University Of Wisconsin-Madison

NIH Research Projects · FY 2025 · 2022-09

We will conduct a 5-year research project that aims to extend and enhance the delivery of comprehensive, community-based prevention services to hard-to-reach people who use opioids and/or stimulants, to reduce their risk of fatal and nonfatal overdose. The main objective of this research is to design, and pilot test a mobile health (mHealth) intervention comprised of a bundle of internet and mobile phone-based tools for reducing overdose risk. We will build upon a productive research partnership with an established, comprehensive community-based prevention services organization operating in 10 cities in Wisconsin to pursue 3 specific aims. First, using a prospective cohort study design and several innovative methods for capturing prevention service engagement, psychosocial variables, and behavioral outcomes, we will characterize the most important mechanisms through which prevention services influence overdose risk behaviors. Second, we will study the role of prevention service engagement in linking clients to other sources of health care and addiction treatment services, making novel use of linkages between administrative datasets in partnership with state government agencies. Third, we will convene a Community Leadership Team consisting of people who use opioids and/or stimulants and collaboratively synthesize our research findings to inform the development and implementation of an innovative strategy for engaging clients who face barriers to accessing prevention services. The community engagement activities will culminate in a single-arm pilot intervention trial that evaluates the feasibility and acceptability of a suite of internet and smartphone-based tools designed to improve access to overdose prevention resources, fill knowledge gaps, and deliver social support through connection to trusted peer networks. This approach will ensure people who use drugs have a real voice in the creation of programs designed to serve them. Our research will generate a robust understanding of the important mechanisms through which prevention services reduce overdose risk, and test promising strategies for extending their reach to under-served clients.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT For older adults, poor communication about serious injury and life-limiting illness has consequences for patients and families, clinicians, and healthcare systems. Afflicting 500,000 older adults annually, treatment for traumatic injury frequently involves burdensome interventions (like prolonged life support), major changes in functional or cognitive status, and high mortality. Surgeons use mortality statistics to communicate about the gravity of illness, but current communication standards often lead to goal discordant care, moral distress for clinicians, and prolongation of the dying process. Given high treatment burdens and frequency of poor prognosis, seriously injured older adults would benefit from communication interventions that clarify patients' goals, alleviate conflict in the ICU, and reduce unwanted invasive procedures for dying patients. A Randomized Clinical Trial of Scenario Planning for Older Adults with Serious Injury is a 5-year R01 Clinical Trial that responds specifically to NOSI-AG-20-041 for evaluation of decision support tools and communication aids for seriously ill older patients and their surrogate decision-makers to achieve goal- concordant care. We have developed a novel communication tool called Best Case/Worst Case-ICU that uses scenario planning—narrative description of plausible futures—and a graphic aid to illustrate information about the patient's care trajectory and overall prognosis for daily use in the trauma ICU. We pilot tested this intervention and found that surgeons can use this tool to support families and it may improve quality of communication and reduce clinician moral distress. We are now ready to test the intervention in a large-scale effectiveness study. We propose a pragmatic multisite randomized clinical trial following 4,500 older adults with traumatic injury. We aim to test the effectiveness of the Best Case/Worst Case-ICU intervention on quality of communication (Aim 1), clinician moral distress (Aim 2), and length of stay in the ICU (Aim 3). This award will allow us to test an intervention that is easily scalable and can be disseminated rapidly for use with older adults with serious illness. The research is innovative because it tests scenario planning —a decision-making strategy that has been successfully applied in business and government—but is not widely used in healthcare. The research is significant because, if we demonstrate effectiveness, it has the potential to transform how surgeons and other ICU clinicians talk with patients and families about treatment and prognosis and dramatically improve care older adults receive in the trauma ICU. Our multidisciplinary team has a long history of collaboration and is well positioned to achieve our objectives. The feasibility of this study is enhanced by support from the Coalition for National Trauma Research, which comprises the major trauma professional organizations in the United States and uses the American College of Surgeons' Trauma Quality Improvement Program as a data collection platform.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Comparative functional genomics offers a powerful framework to study the molecular underpinnings of species- specific traits. Gene regulatory networks (GRNs) which control precise context-specific expression patterns of genes play a significant role in diversifying phenotypes across species. These networks are central to cell type specific function and are often disrupted in many diseases. However, comparison of gene regulatory networks across species has been challenging because of the lack of sufficient number of samples across matched biological contexts. Single cell omic technologies, such as single cell RNA-seq (scRNA-seq) and ATAC-seq (scATAC-seq), are revolutionizing biology enabling researchers to profile the activity of nearly all genomic regions in each individual cell. Single cell omic studies are quickly expanding to multiple species providing unprecedented opportunities to define cell types and their underlying gene regulatory networks and study their evolution. However, computational methods for defining cell-types and cell-specific GRNs across species are in their infancy. In particular, samples in a multi-species scRNA-seq dataset are related by a phylogeny, however, existing integration approaches do not model these relationships. Furthermore, existing approaches are restricted to one-to-one