University Of Pennsylvania

NIH Research Projects · FY 2026 · 2024-07

Project Summary/Abstract Direct recording of neural activity from the human brain using implanted electrodes (intracranial EEG, iEEG) is a fast-growing and high-impact technique in human neuroscience. The NIH has mandated data sharing plans, but useful sharing that promotes neuroscience discoveries from archival datasets faces many obstacles. One obstacle is difficulties in creating shareable iEEG datasets. In Aim 1, we will create new tools based on the iEEG-BIDS standard, created in 2019 to promote sharing by providing specifications for iEEG datasets. Aim 1A will create an iEEG-BIDS compliant solution for anatomical iEEG data, localizing electrodes using the pre-operative MRI and post-operative CT scans. Aim 1B will create an iEEG-BIDS compliant solution for functional iEEG data, the voltage by time data collected from each electrode as a measure of neural activity. Aim 2 will address the problem that it is currently very difficult for iEEG investigators to share the results of their iEEG analyses, and difficult for other scientists to explore them. A new tool will merge an advanced 3D Viewer designed for iEEG with the output of an investigator's existing anatomical and functional iEEG pipelines. The tool will export a single, compact package that can be shared directly with other scientists or uploaded to journals or an archive. By simply opening the package in a web browser, users will be able to interactively explore the dataset, facilitating replication, reliability, and new discoveries.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY/ABSTRACT Nonopioid analgesics are highly effective for pain management following most dental procedures, but data suggest that prescription of opioids by dental clinicians is excessive. Prescription opioid misuse remains a significant public health concern in the US, and ongoing opioid crisis has highlighted the need to optimize pain management with non-addictive analgesics, with non-steroidal anti-inflammatory drugs (NSAIDs) at the forefront. Numerous clinical trials have shown that NSAIDs are effective in the treatment of post-surgical pain following third molar extraction. However, there is substantial inter-individual variability in the analgesic response, with 20-30% of patients requiring opioids in addition to NSAIDs to achieve adequate pain relief. The use of precision medicine approaches to tailor analgesic therapy would enable oral surgeons to rationally prescribe opioids to only those patients who require them and avoid unnecessary opioid prescriptions in those patients who can achieve adequate pain relief with NSAIDs alone. However, the factors that contribute to heterogeneity in analgesic response to NSAIDs and need for opioid rescue medication have not been well-studied, and there is limited evidence to inform which patients should receive opioid prescriptions and how much should be prescribed. The work proposed in this application will identify individual host factors associated with need for opioid analgesics, in addition to NSAIDs, following third molar extraction. We will specifically investigate the role of inter-individual differences in regulation of the acute inflammatory response to surgical trauma and seek to identify an inflammatory response signature associated with need for opioid following third molar extraction. We will also investigate the role of neutrophil subtypes in mediating heterogeneity in analgesic efficacy of NSAIDs. Elucidation of the molecular mechanisms underlying inter-individual variability in analgesic efficacy of NSAIDs may allow the identification of biomarkers that are predictive of response, thereby facilitating a precision medicine approach to pain management. This work will provide a foundation for future clinical interventions to better manage pain following third molar extraction, with the goal to limit opioid prescriptions to only those patients not likely to respond adequately to NSAIDs.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Major depressive disorder (MDD) is a leading cause of disability in the United States and worldwide, yet current pharmacologic treatment options are insufficient for many patients. Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive, FDA-approved treatment that utilizes electromagnetic pulses to modulate brain activity, but clinical efficacy is inconsistent, and the mechanism of action is still being determined. Understanding how rTMS alters glutamate (Glu) signaling and functional connectivity (FC) in the Salience Network (SN), a functional brain network that regulates attentional and emotional processing, could pave the way for improving or personalizing this promising intervention. Previous studies using magnetic resonance spectroscopy or resting- state functional magnetic resonance imaging (rsfMRI) at 3T have reported that rTMS increases Glu and FC in the SN. However, methodological limitations contribute to extensive heterogeneity in this literature. This proposal aims to overcome these limitations by applying robust, cutting-edge, ultra-high field (UHF; 7T) neuroimaging techniques, including Glutamate Chemical Exchange Saturation Transfer (GluCEST) and rsfMRI, that confer unprecedented spatial resolution and sensitivity. We aim to elucidate the manner in which rTMS alters Glu (Aim 1) and FC (Aim 2) in the SN, and how this associates with clinical improvement, as measured by our comprehensive psychological assessment. The overarching hypothesis is that the antidepressant effect of rTMS stems from increased glutamatergic activity and FC in key SN regions that regulate activity across functional networks. Our high spatial resolution, multimodal imaging approach will provide unprecedented mechanistic insights into the effects of rTMS and the role of Glu in the pathophysiology of MDD. To further investigate individual differences in treatment response, we will employ an exploratory multivariate model incorporating Glu, FC, and proximity of the stimulation site to the SN (Aim 2.3, exploratory). This project would both advance our ability to predict outcomes for individuals receiving rTMS therapy and build the applicant’s skills in human neuroimaging, neuromodulation, interventional psychiatry, and scientific communication in an environment with clear expertise in these areas. A world-class mentoring team – Dr. David Roalf, Dr. Desmond Oathes, Dr. Theodore Satterthwaite, and Dr. Kristin Linn – will provide complementary advising and resources in research and clinical arenas as the candidate gains independence over the course of the project. With a detailed training plan aligned with innovative scientific aims, this NRSA is the ideal opportunity for the candidate to develop key skills to undertake not only strong experimental and computational work, but also care for patients with mood disorders while effectively tailoring her research to the needs of this patient population as an interventional psychiatrist-scientist.

