to conduct larger animal experiments and ultimately, apply this treatment to LSCD patients.