relationships across species, which makes it difficult to study some of the major sources of evolutionary innovation (e.g., duplications) in cell type identity. In this project, we will develop novel computational methods to tackle two problems: (a) defining cell types and their lineage relationships across species from scRNA-seq and scATAC-seq datasets, (b) inference and comparative analysis of cell type-specific GRNs across species from single cell RNA-seq and ATAC-seq data. Our tools will be based on machine learning methods, namely, probabilistic graphical models, multi-task and multi-view learning, and matrix factorization, that offer principled frameworks to integrate information across species. We will first test these tools in human and mouse scRNA-seq/ATAC-seq datasets from our collaborators and published studies. We will demonstrate the full potential of our tools on a novel multi-species kidney scRNA-seq/scATAC-seq dataset that we will collect to study normal kidney function as well as compensatory renal growth, which controls how one kidney recovers after surgical removal of another kidney. We will identify conserved and diverged regulatory networks that will be used to prioritize sequence and protein regulators for validation studies with CRISPR and siRNA. Our analysis will reveal key insights into how GRNs evolve across species and how they establish different cell types. Our approaches and novel datasets will provide critical insight into the molecular programs governing kidney structure and function that could have a significant clinical impact for patients with kidney disease. Our methods will constitute a suite of broadly applicable tools that can shed insight into principles of gene regulation and cell fate specification that will be applicable to single cell datasets from diverse multi-cellular systems.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY In 2016, we initiated the University of Wisconsin (UW)-Madison Summer Program in Undergraduate Urology Research (SPUUR) to attract and retain aspiring scientists into the field. Supported by our George M. O'Brien Center in benign urology research, and excellent mentors including NIDDK Multidisciplinary Urologic Research (KURe) Career Development Program (K12) awardees, we trained 28 scholars. Half the scholars co-authored at least two manuscripts and all remained in research-related positions or joined advanced degree programs. We recruited exclusively from UW-Madison and now seek to expand scholar recruitment to the entire US. We will support 10 students per summer. We will use proven active learning modules to introduce scholars to research and clinical challenges from perspectives of the Schools of Medicine and Public Health, Pharmacy, Nursing and Veterinary Medicine. We believe the benign urology workforce is strengthened by diverse perspectives. We will apply best practices in recruiting participants from traditionally underrepresented communities. Our diversity recruitment efforts are effective and SPUUR scholars trained to date are more diverse than the UW-Madison undergraduate population from which they were recruited. We are determined to propel our scholars into advanced clinical and basic urologic research training programs. We will leverage a unique UW-Madison resource, the nation's only U24 Urology Centers Interaction Core, to initiate professional networks between scholars and faculty at urology centers throughout the country and facilitate entry into advanced degree programs. Historically, most NIDDK-KUH-sponsored R25 Programs have had a kidney or hematologic focus. The lower urinary tract is underrepresented and thus remains under-appreciated by prospective biomedical research scholars. Our program will fill the void by intensively focusing on the lower urinary tract. Students will engage in a carefully constructed curriculum to cultivate critical thinking, develop research questions, conduct research responsibly, network virtually with peers from other summer undergraduate programs, learn about the graduate school application process, interact with current graduate students and campus organizations (particularly those representing students of color and other underrepresented communities), and receive one- on-one advising from mentors experienced in developing biomedical research scholars. Our unique focus on the lower urinary tract, our rigorous and evidence-based plan to recruit underrepresented students, our superior faculty partners, our proven experience in summer undergraduate research programs, our learner-centered training and educational approach, and the unparalleled UW-Madison environment and resources portend the success of our program.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY The small nucleotide c-di-AMP is a ubiquitous second messenger produced by thousands of bacterial and archeal species. Many bacteria, especially those in the Firmicutes phylum, require c-di-AMP for growth in standard laboratory media, and the mechanisms for c-di-AMP essentiality have been extensively studied. However, in a wide range of c-di-AMP-producing bacteria, including those for which c-di-AMP is completely dispensable, unregulated c-di-AMP accumulation is toxic, diminishing bacterial growth and stress response. Thus, c-di-AMP homeostasis is critical for bacteria that produce it. Nevertheless, the mechanisms that regulate c-di-AMP levels in the bacterial cell, and the mechanisms by which c-di-AMP regulates different cellular pathways to achieve optimal physiology, are both poorly understood. My lab found that c-di-AMP levels are dynamic in the bacterial cell, and c-di-AMP hydrolysis is a key mechanism to modulate c-di-AMP levels. Furthermore, we have generated bacterial mutants lacking c-di-AMP phosphodiesterases, called pde mutants, and extensively studied their phenotypic defects. In the next five years, we will investigate two major aspects of c-di-AMP signaling: i) the signal detection and regulatory mechanisms of c-di-AMP phosphodiesterase, and ii) the mechanisms by which c-di-AMP coordinates its molecular targets to achieve optimal physiology and stress response. For the second theme, we will take two complementary approaches. One, we will elucidate the biological functions of c- di-AMP molecular targets based on targeted phenotypic, genetic, and biochemical analyses. Two, we will identify the molecular targets underlying the toxicity phenotypes of pde mutants, through genetic fitness assays. Our findings will reveal fundamental mechanisms of c-di-AMP signaling, and these mechanisms will provide a framework to understand the global regulatory roles of c-di-AMP in the physiology of different bacterial species.