NIH Research Projects · FY 2024 · 2024-07

The Third Britton Chance International Symposium on Metabolic Imaging and Spectroscopy Project Summary Elucidation of metabolic processes is critical for a wide range of fundamental physiological research and for clinical applications associated with cancer, diabetes, cardiovascular-, skeletomuscular-, lung-, neurological-, and neurodegenerative-diseases, and brain function. Many researchers aim to elucidate metabolic processes, but they do so with the perspective of a particular biomedical field or imaging technology. Unfortunately, few opportunities exist for researchers dedicated to metabolic imaging and spectroscopy to convene outside of their communities. Thus, the goal of this proposed conference is to bring together clinicians, biomedical scientists, and physical scientists from different communities to collaboratively explore key topics in metabolic imaging and spectroscopy, especially as it relates to biomedical and clinical applications. Emphasis will be placed on clinical translation, latest technical developments, and multi-modal metabolic imaging/spectroscopy. The conference program is aligned with the missions and interests of several NIH institutes including the NIBIB, NCI, NINDS, NIA, and NIDDK. Specifically, the conference will feature: 1) In-depth methodology sessions that focus on developing tools for the study of biological/physiological processes such as hemodynamics, glycolysis, mitochondrial bioenergetics, hypoxia and redox state; these processes are common underlying factors in many diseases; 2) Application sessions focused on the connections between metabolism and pathology in cancer, brain, muscle, and visceral organs including heart/lung/liver/kidney; 3) Panel discussions to identify critical research questions, innovations, clinical translation needs, and regulatory issues (e.g., IRB, IND, etc.); 4) A workshop on career development and grant writing to help investigators, particularly new investigators; 5) Enhanced participation of junior researchers (students, postdocs, research associates, and early-stage investigators) facilitated by travel stipends, flash-highlight talks, poster awards, round-table small group discussions with senior researchers; 6) Enhanced diversity by encouraging participation of minorities and women with dedicated travel scholarships. Dr. Chance was a founding father of both in vivo NMR Spectroscopy and Biophotonic Imaging/Spectroscopy, and he was a pioneer in the study of metabolism and physiology. He remains inspirational for many researchers working in different subfields, due to his emphasis on cross-disciplinary research exchange, innovation, and clinical translation, and his efforts to build the scientific community by tirelessly educating and helping students and young researchers. Branding this event as a “Britton Chance” symposium will help increase conference visibility within this diverse research community; indeed, >200 people participated in each of the first two symposiums (2013, 2018). By reflecting and carrying on the scientific spirit of Dr. Chance, we anticipate that this proposed third symposium will stimulate innovation, collaboration, and deeper involvement of junior researchers in the field. After the symposium, both review and research manuscripts will be solicited and published in focused issues of two peer-reviewed journals.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting over 10 million people worldwide and contributes to over $52 billion yearly in US healthcare costs. Despite investigation into mechanisms of α-synuclein (aSyn) misfolding and aggregation leading to neuronal dysfunction and death, the inciting events of the hallmark of PD pathology in humans are not understood. The systemic manifestations of PD have led to the discovery of αsyn pathology in multiple peripheral tissues, including plasma. Understanding the origin of peripheral aSyn pathology is critical to advancing the understanding of PD and to developing early disease-modifying therapies. In preliminary work, antibodies generated against in vitro strains of aSyn are detectable at higher levels in individuals with PD (iwPD), reliably discriminating them from individuals with Dementia with Lewy bodies, when no other differentiating biomarkers exists Furthermore, one of these aSyn strains is elevated in plasma from iwPD who have a reduced rate of cognitive decline. These strains are not reliably detected in cerebrospinal fluid and do not correlate with levels of strain-specific aSyn found in white or red blood cells implicating an alternative peripheral tissue source. The goals of this proposal are 1) to use extracellular vesicles, membrane-bound structures secreted by cells, to identify the tissue source of plasma aSyn strains in an unbiased manner and 2) determine the functional and pathogenic properties of plasma aSyn strains in biochemical assays and a cellular model of PD. In line with the mission of the NINDS, this work has the potential to improve understanding of fundamental mechanisms of PD pathogenesis and to lead to development of treatments that reduce PD morbidity. Dr. George Kannarkat is a driven, highly qualified physician-scientist who is currently an Instructor and participating in the “Remapping Clinical Neuroscience through Translation and Innovation Training” T32 training program at the University of Pennsylvania. He has a proven track record of productivity in basic, translational, and clinical research, particularly with a background in peripheral and immune mechanisms of neurodegeneration. This five-year mentored K08 award including mentorship, formal and self-directed training, and a rigorous research plan will allow him to broaden his skills in 1) large-scale nucleic acid and proteomics analysis, 2) models of synucleinopathy, and 3) manipulation of induced pluripotent stem-cells. With strong institutional support from the numerous resources at the University of Pennsylvania and the mentorship from Dr. Alice Chen-Plotkin (mentor), a world-renowned expert in bioinformatic approaches to identifying biomarkers of neurodegeneration, and his advisory committee, he is well-poised to achieve his goal of being an independent R01-funded physician-scientist studying the interplay of peripheral and central mechanisms, such as inflammation, environmental exposures, and proteinopathy, leading to neurodegeneration

NIH Research Projects · FY 2025 · 2024-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Type 1 diabetes (T1D) is an autoimmune disease triggered by genetics and environmental factors. In particular, the gut microbiota has been implicated in T1D pathogenesis. Bacterial colonization modifies islet autoimmunity in mice. Changes in microbial composition and diversity have also been reported in children who developed T1D. Microbes have been proposed to drive autoimmunity via molecular mimicry, where microbial exposure triggers an inappropriate response from self-reactive T cells recognizing a similar appearing microbial antigen. However, autoreactive T cells and cross-reactive recognition are not limited to disease; they are also present in a healthy immune repertoire. How autoimmune pathology arises from these physiologic interactions remains unknown. In collaboration with the NIDDK Human Pancreas Analysis Program (HPAP) at the University of Pennsylvania, we examined CD4+ T cells isolated from the duodenum and pancreatic islets of recent onset T1D and healthy organ transplant donors. CD4+ lamina propria lymphocytes (LPL) in T1D patients, including those recognizing commensal bacteria, exhibited altered responses to stimulation. Concurrent TCR sequence analyses on commensal-reactive T cells further showed a broader pattern of antigen-recognition in T1D patients. Based on these findings, we hypothesize that T cells in autoimmune patients are more cross-reactive, potentially enabling inappropriate commensal responses to target self-antigens. Because T1D-associated gene signatures can be detected before clinical diagnosis and are present in circulating T cells, we further propose that tissue pathology can be detected in peripheral blood to offer new opportunities for early detection and intervention. In Aim 1, we will define cross-reactivity between microbial and self-antigens and test the hypothesis that T1D and at-risk individuals have a broader breadth of T cell recognition. In Aim 2, we will build on our findings in the LPL to identify key properties of gut-associated T cell responses in the blood. Our goal is to identify novel cellular biomarkers for T1D. PBMCs from TrialNet, collected at various stages of islet autoimmunity before disease onset, will be used to identify key immune signatures that can predict disease onset and progression. Data generated from this study will provide fundamental knowledge on the underlying immune pathology in T1D and may uncover new biomarkers for islet autoimmunity. The proposed research will therefore have a broad impact on T1D research.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Insomnia is the most common sleep disorder and negatively impacts daily functioning, quality of life, and work productivity. Despite the high prevalence of insomnia, particularly in women, and >40 years of extensive research efforts, the field still lacks a solid understanding of how insomnia should be assessed and defined. For example, polysomnography (PSG) has failed to yield a consistent pattern of objective impairment and, in fact, often fails to distinguish individuals with insomnia from good sleepers. Clinical diagnosis of Insomnia Disorder is therefore based solely on subjective report of difficulty initiating or maintaining sleep (DIMS) associated with perceived distress or impairment. Earlier diagnostic editions differentiated multiple subtypes based on patterns of clinical symptoms that failed to demonstrate reliability and validity and have since been abandoned. Insomnia is now operationalized as a monolithic subjective entity despite the likelihood that it is instead a heterogeneous disorder with multifactorial etiology and presentation with complexity akin to depression. Heterogeneity significantly reduces the likelihood of understanding the pathophysiology of insomnia or realizing a goal of personalized insomnia treatment. We propose that the time is ripe for a reconceptualization that combines knowledge about subjective and objective DIMS with mechanistic factors known to be associated with insomnia: hyperarousal and circadian misalignment (i.e. sleeping out of phase with endogenous rhythms). To that end, this proposal seeks to revisit the operationalization of insomnia to derive a classification system with high reliability and validity. We will recruit n=400 individuals with subjective DIMS, defined as sleep onset latency, wakefulness after sleep onset, and/or early morning awakenings >30 minutes. Following home sleep apnea screening, in depth phenotyping will assess four constructs of insomnia: 1) subjective DIMS based on sleep diaries and retrospective self-report; 2) objective DIMS with actigraphy and home PSG; 3) physiologic (heart rate variability), cognitive (self-report) and cortical (beta EEG) hyperarousal; and 4) circadian misalignment defined as the phase angle between dim light melatonin onset and midpoint of sleep along with subjective and actigraphic measures of rhythmicity. Structural equation modeling and latent profile analysis will be used to both derive latent factors underlying each dimension (dimensional model) and to categorize individuals into distinct subgroups based on these inputs (categorical model). Reliability will be examined in a subset (n=200) by conducting a second assessment one month later to assess for temporal stability of categories and dimensions. Concurrent validity of each dimension and category will be assessed by examining associations with validators of subjective distress, mental health symptoms, quality of life, neurocognitive performance and peripheral inflammation.

NIH Research Projects · FY 2026 · 2024-07

Abstract The proposed work in this MIRA application focuses on my long-term goal to create programmable toolkits for proteome editing. Inspired by research into developing broad-targeting CRISPR enzymes for programmable genome editing, my laboratory designs analogous peptide-guided enzymes for post-translationally modifying target proteins from sequence alone, enabling targeting of both structurally-stable and disordered proteins. To this point, we leverage generative artificial intelligence approaches to design these peptides, including protein language model (pLM)-based interface predictors and diffusion models for de novo peptide binder generation. Integrated with these efforts, we experimentally engineer peptide-E3 ubiquitin ligase fusions (termed ubiquibodies or uAbs) that can degrade diverse pathogenic proteins. Building upon this work, we will combine our current technologies and expand our research into three new areas to enable broad-scale proteome editing applications via generative protein language modeling. First, we will build upon our current experimental efforts to enable peptide-guided deubiquitination, thus enabling both protein degradation with our uAbs and now protein stabilization of target proteins. To accomplish this goal, we will explore fusion of generated peptide binders to deubiquitinase domains, as well as to designed recruiter domains of endogenous deubiquitinases. The results of this research will establish easy-to-design “on” and “off” switches for controlling protein states in cells. Second, we will develop a new line of investigation, specifically to enable mutant-specific targeting of protein variants. Here, we will leverage the variant prediction capabilities of state-of-the-art pLMs, alongside Gaussian Process-based predictors, to prioritize binders that discriminate between wild-type proteins and highly-similar mutant variants that confer pathogenic properties. These binders can then be integrated into our degradation and stabilization architectures for experimental validation. We envision that this new line of investigation will enable selective degradation of mutant, disease-causing proteins, such as mutant Huntingtin protein or mutant KRAS. Third, we will establish a specific focus on selectively targeting dysregulated, post-translationally-modified proteins, which have numerous implications in various diseases, such as aging and cancer. To enable discrimination between wild-type proteins and their modified counterparts, we will train new PTM-specific protein language models via both masked language modeling and autoregressive training tasks. By integrating SwissProt annotations with unique per amino acid-based tokenization strategies, the results of this research will enable the design of degraders that are selective to modified proteins, such as phosphorylated oncogenic drivers, leaving wild-type isoforms intact. Together, the work we propose in this MIRA application promises to yield robust, scalable, and most importantly, programmable tools for protein targeting and editing applications.