NIH Research Projects · FY 2025 · 2022-09

Accurate non-invasive biomarkers are urgently needed to identify which patients with hormone receptor positive (HR+) breast cancer will respond to neoadjuvant endocrine therapy. Lack of direct knowledge of the endocrine sensitivity of each patient’s breast cancer impedes optimal, tailored therapy. Without a more personalized approach, many women and men will continue to suffer from the current morbidity and mortality of breast cancer. The overall objective of the proposed clinical trial is to investigate the ability of quantitative, hybrid functional imaging for assessing hormonal sensitivity, estrogen receptor (ER) functional inhibition, and early response to neoadjuvant endocrine therapy. The long-term goal is to develop functional imaging approaches to directly test tumor sensitivity to endocrine therapy in breast cancer patients for individualized treatment plans and improved outcomes. The proposed research will investigate early changes in expression of a classic estrogen-regulated target gene as a surrogate measure of endocrine sensitivity: progesterone receptor (PR) using a progestin-based radioligand, 21- [18F]fluorofuranylnorprogesterone (FFNP) and quantitative simultaneous breast positron emission tomography/magnetic resonance imaging (PET/MRI). The central hypothesis is that FFNP uptake in primary breast tumors will show dynamic changes in response to presurgical endocrine therapy, which will correlate with treatment response and exceed inherent technical variability. The proposed clinical trial is a prospective, single-center study that will enroll women with newly diagnosed ER+/PR+/HER2- invasive breast cancer who will undergo simultaneous breast PET/MRI with FFNP before and after a short course of endocrine therapy prior to surgical excision. The study aims to determine 1) the efficacy of FFNP PET/MRI for predicting response to presurgical endocrine therapy and 2) the quantitative reliability of FFNP breast PET/MRI. The proposed research is innovative because it will use functional imaging with simultaneous breast PET/MRI to improve the success of neoadjuvant endocrine therapy. Imaging treatment-induced changes in estrogen-regulated signaling events, using FFNP PET/MRI, will have a significant positive impact by enabling early assessment of endocrine therapy response mediated through ER before changes in tumor size can be measured using conventional techniques such as mammography, ultrasound, and palpation. Once validated, this approach can easily be integrated into the preoperative evaluation of patients with primary HR+ breast cancer to individualize neoadjuvant and adjuvant treatment plans for improved patient outcomes.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT: Quantitative relaxometry is a promising method for quantifying brain changes with early development and brain tissue differences as a function of genetics, environment or pathology. Relaxometry may be useful for assessing abnormal white matter damage in infants at risk for cerebral palsy, which is the leading movement disorder in children. Head motion is a significant challenge for MRI studies in young children, resulting in image artifacts and errors in quantitative imaging measures. Long and loud scans also adversely affect imaging compliance in young children. Consequently, there is a critical need to develop imaging methods that are robust to motion, faster, and quieter. This project will develop, optimize and evaluate novel 3D radial imaging technologies for multimodal structural imaging and quantitative relaxometry for studies in sleeping infants and toddlers. The outcome will be a fast, ultra-quiet imaging technique capable of providing imaging maps of quantitative relaxation times that are robust to nearly all motions. These optimized, motion-corrected, quantitative relaxometry technologies will be applied to a cohort of infants and toddlers without sedation to generate developmental relaxometry templates for normative studies from 0 to 2 years of age. The normative relaxometry framework will be applied to lesions and abnormal brain development of infants at risk for or diagnosed with cerebral palsy, The normative framework will also be used to investigate individual differences in brain and sensorimotor development in both typical development and cerebral palsy. Ultimately, this project will provide a set of robust, reliable and accurate image acquisition methods, software tools, and strategies for investigating healthy and abnormal brain development in both clinical and research pediatric populations without sedation.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Epstein-Barr virus (EBV) causes a wide spectrum of lymphomas including Hodgkin, Burkitt, and diffuse large cell lymphomas (DLBCL). The proportion of EBV+ lymphomas rises sharply as does morbidity and mortality in HIV-infected persons despite combined antiretroviral therapy (cART), suggesting specific failure to control viral driven lymphomas. However, the mechanistic processes contributing to EBV-driven lymphomagenesis remain poorly understood. This proposal will investigate a conceptually novel hypothesis that the LMP2A viral oncogene interacts with the B cell receptor (BCR), serving to maintain it in its growth promoting IgM isotype. This hypothesis predicts that EBV transformed cells expressing LMP2A are dependent upon their constitutive activation of cellular mediators of antigen-induced signaling, and thus will be susceptible to agents that target this signaling cascade, whereas EBV-transformed cells that do not express LMP2A will be resistant to such drugs. Our proposal stems from two exciting observations that we have made about lymphoblastoid cells (LCLs) transformed by an EBV mutant that lacks LMP2A (∆LMP2A-EBV). First, we discovered that, unlike normal LCLs, the growth of ∆LMP2A-LCLs is not dependent on mediators of antigen-induced signaling like BLNK and BTK. Second, in contrast to normal LCLs, ∆LMP2A-LCLs fail to maintain the growth promoting IgM form of the BCR and instead express the IgG-BCR. Since antigen signaling through IgG-BCR promotes differentiation into plasma cells which secrete high levels of antibodies but have limited proliferative potential, our central hypothesis is that LMP2A contributes to lymphomagenesis by facilitating outgrowth of EBV transformed B lymphocytes expressing the growth promoting IgM-BCR. This proposal will analyze mechanistic pathways and gene expression profiles in EBV-transformed LCLs and test lymphoma formation in humanized mice in order to distinguish the direct contributions of LMP2A from those mediated by the IgM-BCR, and assess the therapeutic vulnerability that arise from each. Specifically, we will (1) Investigate intersection of LMP2A and BCR Ig isotype on B lymphocyte transformation, (2) Characterize impact of antigenic stimulation in the presence or absence of LMP2A expression, and (3) Determine LMP2A-mediated susceptibility to therapeutic inhibition of signaling molecules. Together these studies provide a framework for how LMP2A interacts with the BCR to promote EBV transformation and provide new opportunities for therapeutic intervention in EBV-associated lymphomas.

NIH Research Projects · FY 2025 · 2022-09

UW-Madison seeks to establish a one-year Postbaccalaureate Research Education Program (UW-PREP) to develop skills for outstanding recent graduates that have experienced barriers in obtaining meaningful research experiences to be successful in gaining leadership roles in the biomedical workforce. The goals of this program are to prepare these scholars to apply, matriculate, and then graduate from outstanding PhD programs from the UW and throughout the country. UW-PREP’s mission is threefold: 1) to provide opportunities and evidence-based platforms for participants to learn how to design and conduct impactful, rigorous and reproducible scientific research; 2) to foster participants’ growth in areas of professional development that promote self-awareness and advocacy, build science communication skills, and reinforce an identity as a scientist; and 3) to advance a culture of mentoring, research integrity, and life-long learning. The UW-PREP will be established with a primary focus on a mentored research experience in which participants will be immersed in developing skills needed to design and conduct rigorous, ethical research. The UW has outstanding resources and infrastructure in place to support innovative and impactful research. PREP Research Education Activities will be designed on published UW Entering Research curricula, a collection of evidence-based active learning activities. Activities will reinforce rigor and reproducibility and foundations of responsible conduct of research both being inherent to their mentored laboratory experience. They are also designed to help appointed scholars devise their Individual Development Plan (IDP) and will incorporate Professional Development activities to build STEM-specific social capital to navigate the hidden curriculum of academic research spaces in a large research institution. PREP Leadership and funded faculty representing all aspects of life sciences enjoy outstanding institutional support and coordination of evidence-based curricula to build a PREP that will help to launch exceptionally trained and confident scholars into the biomedical workforce.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT It is increasingly apparent that both common and rare genetic variation influences the risk and severity of syndromic autism spectrum disorders (ASD). Among patients with highly penetrant ASD associated mutations there is often substantial heterogeneity in the severity of physical, behavioral, and cognitive deficits. However, precisely which regions of the human genome affect disease outcomes in the context of any high confidence ASD mutation are currently unknown. This critical gap in knowledge prevents the rational design of therapies that target underlying molecular and cellular deficiencies. Here, I will leverage genetically diverse iPSC lines to test the hypothesis that common genetic variation shapes the severity of hyperproliferation phenotypes caused by mutations in PTEN, a highly penetrant ASD associated gene. I will employ a novel pooled cell culture method, which allows rapid and efficient phenotyping of dozens of distinct lines in the same culture dish, to quantify the variation in hyperproliferation induced by PTEN haploinsufficiency. This will allow me to identify regions of the genome which modulate this ASD relevant phenotype, and test for the presence of sex specific genetic effects. I will then mechanistically validate associated loci with three distinct approaches. First, we will expand and validate our culture-based findings in patients with 1) idiopathic autism with brain overgrowth with longitudinal MRI and neurocognitive profiling, from whom these lines were generated, 2) ~600 deeply phenotyped patients with a variety of PTEN mutations and clinical presentations. Second, we will correlate our findings with those from other genome wide association studies to identify causal genes, and loci with shared genetic risk with other diseases. Third, we will validate associated loci in isogenic PTEN(+/-) lines, and test hypotheses aimed at the mechanisms by which genetic variants protect/exacerbate the effects of PTEN mutations. In all, the proposed studies will identify common genetic modifiers of phenotypic severity and patient outcomes in a genetically defined ASD subtype. To develop the expertise necessary for this cross-disciplinary project, I will undergo comprehensive training in bioinformatics, statistical genetics, and molecular neurodevelopment under the guidance of my primary mentor Jason Stein. I will also undergo supplemental training from my advisors in iPSC differentiation methods (Beltran), MRI image analysis (Piven/Styner), ASD pathogenesis (Piven/Zylka/Eng), and approaches to correlate genetic and clinical data (Stein/Piven/Eng). They will train me in the methods and principles required to successfully complete this project. This training will facilitate a successfully transition into my independent scientific career, where I will study genetic modifiers of neurodevelopmental disorders.