NIH Research Projects · FY 2026 · 2024-07

Project Summary Sepsis is a syndrome characterizied by a dysregulated host immune response to an infection that leads to organ dysfunction. Because sepsis is among the leading causes of death among hospitalized patients and accounts for substantial harms, costs, and loss of quality of life, many efforts have been made to improve sepsis care. The mainstay of treatment is timely recognition and prompt initiation of broad-spectrum an- timicrobial therapy. However, identification of sepsis is fraught with uncertainty in busy and complex clinical environments and treatment delays are common in the emergency department, hospital ward, and intensive care unit. As a result, the use of artificial intelligence (AI) and machine learning (ML) methods to provide timely clinical decision support (CDS) has good face validity to improve care. Despite hundreds of published papers on predictive sepsis systems, there is very little evidence that such systems actually improve care processes or patient outcomes. Therefore, this proposal outlines three important knowledge gaps at the in- tersection of AI/ML methods and clinical care that have so far hindered the development of successful sepsis CDS systems. First, the optimal outcome (i.e., training label) on which to develop predictive systems for sep- sis is unknown. Current sepsis definitions were designed primarily to standardize clinical trial enrollment and epidemiologic surveillance rather than to support bedside treatment decisions. Second, although sepsis is currently defined by changes in organ function from baseline, the optimal approach to capture time-varying changes in clinical parameters remains unknown. Many AI/ML methods are uniquely suited to learning such important patterns in the data but their use in predicting sepsis remains under-explored. Third, there are significant differences in patient outcomes and clinical presentation between community- and hospital-onset sepsis. However, how these differences might affect predictive accuracy, estimates of variable importance, timing and use of predictive alerts, among other important considerations for CDS development, remains un- known. Thus, this proposal seeks to answer these fundamental questions to overcome key knowledge gaps and realize the promise of AI/ML methods for improving sepsis care. Broadly speaking, we will consider sev- eral state-of-the-art approaches to answer these questions, including the use of i) informatics methods such as active learning to facilitate efficient and large-scale clinician review of patient data, ii) advanced causal inference methods such as target trial emulation to compare the clinical effects of treatment according to different sepsis definitions, and iii) AI/ML methods such as convolutional neural networks and denoising autoencoders to determine the optimal representations of complex and time-varying clinical features. An- swering these questions will pave the way for the development of AI/ML CDS systems that are relevant at the bedside, scalable across a diverse range of care contexts and patient populations, and most importantly that improve clinical care and patient outcomes.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Ineffective pain management is an urgent medical crisis impacting the lives of over 50 million Americans and millions more chronic pain patients around the world. Opiate analgesics can provide robust pain relief but can also produce life-threatening side effects and high rates of misuse, which contributes to the ongoing Opioid Epidemic. Unlike the off-target effects of opiates that act widely throughout the body to indiscriminately bind mu-opioid receptors (MOR) on many cell-types, endogenous opioid peptides mediating antinociception undergo controlled release at specific synapses from specific nociception-related cell-types. Identifying the precise noci-ceptive cell-types that express MORs, which are regulated by endogenous opioids, may lead to new research developments for effective circuit-targeted analgesic treatments to minimize the need for traditional opiate anal-gesics and reduce abuse liabilities. The rostral intralaminar thalamus {rlLN) is critical in this regard as it is known to relay nociceptive information from spinal cord and brainstem structures to cortical regions, such as the rostral anterior cingulate cortex (rACC). rlLN neurons express high densities of MORs {rlLNMOR), yet it remains unknown whether endogenous forms of pain relief modulate nociceptive activity within this rlLNMOR ➔ rACC circuit. To enhance our investigations into thalamocortical circuits in endogenous analgesia processes, we have developed an operant conditioning assay that leverages expectation-induced antinociception, i.e. placebo analgesia, in a drug-free manner. The F99 phase entails two subaims. In Aim 1a, the applicant will leverage in vivo fiber photometry calcium imaging to determine nociception and analgesia-related responses in the axonal projections of rlLNMOR ➔ rACC. In Aim 1b, to determine the necessity of MOR signaling in thalamocortical neurons for the endogenous analgesia response, the applicant will use an intersectional genetic and viral approach to selectively delete MORs from rlLN ➔ rACC neurons. The KOO phase, Aim 2, will help prepare the applicant for a successful academic research career investigating neurobiological mechanisms of gastrointestinal pain. This postdoctoral period will provide the applicant with expertise in techniques like 2-photon calcium imaging of neural population dynamics in the brain during visceral pain models, as well as sharpen managerial and mentoring skills necessary to succeed and thrive as an independent research scientist. In total, successful completion of this proposal will provide insight into neurobiological mechanisms underlying pain and analgesia, while also equipping the applicant, Lindsay Ejoh, to uncover brain circuit mechanisms of severe and chronic gastrointestinal pain disorders under her own independent research program.

NIH Research Projects · FY 2025 · 2024-07

SUMMARY This proposal aims to translate our engineered extracellular vesicles (EVs) into an effective cancer therapy. Major barriers to effective chimeric antigen receptor (CAR)-T cell therapy in solid cancers include limited CAR-T cell tumor infiltration, loss of their function in the tumor microenvironment (TME), and severe life-threatening toxicities. Designer EVs from cytotoxic immune cells have the potential to overcome these crucial issues and become effective cell-free immunotherapy for solid cancers. In response to PAR-22-071, we will optimize our designer EVs derived from engineered Nature Killer (NK) cell line NK92 to treat melanoma, the deadliest skin cancer. Death Receptor 5 (DR5) is highly expressed in cancer cells, myeloid-derived suppressor cells (MDSCs), and cancer-associated fibroblasts (CAFs). MDSCs and CAFs are the major components of the immunosuppressive TME. We fortuitously discovered that DR5-agonistic single-chain variable fragments (scFvs) were highly effective in killing DR5+ tumor cells when expressed on non-cytotoxic lymphoblastic lymphoma Sup-T1 cells. We developed a new lentiviral vector to deliver more DR5-scFvs to the surface of EVs and infected NK92 cells with the vectors. EVs from the engineered cells (DR5-scFv EVs) were more effective in killing DR5+ melanoma than DR5 antibodies and DR5-CAR-T cells. Systemic delivery of DR5-scFv EVs significantly inhibited DR5+ tumor growth in vivo in multiple cancer models. A unique feature of DR5-scFv EVs is that they significantly inhibited MDSCs and CAFs in vitro and in vivo and increased CD8+ T cell functions in patient-derived organotypic cultures. Loading siRNA to mutant NRAS into DR5-scFv EVs further enhanced their tumor-killing effect on melanoma with mutant NRAS. We will further develop DR5-scFv EVs for clinical translation. In Aim 1, we will optimize cytotoxicity and immune stimulatory functions of DR5-scFv EVs using different approaches and test these designer EVs in state-of-the-art models, such as patient-derived organotypic melanoma cultures and genetically annotated melanoma patient-derived xenografts (MPDXs). Aim 2, we will test the designer EVs in mouse clinical trials using genetically annotated MPDXs in humanized mice. In Aim 3, we will optimize bioprocessing and standardize workflow for the engineered EVs production to increase DR5-scFv-EVs yields and develop Standard Operating Procedures (SOPs) for upstream production, downstream process, and quality controls. This proposal will prepare our designer EVs for GMP production and to be explored as an alternative therapy for CAR- T cells. We expect that our designer EVs will be qualified for NCI translational programs to continue a path toward the clinic upon completing the proposal.

NIH Research Projects · FY 2025 · 2024-07

PROJECT ABSTRACT: Diffuse midline glioma (DMG) is a universally lethal form of brain cancer that affects both children and adults, with most patients passing away within 1 year of diagnosis following standard of care radiation therapy. Not a single drug has been FDA approved despite a multitude of clinical trials, with most agents tested not designed specifically for this disease. This highlights the need for identification of therapeutic targets directly linked to the pathogenic mechanism in DMGs. The hallmark of DMGs are dominant negative mutations in histone H3, a key structural and regulatory constituent of the chromatin structure in which DNA is packaged. Mutant H3 drives tumorigenesis by promoting transcriptionally silent chromatin at specific tumor suppressor genes including CDKN2A. How transcription is silenced at these genomic sites is poorly understood and is a key knowledge gap in the field. Our preliminary data implicates a mechanism in which mutant H3 induces silent chromatin at tumor suppressor genes by hijacking the activity of a specific configuration of the polycomb repressive complex 1 (PRC1). PRC1 is an epigenetic silencing complex with numerous protein subunits which can adopt highly heterogeneous configurations (>150 combinations), making it particularly challenging to study. Leveraging pooled CRISPR screens integrated with quantitative proteomics, we have implicated a PRC1 complex containing chromobox 4 (CBX4-PRC1) as the mediator of inappropriate transcriptional silencing in DMGs. Our central hypothesis is that CBX4-PRC1 is co-opted in DMGs to drive tumor growth by transcriptionally repressing tumor suppressor genes including CDKN2A. The goal of this proposal is to define CBX4-PRC1 as the functional configuration which mediates inappropriate transcriptional silencing in DMGs and determine whether targeting CBX4 can be combined with standard of care radiation treatment in DMG models. We will leverage our strong preliminary data, multi-disciplinary team, and novel patient-derived DMG models to test our hypothesis through the following aims: 1) Define the functionally essential PRC1 complex configuration in DMGs. 2) Elucidate the role of CBX4 in PRC1 chromatin recruitment and silencing function in DMGs. 3) Evaluate the therapeutic potential of targeting CBX4 in conjunction with standard of care radiation therapy in DMGs. This proposal will lay the mechanistic framework for CBX4 as a novel therapeutic target in DMGs, a currently incurable brain cancer. We will reveal the specific mechanism by which mutant H3 promotes inappropriate transcriptional silencing to drive DMG growth and determine the relevance of this mechanism across DMG subtypes to guide patient selection in the future.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT Preexisting obesity and non alcoholic steatohepatitis (NASH) are strongly associated with COVID- 19 cytokine storm and severe outcome. SARS-CoV-2 virions are present in the patient adipose tissue and liver at low levels and it is not known if they play a major functional role in severe COVID-19 pathology. The Raabe lab has derived for the first time organoids from livers of patients with NASH and shown that they are senescent and exhibit a senescence associated secretory phenotype (SASP) and thus model the known senescence of hepatocytes and other liver cells in end stage NASH liver. Further, SARS-CoV-2 has been shown recently to induce senescence and an SASP in infected lung cells that spreads through senescence associated secretion of chemokines and cytokines including chemokine CXCL10, interferon IFN and cytokine TNF to attract macrophages and activate them to a pro inflammatory state. The Raabe lab will study the hypothesis that genetic or chemical removal of senescent cells in SARS-CoV-2 infected obesity or NASH mouse models or in human adipose tissues derived cells and NASH patient liver derived organoids will alleviate NASH related severe COVID-19 symptoms. In Aim 1 we will induce obesity and NASH senescence in mouse models and infect these with SARS-CoV-2. Prior or after infection we will remove senescence promoting factor p16 expressing cells either genetically or chemically and then determine if this alleviates severe obesity or NASH COVID-19 symptoms. In Aim 2 primary human lung cells will be infected with SARS-CoV-2 and the supernatant containing the SASP and virus will be added to primary human adipocytes or NASH liver derived organoid cultures. We will remove senescent cells chemically using established senolytics to study if their removal alleviates inflammatory responses. Readout will be RNA-Seq, IHC and ELISA to study the effect on senolytics on the SASP.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY / ABSTRACT Research Plan: Tumor evolution is a fundamental obstacle to cancer treatment, as malignant cells adapt to treatment and change during progression. Understanding these adaptations is key to not only identifying vulnerable gene targets, but also intervening during the optimal time window, before the targets become obsolete. Charting this evolution requires understanding the genetic and epigenetic events that drive or accompany disease progression, but these are largely unmapped in many malignancies. A case in point is diffuse glioma, an incurable brain cancer composed of distinct cellular states that dramatically change in relative abundance during glioma progression, ultimately changing the response to treatment. Efforts to decode glioma evolution have been limited: while we have characterized the distinct glioma cell states, we have not found their genetic and epigenetic drivers or measured how these affect cell states abundance throughout progression. To this end, we have developed novel single-cell technologies to concurrently profile in the same cell i) the transcriptome and epigenome (specifically, DNA methylation) and ii) the transcriptome and genome (DNA mutations) at an unprecedented detection rate. First, by combining genotype with cellular state data, I will define how somatic mutations dysregulate transcriptional patterns, resulting in cellular states (Aim 1a), and characterize how somatic mutations dictate glioma lineage structure (Aim 1b). By combining this with my postdoctoral work – characterizing how epigenetic diversification contributes to transcriptomic diversity – I will create the first multi-dimensional model of glioma evolution. Second, I will use our method to measure single-cell DNA methylation and transcriptional profiles in samples taken longitudinally from the same patients. This will test the hypothesis that dysregulated epigenetic patterns contribute to glioma progression, and it will not only provide a multi-dimensional model of glioma progression, but will also provide epigenetic markers to track progression in the clinic (Aim 2). Finally, I will improve diagnosis in the clinical setting by designing a tool that infers cell state composition, progression and likely response to treatment based on data from the bulk DNA methylation assay that is routinely done in the clinic (Aim 3). These studies will pave the way towards novel glioma treatments, accurate diagnoses and optimal timing of treatment. Career Development Plan: I have outlined a 5-year career development plan to meet my goal of becoming an independent investigator in cancer biology who focuses on intrinsic determinants of tumor progression and response to treatment. I have assembled a Mentorship Committee of leaders in the field, with whom I will train to close remaining knowledge gaps in multi-omics evolution and how to leverage my results to support clinical care. Finally, the Broad Institute constitutes the ideal environment for attaining my scientific and career goals, given their outstanding research communities and track records of training independent scientists.