NIH Research Projects · FY 2026 · 2022-09

ABSTRACT The purpose of this epidemiologic study is to determine if sensory changes (hearing, vision, olfaction) and an emerging biomarker of aging (PhenoAge) measured in midlife are strong predictors of long-term risk for early Alzheimer’s disease and related dementias (ADRD), and to study the shared etiology of co-occurring sensory and cognitive changes. Subjects are previous participants in a prospective longitudinal cohort study, the Beaver Dam Offspring Study (BOSS) and were 21-84 years of age at the baseline examination (2005-2008). The proposed study (BOSS–Neurocognitive Aging Study) will apply standardized protocols used in the BOSS baseline, 5-year and 10-year follow-up examinations including a hearing evaluation (otoscopy, audiometry and word recognition in quiet and in competing message), eye examination (refraction, visual acuity and contrast sensitivity) and olfaction testing (San Diego Odor Identification Test) and conduct an extended cognitive test battery (Trail Making Test, Auditory Verbal Learning Test, Verbal Fluency Test, Digit Symbol Substitution Test, Digit Span, Stroop Test, Mini-Mental State Examination). We will further enhance the evaluation of early ADRD status by incorporating medical records, caregiver interviews and blood-based biomarkers of AD and neurodegeneration (amyloid b40, amyloid b42, total Tau and phosphorylated Tau) in a clinical review that will be conducted by a multidisciplinary neurocognitive expert panel in a diagnosis consensus conference. Cardiovascular risk factors will be assessed and standardized questionnaires on medical history, medication usage and lifestyle and environmental factors will be completed. We will measure longitudinal changes in PhenoAge using new and stored baseline blood samples. The proposed epidemiological study will provide important information about early biomarkers for the risk of developing cognitive decline and early ADRD and will inform about relationships between changes in sensory and cognitive function. This will contribute to developing clinically useful methods to identify high-risk people in midlife and to understanding the shared etiologies of sensory and cognitive changes. This project’s results will inform about potential pathways for prevention of ADRD and the promotion of healthy brain aging.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Many diseases of aging, including Alzheimer’s disease (AD) and AD related dementia, have been linked with significant metabolic changes that are regulated by diverse molecular classes. Nodes of the aging- and AD-associated metabolic signature include: (i) General proteomic profile as reflecting changes in protein homeostasis and/or an ongoing neurodegenerative event; (ii) Glycosylation of glycoproteins, as reflecting changes in engagement and trafficking along the secretory pathway, as well as “post-delivery” processing cell- surface glycoproteins; and (iii) Biological membrane lipid composition and general bioactive lipid metabolism. Several studies have shown changes in brain metabolism are not uniform throughout AD progression, with parietal, posterior temporal, and anterior occipital lobes most severely affected. Because of this, broad conclusions about the AD brain following analysis that does not include spatial information may be painting an incomplete picture of AD pathogenesis. Due to the complexity of the brain, spatial distribution as well as functional integrations of the above nodes are warranted to understand both aging of the brain and AD pathophysiology. To perform a more comprehensive analysis of region-specific molecular pattern changes in the AD brain, we propose to employ matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI MSI) technology to examine spatial distribution changes of several molecular classes in animal models of AD as well as postmortem brain tissue of late-onset AD (LOAD) patients. The GENERAL HYPOTHESIS of this research is that alteration in the molecular pattern of various biologically relevant molecular classes can reflect or influence the onset and progression of AD. Aim 1 will map region-specific glycan and glycoprotein expression pattern changes in the whole brain tissue sections of AD mouse models and LOAD patient tissue samples. We will create an atlas of the glycoproteome and illuminating changes in glycosylation that could be key in understanding AD pathogenesis. Aim 2 will map lipidome and unsaturated lipid isomers and changes in metabolic signature in the whole brain tissue sections of AD mouse models and LOAD patient tissue samples. The use of innovative double-bond localization chemistry will expand our current understanding of the AD lipidome, revealing a molecular map of not just lipid classes, but specific lipid isomers within the AD brain. Aim 3 will develop technology- and computationally- driven approaches for biomolecule validation, co-localization, and multidimensional correlation. Our proposed machine learning algorithms will enable simultaneous and region-specific correlation of multiple classes of molecules. Our collaborative team’s orthogonal research foci and interdisciplinary expertise will enable us to generate novel mechanistic and translational data that will inform the research community on the progression of aging and AD. The mechanistic component has the potential to yield significant knowledge that can be used to target specific biomolecules and expand our research beyond this RFA.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY TARGET OF RAPAMYCIN (TOR) is a deeply conserved protein kinase that regulates eukaryotic metabolism. TOR senses and integrates upstream signals, especially nutrient availability, to coordinate metabolism and promote growth only when conditions are favorable. TOR dysregulation causes or contributes to a broad range of human diseases, including cancers, age-related health disorders, and metabolic disorders, which are the major causes of morbidity and mortality in the United States. Therefore, a major goal for biomedical research is to develop therapeutic treatments that specifically target components of the TOR signaling network without broadly disrupting metabolism and homeostasis in healthy cells that rely on TOR. Recently, there have been significant advances to that goal with the discovery of several putative amino acid sensors for TOR. Conflicting reports about the relative contributions, importance, and molecular mechanisms of these sensors have stymied these advances, however. This project uses an innovative approach to bring fresh perspective to these ongoing debates by shifting focus to the other major eukaryotic lineage, plants. In my lab’s ongoing work to elucidate the TOR signaling network in plants, I discovered a novel amino acid sensor for TOR, an aminoacyl tRNA synthetase (aaRS). This aaRS is necessary to maintain TOR activity and sufficient to stimulate TOR in plant cells. Using a combination of biochemical, molecular, genetic, and systems-level approaches, I propose to precisely define how the aaRS activates TOR in plant cells through three independent aims. In Aim 1, I propose to mutate key enzymatic residues and structural features of the aaRS to determine the molecular features it requires to activate TOR. In Aim 2, I propose to map the signal transduction pathway mediating aaRS-TOR activation using robust orthogonal interactomic approaches. Putative signal transduction components will then be validated using reciprocal assays and functional genetics to comprehensively define how aaRS-TOR interactors contribute to TOR regulation. In Aim 3, I propose to establish the selective sensitivity of TOR for specific amino acids and determine whether the aaRS is a bona fide amino acid sensor for TOR. Taken together, these three aims will define the molecular mechanisms underlying the putative amino acid-aaRS-TOR signaling axis and open new directions for future research on metabolic regulation in eukaryotes. Moreover, this pathway will serve as a model for understanding how tRNA synthetases have evolved functions beyond translation in signal transduction pathways and illuminate how the complex TOR signaling network evolved to integrate diverse physiological cues in humans. Long-term, our findings will make significant contributions to a major goal of contemporary biomedical research: fine-tuning TOR signaling networks to improve and lengthen healthy human lifespans.