NIH Research Projects · FY 2025 · 2024-07

ABSTRACT The use of checkpoint inhibitors has altered the treatment landscape in oncology, leading to cures in previously untreatable diseases. Checkpoint inhibitors block the function of the PD-1 and CTLA4 inhibitory receptors on T cells to enhance the activation of T cells, particularly tumor-specific lymphocytes. However, non-specific immune activation has no antigenic specificity and also induces immune-mediated adverse events (irAE) that resemble spontaneous autoimmunity and involve any and all tissues. IrAEs cause significant morbidity and disruption in the cancer treatment of oncologic patients and, at times, can be life-threatening. However, the occurrence of irAEs is also associated with improved tumor regression. This challenge has created a pressing need to understand the pathogenic mechanism of irAEs to allow for proper therapeutic management and better predictive algorithms to identify patients that may develop these autoimmune toxicities. However, dissecting the immunopathology of irAEs is limited by a lack of understanding of the antigenic targets of the autoimmunity. To address this gap in knowledge, we have established a prospective cohort of patients treated with PD-1 blockade. Unlike cross-sectional studies, our cohort is unique in its (1) size, (2) prospective nature (before and after PD-1 immunotherapy), and (3) longitudinal design (before and after irAE incidence). We find that almost all subjects that develop irAEs after PD-1 inhibition have an increased abundance of naïve CD4 T cells at baseline, as well as more pronounced activation of these naïve T cells following PD1 inhibition. No changes in CD8+ phenotypes correlate with irAE’s. Secondly, we utilized a whole exome autoantigen screening array to identify multiple non- tumor related targets unique to each subject’. Together, these data lead to our parallel hypotheses that 1) immune mechanisms of CD4-dependent autoimmunity are similar between subjects and independent of the target tissue except when 2) the target of autoimmunity is coexpressed in the tumor and the target tissue. In our first Aim, we will focus on the phenotype of the CD4+ T cells involved in the development of post-treatment autoimmune inflammatory arthritis. Single cell multiome assays paired with T cell receptor sequencing will identify and characterize oligoclonal expansions. In parallel, putative autoantigens identified by autoantibody screening will be used in activation-induced marker (AIM) assays to validate the presence of autoantigen-specific CD4+ T cells. We hypothesize that autoantigens are specific to the individual rather than universal; however, the phenotype of the autoreactive CD4+ T cells will be similar amongst individuals. Secondly, we will examine the antigen-specificity and CD4+ T cell responses in subjects with lung cancer that develop pneumonitis or a peripheral irAE. We hypothesize that pneumonitis will reflect preexisting target tissue damage and antigen cross- reactivity with tumor; whereas other tissues reflect de novo autoimmunity induced by PD-1 blockade. These experiments will define the biology of irAEs and establish a pathway for the analysis of antigen-specific autoreactive CD4+ T cells and prediction of AEs.

NIH Research Projects · FY 2026 · 2024-07

PROJECT SUMMARY Coronary Microvascular Disease (CMVD), defined as disease of the coronary pre-arterioles, arterioles, and capillaries, accounts for 30-50% of the burden of Ischemic Heart Disease (IHD). However, little is known about the pathogenesis and there are no targeted therapies. Friend of Gata 2 (FOG2) is a cardiomyocyte transcriptional co-regulator which is crucial for both the development and maintenance of the coronary microvasculature. FOG2 promotes expression of angiogenic genes and inhibits expression of anti-angiogenic genes, however, there are two predominant isoforms of FOG2, and the mechanism by which FOG2 promotes an angiogenic program is not known. Our central hypothesis that cardiomyocyte FOG2S is the isoform which regulates a pro-angiogenic gene program to support coronary microvasculature, via interactions with HIF1a. Our objectives in this proposal are to define the structure-function relationship of FOG2 isoforms and HIF1a and establish the paracrine effects of cardiomyocyte FOG2 and FOG2S on heterotypic vascular cells phenotypes, as outlined in the following two aims. In Aim 1, we will determine the structure-function relationship between FOG2 and HIF1a using co-immunoprecipitations studies and luciferase constructs for angiogenic gene promoters. We will also establish the genome-wide cistrome of FOG2 isoforms. In Aim 2, we will determine the paracrine effects of cardiomyocyte FOG2S on vascular cells in vivo and in vitro. First we use a new isoform-specific inducible knockdown model we have developed to define the role of FOG2S in maintaining the coronary microcirculation in vivo. We then establish the ability of cardiomyocyte FOG2S, as opposed to FOG2, to regulate endothelial and smooth muscle cell proliferation, migration, and angiogenic potential. Our studies will provide novel insight into mechanisms of coronary microvascular homeostasis. The proposal's outcomes will fill a critical gap in understanding the molecular factors maintaining the coronary microvasculature and shed light on mechanisms relevant to a significant portion of IHD.

NIH Research Projects · FY 2025 · 2024-07

Project Summary/Abstract: The purpose of this individual National Research Service Award (NRSA) application is to provide the applicant with rigorous research training to develop into an independent investigator focusing on factors that influence healthy infant feeding practices. This award will ensure the applicant achieves competence in establishing a conceptual understanding of the impact of social determinants of health (SDOH) on infant feeding practices, develops foundational skills needed to launch an independent program of rigorous and ethical research, and gains professional development skills needed to advance in a research-focused academic setting. This training will occur in a resource-rich academic environment with support from a world-renowned advising team ideally suited to the applicant’s topic and training plan. This intensive research training will extend the applicant’s clinical expertise and facilitate rapid translation of evidence into clinical practice. Only about one-fourth of Americans are exclusively breastfeeding at six months of age. Social Determinants of Health (SDOH), including low socioeconomic status (SES), early maternal return to work, poor feeding support in childcare facilities, housing instability, food insecurity, and low-resource neighborhoods have all been cited as potential influential factors to initiating or meeting breastfeeding recommendations. Additionally, the introduction to solid foods before four months of age and poor maternal mental health have both been associated with suboptimal breastfeeding durations. Prior research has focused on the relationship between each risk factor and infant feeding practice in isolation. To date, little is known about the additive effects of multiple adverse SDOH on the impact of infant feeding practices and whether their impact is mediated by maternal mental health status or the timing of introduction to solid foods. This NRSA application proposes to fill this gap through the following aims: 1) identify the impact of select SDOH (SES, employment, food access, housing stability, child care support) on breastfeeding outcomes, 2) examine if the relationship between SDOH and infant feeding practices is mediated by maternal mental health and the timing of introduction to solid foods, and 3) investigate the impact of Black mothers’ experiences with adverse SDOH and their perceived lactation resource availability on infant feeding practices. This multi-methods research project will provide the applicant with training in multivariable regression models (Aim 1), structural equation modeling (Aim 2) and grounded theory qualitative research methods (Aim 3). This study is expected to generate critically important new knowledge about the relationship between breastfeeding outcomes and adverse SDOH. The proposed research also aligns with NINR’s strategic plan to conduct nursing research that will solve pressing health challenges and improve health.