NIH Research Projects · FY 2025 · 2022-09

Modified Project Summary/Abstract Section – Overall Program This P01 project grant will establish a fully integrated interdisciplinary program of research Projects and Scientific Cores that are essential to develop a fundamental understanding of the complex interplay between adolescent health and development, and technology and digital media (TDM). Previous evidence has illustrated TDM’s connections to adolescent risk behaviors such as increased alcohol behavior and social media exposure, as well as relationships to adolescent well-being such as improved socioemotional health and peer social media connections. The goal of the Projects described in this proposal is to address the urgent need to understand how TDM exposure and usage affect multiple developmental domains and health outcomes. The three PIs are all located at the University of Wisconsin-Madison, ensuring close collaboration and synergy, in addition to outstanding institutional support and resources, including matching funds. The Projects include: Project 1: Using TDM to understand mechanisms in adolescent health and risk behavior. Project 2: Using functional magnetic resonance imaging to understand how positive and negative TDM experiences relate to mental and behavioral health. Project 3: Using mixed methods to evaluate self- and other-generated TDM content as predictors of adolescent socioemotional well-being. Each Project utilizes a 2-year longitudinal design and draws from a shared participant pool. Data collection approaches across Projects include observed/measured data including observed social media content and fMRI data, self-reported participant experiences and perceptions via surveys and interviews, and Ecological Momentary Assessment to capture real-time TDM exposures. To support this program, the Administrative Core (Admin Core) will provide organizational and management support to arrange regular meetings across Program collaborators, involve students in the research, leverage biostatistical support and promote dissemination. This P01 proposal will include a Recruitment and Retention Core (R&R Core), supporting a shared participant pool across Projects, and ensuring retention over time. This P01 program will promote synergy in these research efforts through integrated data collection processes over a synergistic longitudinal design, aligned measures and a shared participant pool so that analyses can be structured within and across Projects. This P01 proposal includes a priority on dissemination of findings, both to scientific audiences and to the communities across Wisconsin to reach those who participated in this research. Thus, this P01 program will enhance the scientific knowledge, ideas and outcomes obtained through the interactions of the 3 Projects, the Admin Core and the R&R Core. This proposed program will provide both the infrastructure support and the scientific approach necessary to advance data-informed theories and conceptual models addressing how TDM exposure and usage impact developmental trajectories and health outcomes of adolescents. Because of the broad potential for advancing research and possible clinical translation of results, these connected Projects portend an opportunity to improve prevention and intervention approaches for adolescent health and TDM.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Autism spectrum disorder (ASD) is a lifelong disorder that has consequences throughout adulthood. Recent population health studies indicate that aging autistic adults have shorter life expectancy and increased rates of physical and mental health problems. However, there is a paucity of studies that have focused on the progression of health and wellness with aging in ASD and factors that can contribute to better or worse outcomes. In this Autism Center of Excellence (ACE) Network project, the Interdisciplinary Science to Learn about Autism – Aging (ISLA-A) Network will deploy a harmonized, optimized, and innovative protocol to investigate the effects of aging in autism in one of the largest prospective longitudinal cohort studies of autistic adults to date. The aims of the ISLA-A center are 1) to establish and follow a large cohort of autistic male and female adults, siblings and age- and sex- matched non-autistic adults with a comprehensive harmonized research protocol to investigate multi- modal aspects of aging, including measures of clinical severity, physical and mental health, cognitive aging, brain structure and function, and epigenetic measures of biological aging; 2) to characterize both group and individual age-related changes in autism severity, health, wellness and brain measures with aging; 3) to investigate the relationships between clinical, health, and brain imaging measures; and 4) to investigate whether biological aging is accelerated in autism using new epigenetic measures of aging. The overarching goal of the ISLA-A Network is to create a comprehensive, harmonized, and high-dimensional dataset that will characterize trajectories of aging in autism that may be used to investigate whether early or accelerated aging is a hallmark feature of autism, and how aging in autism influences health and brain outcomes. The inclusion of siblings, who share genetics with the autistic adult cohort, will help to identify autism-specific factors related to aging and outcomes. The ISLA-A study results will identify candidate factors that are predictive to autism aging outcomes and will guide the development of interventions and services to improve outcomes. This new large, multi-modal, longitudinal, and generalizable dataset will be shared with the autism research community for independent studies. Overall, the ISLA-A Network study will generate a rich, high-impact resource for better understanding of aging in autism, with the ultimate goal of improving the health and support of autistic adults.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT Autism spectrum disorder (ASD) is a lifelong disorder that has consequences throughout adulthood. Recent population health studies indicate that aging autistic adults have shorter life expectancy and increased rates of physical and mental health problems. However, there is a paucity of studies that have focused on the progression of health and wellness with aging in ASD and factors that can contribute to better or worse outcomes. In this Autism Center of Excellence (ACE) Network project, the Interdisciplinary Science to Learn about Autism – Aging (ISLA-A) Network will deploy a harmonized, optimized, and innovative protocol to investigate the effects of aging in autism in one of the largest prospective longitudinal cohort studies of autistic adults to date. The aims of the ISLA-A center are 1) to establish and follow a large cohort of autistic male and female adults, siblings and age- and sex- matched non-autistic adults with a comprehensive harmonized research protocol to investigate multi- modal aspects of aging, including measures of clinical severity, physical and mental health, cognitive aging, brain structure and function, and epigenetic measures of biological aging; 2) to characterize both group and individual age-related changes in autism severity, health, wellness and brain measures with aging; 3) to investigate the relationships between clinical, health, and brain imaging measures; and 4) to investigate whether biological aging is accelerated in autism using new epigenetic measures of aging. The overarching goal of the ISLA-A Network is to create a comprehensive, harmonized, and high-dimensional dataset that will characterize trajectories of aging in autism that may be used to investigate whether early or accelerated aging is a hallmark feature of autism, and how aging in autism influences health and brain outcomes. The inclusion of siblings, who share genetics with the autistic adult cohort, will help to identify autism-specific factors related to aging and outcomes. The ISLA-A study results will identify candidate factors that are predictive to autism aging outcomes and will guide the development of interventions and services to improve outcomes. This new large, multi-modal, longitudinal, and generalizable dataset will be shared with the autism research community for independent studies. Overall, the ISLA-A Network study will generate a rich, high-impact resource for better understanding of aging in autism, with the ultimate goal of improving the health and support of autistic adults.

NIH Research Projects · FY 2025 · 2022-09

Summary Telomeres are end-capping protein-DNA structures at the ends of the linear human chromosomes. They protect our genome integrity by sacrificing their repetitive DNA when the ends of the chromosome suffer attrition during DNA replication and camouflaging the chromosome ends from wrong DNA breakage recognition. Deregulation or loss of telomeres results in genome instability and leads to human diseases such as cancer and premature aging. The telomere's repetitive DNA nature provides a unique challenge in understanding their biological processes. This is because telomeric proteins can bind the repetitive telomeric DNA in many ways, leading to complexity and diversity in the telomere chromatin landscape; there are functional consequences to how telomeric proteins decorate a telomere chromatin landscape because these proteins directly participate in telomere protection and length maintenance. Thus, our understanding of telomeres is like a "black box". We know the inputs (proteins and lncRNA) and outputs (telomere length and end-protection) and understand how variations of inputs transform to output changes. However, we do not know what is going on inside the "black box". This "black box" is the telomere chromatin landscape. Characterizing the telomere chromatin landscape has been an insurmountable task for the telomere research field for decades. The ChIP-Seq technique has revolutionized chromosome biology research, but repetitive genomic regions such as the telomeres are left behind. This is because the relative positional information of the protein-DNA interactions is lost upon the fragmentation step in ChIP-Seq, preventing us from reconstructing the chromatin landscape of interest. This proposal seeks to innovate new tools to map the human telomere chromatin landscape at a single-telomere level. These tools will then use to study how the telomere chromatin landscape regulates telomere end-protection and length maintenance. First, I will establish the proof-of-concept experiments for using non-native DNA methylation to mark protein-DNA interactions at the repetitive telomeric DNA regions and reconstruct the chromatin landscapes with structural details. These tools will then be used to tackle two major research areas: (1) What is the human telomere chromatin landscape and how it changes across the cell cycle from a resting protective state to one permissive to DNA replication progression. (2) How changes in the human telomere chromatin landscape drive telomere length maintenance. This proposal thus consists of both technological and conceptual innovations. The new tools will provide a new way to investigate chromosome biology at repetitive genomic DNA regions; thus, its impact extends beyond the telomeres. We will get an unprecedented first look into the telomere chromatin landscape. Hence, this proposal has enormous potential to open multiple new research directions in telomere biology; a paradigm shift in our telomere knowledge is expected. Because of the biomedical importance of telomeres, the outcome of this proposal can provide novel avenues to tackle telomere-related human diseases.

NIH Research Projects · FY 2025 · 2022-09

Project summary/Abstract Two existential threats to female fertility include the premature loss of the ovarian reserve and early implantation/placentation failure. We recently discovered that Irx3 and Irx5, two of six members of the Iroquois homeobox (Irx) transcription factor family, show remarkable conservation of molecular and cellular actions associated with cognate cell-cell transitions critical for germline nest breakdown and primordial follicle formation (ovarian reserve) during ovary development and embryo-endometrial interactions leading to vasculogenesis during implantation and establishment of pregnancy. Previously, we employed a series of Irx3/5 mutant mouse models, including an Sf1-Cre/Flox strategy to highlight autonomous roles for Irx3 vs Irx5, especially in somatic cells. The fertility profiles for these mice also indicated that developmental expression of Irx3 contributes to the adult follicle’s response to external growth and ovulation signals and suggested that Irx3 has a critical role in oocyte integrity. AIM 1 is designed to discover the roles for Irx3 in the oocyte and uncover downstream targets and regulation profiles over time in ovary development. Ovarian histology and follicle counts identified oocyte deficits, but reproductive data suggested that other factors were also contributing to the subfertility phenotype in Irx3 knockout mice (Irx3 KO). Indeed, ablation of Irx3 in female mice resulted in a subfertile phenotype with fewer pups born in each litter. Our studies indicated a marked induction of Irx3 at the onset of decidualization and we observed a progressive loss of embryos starting at the decidual phase and extending through the end of pregnancy indicating a disrupted uterine response to the embryo that caused deficits in placentation. AIM 2 will test the hypothesis that Irx3 integrates stromal-endothelium and stromal-trophoblast interactions to ensure successful establishment of pregnancy. Finally, we observed punctate staining for IRX3 within cytoplasm of oocytes and uterine decidual cells. Based on our observations that IRX3 plays an important role in mediating cell-cell interactions, AIM 3 is designed to examine cytoplasmic signaling and localization of cytoskeletal machinery with respect to IRX3 presence. Altogether, our studies are designed to discover how Iroquois homeobox proteins mediate germline nest breakdown and primordial follicle formation to establish responsive follicles with healthy oocytes and decidualization that promotes a successful implantation and placentation program. Successful outcomes will increase our understanding of the biology that results in a healthy ovarian reserve and optimal uterine conditions for early embryo survival.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Each year, nearly 20,000 patients in the United States are diagnosed with acute myeloid leukemia (AML), and 5-year overall survival rates remain dismal despite high intensity chemotherapy and, in many cases, stem cell transplant. Success of chimeric antigen receptor (CAR) T cell therapy in patients with B cell malignancies has prompted intense interest in applying this revolutionary type of immunotherapy to AML. However, clinical translation has been limited by overlap of AML target expression on indispensable, healthy tissues, which increases their susceptibility to CAR T cell-mediated cytotoxicity. Endothelial cell toxicity is of particular concern for some AML targets under investigation. To advance CAR T cell therapy for AML, it is critically important to devise strategies to preserve anti-leukemic efficacy while simultaneously sparing normal tissue from on-target, off-tumor toxicity. The central hypothesis of this proposal is that antigen-specific inhibitory CARs (iCARs) can be engineered to modulate CAR T cell activation signals and will decrease on-target, off-tumor toxicity of AML CAR T cells. Guided by strong preliminary data, this hypothesis will be tested with three specific aims: 1) Identifying optimal inhibitory motifs to incorporate into iCAR-containing NOT-gated CAR T cells; 2) Defining mechanisms by which those inhibitory motifs within iCARs can interrupt CAR T cell signaling using advanced proteomic techniques; and 3) Determining endothelial-specific NOT-gate surface targets by integrating transcriptomic and proteomic data. Key innovations of this proposal include implementation of a targeted screen to identify a best- in-class iCAR that will be generalizable across targets and application of mass cytometry (CyTOF) to interrogate fundamental inhibitory signaling mediators in CAR T cells. While the immediate focus of this proposal is designing a NOT-gated CAR for AML, principles defined by these experiments will provide the framework for applying this technology broadly to other tumor types. The proposed research activities are part of a comprehensive career development plan that will build on past expertise of the applicant and are crucial to her development as an independent investigator focused on translational immunotherapy. In particular, the applicant will gain expertise in high dimensional proteomics and bioinformatics by taking advantage of the world class scientific and mentorship environment at Stanford University. The applicant will be mentored by Dr. Crystal Mackall, renowned for her work on fundamental CAR T cell immunology and translational immunotherapy, and co-mentored by Dr. Ravi Majeti, an expert on therapeutic targeting of AML stem cells. Under their guidance, along with the assembled advisory committee (including Drs. Kara Davis, Ansuman Satpathy, and Kenneth Weinberg) and specific educational plan, the applicant will receive the necessary support and resources to accomplish the proposed aims and efficiently transition to independence following the K08 training period.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT CT imaging is one of the primary diagnostic tools utilized in modern radiology departments, and its importance to modern medicine cannot be overstated. In recent years, spectral CT technologies have been developed to address one of the long-standing technical limitations associated with conventional single-kV CT imaging: anatomical structures with different material compositions may have the same CT number for a given acquisition. Current spectral CT imaging systems have been implemented using advanced x-ray source and/or detector technologies that enable image objects to be rapidly scanned using two distinct x-ray spectra. However, these hardware-based spectral CT systems are not without their own intrinsic limitations. Such limitations include a reduced field-of-view, slower scan speeds, misregistration between the high kV and low kV measurements in dual source systems, lack of tube current modulation for many fast kV-switching systems, and having other clinical workflow/efficiency challenges. In this application, we propose to implement and translate a novel single- kV spectral CT imaging method for abdominal CT applications that overcomes the limitations associated with current spectral CT imaging methods. With this new method, all conventional contrast-enhanced single-kV abdominal CT exams can be used to generate a deluxe series of CT images with the desired spectral CT functionalities including virtual non-contrast CT images, virtual mono-energetic CT images, and quantitative material basis images (i.e., one-stop-shop). This new method enables an integrated clinical workflow to improve clinical diagnostic accuracy while reducing both radiation dose and contrast dose to patients, all while reducing overall healthcare costs. Three specific aims will be carried out to accomplish the overarching objective of this project: 1) Implement and optimize techniques to achieve one-stop-shop single-kV spectral CT imaging for abdominal applications; 2) Validate the proposed one-stop-shop single-kV spectral CT imaging method using the hardware based spectral CT imaging methods; and 3) translate the one-stop-shop single-kV spectral CT imaging method to the clinical environment for clinical performance evaluations. Upon the completion of this project, a unique one-stop-shop CT imaging paradigm will have been implemented and translated to clinical abdominal CT exams to enable one-stop-shop abdominal CT diagnoses with just a single-kV contrast-enhanced CT acquisition. This new technique can be made available to all types of imaging facilities including community hospitals or clinics in developing nations, which is in stark contrast to hardware dual-energy CT and photon counting CT imaging which has been traditionally reserved to high profile academic medical centers that can afford the latest technological platforms.

NIH Research Projects · FY 2026 · 2022-09

Theranostic isotope pairs—combining diagnostic and therapeutic radionuclides that target the same biochemical pathway—are transforming personalized medicine. Diagnostic imaging (via SPECT or PET) enables non- invasive disease localization, patient stratification, and treatment monitoring, while therapeutic isotopes deliver targeted cytotoxic radiation. Clinically, these pairs have improved treatment efficacy, reduced side effects, and enhanced patient outcomes. The positron-emitting rare earths 44Sc (Eβ⁺ avg = 632 keV, t1/2 = 4 h) and 86Y (Eβ⁺ avg = 660 keV, t1/2 = 14.7 h), along with the beta-emitting 177Lu (Eβ⁻ avg = 134 keV, t1/2 = 159.6 h) and 161Tb (Eβ⁻ avg = 154 keV, t1/2 = 166 h), have shown growing clinical impact and global demand. These isotopes are accessible: PET isotopes can be produced in commercial low-energy cyclotrons, and therapeutic isotopes are already available from high-flux reactors. To fully realize their potential, next-generation chelation strategies are urgently needed. Current radiochelation methods are incompatible with one-step, kit-based radiosynthesis or require different chemical precursor materials to accommodate isotopes of interest. This challenges clinical adoption, patient access and prevents accurate prediction of therapeutic dosing regimens based on diagnostic imaging. During the previous funding period, our research group has pioneered coordination chemistry approaches that are compatible with one-step, room temperature radiosynthesis of 44Sc, 86Y, 177Lu and 161Tb chelates, while simultaneously accommodating aqueous radiofluorination with the clinical PET isotope 18F (E<β+> = 476 keV, t1/2= 1.82 h). Together, this establishes an unprecedented theranostic pentad consisting of 18F, 44Sc, 86Y, 177Lu and 161Tb. Building on these advances, we propose a systematic, coordination chemistry-driven strategy to create functional radiopharmaceuticals for the F/Sc/Y/Lu/Tb pentad. Our specific aims are to: 1.) Identify chemical linkage strategies to conjugate cancer-targeting peptides without disrupting favorable radiochelation properties; 2.) Implement a photochemical solid-phase radiosynthesis platform to afford ready-to-inject radiopharmaceuticals 3.) Apply self-immolative amide bond chemistry to fine-tune pharmacokinetics through selective, in vivo hydrolysis. Validation of each Aim will be pursued using relevant preclinical mouse models of prostate, neuroendocrine, pancreatic cancer/glioblastoma.

NIH Research Projects · FY 2024 · 2022-09

Project Summary Toxoplasma gondii is the causative agent of toxoplasmosis, a leading cause of death due to foodborne illness that causes serious disease in immunocompromised individuals. The parasite develops asexually and sexually, and asexual parasite development is well-studied. However, the parasite completes its sexual stage only in the cat intestinal epithelium, presenting a significant ethical and logistical barrier to sexual stage research. To remove the need for companion animal research and study the unknown biology of sexual stage T. gondii, our lab developed murine and tissue culture models that support T. gondii sexual development. The polyunsaturated fatty acid linoleic acid is a critical factor for T. gondii to complete sexual development that is uniquely elevated in the feline intestine. The mechanisms by which linoleic acid acts on host or parasite remain unknown. The goal of this proposal is to determine the mechanism of linoleic acid in promoting T. gondii sexual development in cell culture and in vivo. Preliminary data from our group and others suggests that linoleic acid acts on both host and parasite to promote a permissive environment for T. gondii sexual development. I hypothesize that intracellular accumulation of linoleic acid activates T. gondii lipid signaling pathways to promote sexual development. I further hypothesize that sexual development relies on activation of similar signaling pathways in the host. In Aim 1, I will determine the mechanism of linoleic acid action on the parasite by measuring parasite transcriptional responses to linoleic acid treatment. I will also test our new mouse model of linoleic acid accumulation for its ability to promote efficient T. gondii sexual development in vivo. In Aim 2, I will use automated image analysis to determine the importance of host cell type in T. gondii sexual development. siRNA-mediated ablation of linoleic acid-responsive host factors will identify host genes that influence sexual development. Successful completion of these Aims will better define how linoleic acid enables growth of T. gondii sexual stages. The long-term implication of this work is a shareable model of T. gondii sexual development that will help reduce the burden of toxoplasmosis.

NIH Research Projects · FY 2025 · 2022-09

Project summary/abstract Spatial organization and temporal dynamics are inherent properties of eukaryotic cell signaling pathways. Dynamic changes in the subcellular localization of proteins are programmed by post-translational modifications (PTMs), such as phosphorylation and proteolysis, which occur in response to biological stimuli. Eukaryotic signaling pathways rely on the introduction of PTMs to specific proteins to rapidly change protein function and localization, enabling cells to respond to changing internal or environmental conditions. However, despite the importance of spatial organization and temporal dynamics in biological signaling, current technologies are unable to provide a systems-level experimental mapping of the dynamic subcellular localization of post-translationally modified proteins. To meet this challenge, we propose to develop new methods for PTM proteomics with subcellular spatial and temporal resolution by engineering genetically targetable, PTM-selective proximity labeling enzymes. These enzymes will tag post-translationally modified proteins in specific subcellular locations, enabling their enrichment and analysis with mass spectrometry-based proteomics experiments for mapping PTMs with spatial and temporal resolution. We will initially focus on two pervasive PTMs, proteolysis and phosphorylation, both of which play critical roles in numerous biological signaling pathways relevant to human health and disease. We will apply protein engineering approaches to develop three distinct classes of enzymes for spatiotemporally resolved capture of proteolytic neo-C termini, phosphoserine/phosphothreonine, and phosphotyrosine, respectively. We will deploy these tools to dissect the spatiotemporal dynamics of proteolysis during apoptosis; phosphorylation during growth factor signaling; and crosstalk between proteolysis and phosphorylation during the cellular decision between life and death. The biological pathways and states that can be probed with the tools that we will develop are nearly limitless, ensuring that they will have a broad and transformative impact across the biomedical sciences. Completion of the proposed work will transform our understanding of how cellular signaling unfolds across space and time and has the potential to reveal new paradigms for therapeutic intervention in PTM-based signaling pathways.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Collective cell migration is the most prevalent form of cell migration in the body; it is involved in the construction and repair of almost all human tissues. Previous work by the PIs has provided novel insight on the hierarchy of individual cell behaviors and intracellular signals that drive collective migration. This work identified activation of the intracellular signal PLCg1 as strongly correlated with increased collective migration and cell migration persistence. The proposed study aims to elucidate the mechanisms by which PLCg1 drives collective migration and will yield both novel tools and knowledge that may be applied to control this process. We will achieve this goal by: 1) Determining the mechanism(s) by which PLCg1 activation yields cytoskeletal remodeling. We will develop a FRET-based PLCg1 reporter and identify the downstream effectors of PLCg1 that drive cytoskeletal rearrangement, a critical component of cell movement. 2) Elucidating the biophysical mechanisms by which PLCg1 activation translates into directed collective migration. We will develop cellular models with varying levels of constitutively active PLCg1 and apply traction force microscopy to analyze how cytoskeletal forces are transmitted to yield collective cell movement. 3) Determining optimal patterns and extent of PLCg1 activation needed for robust collective migration. We will develop an optogenetic approach to stimulate different spatio-temporal dynamics of PLCg1 activation during directed keratinocyte migration. By employing a combination of innovative molecular and biophysical tools, we aim to uncover mechanistic knowledge that allows us to gain control over collective epithelial migration. Identifying the pathway(s) by which PLCg1 activation connects to downstream signals, as well as its spatial and temporal dynamics within the collective sheet, provides opportunities to regulate both healthy and pathological collective migration across a range of tissues and pathologies. Elucidation of these pathways and cues provides an important foundation for targeting specific elements to induce collective movement in conditions where it is impaired (e.g., chronic would healing) or to halt collective movement in conditions where it is pathological (e.g., cancer).

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Birth defects cause tremendous individual, familial, and societal burdens, and the development of targeted prevention strategies has been stymied by biological and etiological complexity. Exemplary of multifactorial birth defects thought to be substantially modulated by the environment are orofacial clefts (OFCs) and holoprosencephaly (HPE), common human malformations of the face and brain. The pathogenesis of both OFCs and HPE is directly linked to embryonic disruption of Sonic hedgehog (Shh) signaling in animal models, supporting a pathway-based investigation of environmental contributions to birth defect etiology. That Shh signaling comprises a multi-step process inherently sensitive to modulation across multiple steps of its signaling cascade makes the pathway especially germane for examining the impact of co-exposures on etiologically complex, multifactorial disease. The studies proposed in this application are designed to test the central hypothesis that structurally diverse Shh inhibitors synergistically interact to reduce pathway activity at the cellular level and exacerbate Shh-associated craniofacial malformations. To test this hypothesis, I have leveraged NIEHS- and EPA-supported high-throughput chemical screens to create a prioritized list of environmentally relevant, putative Shh pathway disruptors and developed a novel Shh pathway-complete cell culture system that is sensitive to inhibition throughout the Shh pathway. I will utilize this system and other mechanism-based in vitro assays to validate bona fide pathway antagonists, delineate molecular targets within the Shh pathway, and evaluate additive and synergistic interactions resulting from co-exposure to compounds with diverse pathway targets. The developmental toxicity of these compounds will then be examined, both individually and in combination, by targeting dose administration to critical periods of Shh pathway activity during craniofacial development. The known Shh antagonists cyclopamine, vismodegib, and piperonyl butoxide will be used throughout the proposed studies as positive controls for pathway-specific effects. These rigorously designed experiments are expected to reveal environmental factors that adversely impact development and elucidate mechanisms of Shh pathway disruption that promote synergistic interactions. In completing the proposed studies, the applicant’s professional development will be advanced through the establishment of proficiency in experimental design, evaluating the toxicity of mixtures, utilizing animal models of developmental toxicology, and effectively communicating scientific concepts and results. These works will also facilitate the applicant’s goal of becoming an independent investigator at a government agency within the United States.