NIH Research Projects · FY 2025 · 2024-07

The purpose of this renewal application is for funding of the existing Occupational and Environmental Medicine (OEM) Residency at the University of Pennsylvania (UPENN). The largest civilian OEM residency program in the nation, this unique, innovative, train-in-place OEM program, with measureable outcomes, in existence since 1997, was created in response to the National Academy of Medicine's call in 2000 to develop new routes to OEM certification. Designed to train midcareer physicians it was recently expanded to also train recent medical school graduates. Aligned with Healthy People 2020, the program trains physicians to promote the health and safety of people at work through prevention and early intervention. The program is also aligned with the National Occupational Research Agenda, now in its third iteration, to implement improved workplace practices, training qualified physicians to carry out the purpose of the Occupational Safety and Health Act. It is competency-based and helps address the national shortage of OEM physicians in the US, allows trainees eligibility for the American Board of Preventive Medicine Examination (ABPM) and equips them with improved clinical, administrative, teaching, population management and research skills. The overall purpose is to graduate 30 trainees over the 5-year grant period, including 5 NIOSH supported. Qualified applicants have completed at least 1 clinical year and have a Master of Public Health or equivalent degree, or a plan for completing the degree before graduation, in keeping with American Council of Graduate Medical Education (ACGME) requirements. There are two tracks: Internal Track (IT) and External Track (ET). The UPENN External, train-in-place, track helps overcome barriers that deter otherwise motivated physicians from specialist training. ET residents work full-time as OEM physicians at an approved clinical training site (CTS) supervised by an ABPM certified physician. IT residents, usually more recent medical school graduates, rotate at the UPENN and affiliated hospitals, governmental agencies and industry. The program has two interrelated are: the applied component at the CTS and the didactic component that consists of monthly 3-day sessions at PENN: 12 sessions during the first year and 5 during the second year. Both components work in tandem to allow acquisition of the ACGME milestones, program and American College of OEM competencies, and success on the certifying examination. Faculty are national experts in the specific area they teach and are ABPM diplomats. The program has graduated 134 residents since inception. Graduates comprise a diverse workforce, are geographically dispersed representing every US region with most working outside of the 25 largest standard metropolitan statistical areas. They express satisfaction with training, achieve all milestones, score competitively on the certifying examination. Graduates comprised 8% of new ABPM diplomates over the past decade. More than 95% have remained in the field.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY Tumor-Associated immune Macrophages (TAMs) are a metabolically and functionally heterogeneous population of cells that play critical roles in both anti-tumor immunity and tumor growth.Importantly, the type of TAM infiltrate within a neoplasm has been shown to be a prognostic factor for tumor growth and sensitivity to cancer immunotherapies. Hence, elucidating the mechanisms that determine the differentiation of pro-tumor and anti- tumor TAMs within the tumor microenvironment (TME) can lead to novel therapeutic approaches. For nearly 40 years, it has been known that inhibition of the mitochondrial electron transport chain (ETC), which is composed of five multiprotein complexes, is critical for the anti-tumor activity of TAMs. However, the mechanisms by which the activity of the ETC is controlled in TAMs and how this process determines the functions of these immune cells within the TME are largely unknown. Interestingly, the terminal enzyme of the ETC, Complex IV (CIV), is the only ETC complex in which its core protein subunits are replaced by closely related isoforms to tune its activity in response to signals from the tissue microenvironment. Using single-cell transcriptomics and 7 novel mouse strains that we generated, we demonstrated that type I and II interferons (IFNs) potently induce a single transcript encoding the peptide NDUFA4L3 and the microRNA miR-147 in human and mouse TAMs from melanoma tumors. Notably, we also showed that NDUFA4L3 and miR-147 work in concert to remodel CIV protein subunit composition in TAMs through the degradation and subsequent replacement of a core component of CIV, NDUFA4. Importantly, NDUFA4 degradation as a result of NDUFA4L3 and miR-147 induction by IFNs, or its genetic deletion, blocks tumor growth and leads to a dramatic accumulation of anti-tumor TAMs. Therefore, we hypothesize that regulation of CIV subunit composition and activity in TAMs through the induction of NDUFA4L3 and miR-147 by IFNs is a key evolutionarily conserved metabolic checkpoint by which TAM differentiation is controlled, which could be targeted in the context of cancer immunotherapies. Thus, in aim 1 of this project, we will use two novel conditionally knock-out strains for NDUFA4 and NDUFA4L3 that we generated, single-cell and spatial transcriptomics, and the humanized mouse MISTRG-6 that supports the development of human TAMs, to establish how CIV subunit composition in TAMs regulate anti-tumor immunity and responses to checkpoint blockade inhibitors. In aim 2, we will use a molecular model of CIV with an atomic resolution that we generated, metabolomics, and novel technologies to measure cellular metabolism and mitophagy rates at the single cell level to determine how CIV protein subunit composition and its activity contribute to the metabolic and functional states of TAMs. As recent reports demonstrate that macrophages from patients with loss of function mutations in NDUFA4 show a striking pro-inflammatory gene signature and several ETC inhibitors and engineered chimeric antigen receptor (CAR)-expressing macrophages are currently undergoing clinical trials for cancer, our studies may lead to novel therapeutic strategies to complement existing cancer immunotherapies.

NIH Research Projects · FY 2025 · 2024-07

This new T32 training program in Immune Dysregulation is designed to successfully train the next generation of Physician-Scientists, working at the interface of normal and pathologic immune function with end organ damage to elucidate the root causes of immune dysregulation syndromes and to develop novel approaches towards their diagnosis, monitoring, and treatment. The program requests the support of 4 physician-scientist postdoctoral trainees, who are competitively selected from various clinical fellowship programs at the Hospital of the University of Pennsylvania (Penn) and the Children’s Hospital of Philadelphia (CHOP). Selection criteria include a high standard of competence, motivation and perseverance, and a strong commitment to a research career in immunobiology. This program uniquely provides training in both adult and childhood immune diseases as it sits at the nexus of Penn and CHOP and takes advantage of the Institute for Immunology, specialized centers in Autoimmunity, and the Frontier Program in Immune Dysregulation to offer multi-disciplinary training in both basic and translational research related to immune dysregulation. The program will be directed by 3 physician scientists (Drs. Taku Kambayashi, Edward Behrens, and Warren Pear). Dr. Kambayashi will be responsible for trainees at Penn. Dr. Kambayashi is a physician scientist in Transfusion Medicine and serves as a program advisor for the MSTP program, the chair of the Immunology Graduate Group, and the director of the Physician Scientist Program for Pathology residency. Dr. Behrens will be responsible for trainees at CHOP. Dr. Behrens is a physician scientist in Rheumatology and serves as the Chief of Pediatric Rheumatology at CHOP. Dr. Pear will provide general program oversight as the most senior member of the co-program directors. He is a physician scientist in Molecular Pathology and serves as the Deputy Director of the IFI, Vice Chair of Research for the Department of Pathology, and co-leader of the Immunology Program in the Abramson Cancer Institute. All co-program directors run a successful NIH-funded lab in basic and translational immunology. The program will also consist of an administrative structure that oversees the needs and improvement of the training program, assisted by an Executive Committee, Internal Advisory Board, and an External Advisory Board. There are 25 faculty trainers to match the 4 requested postdoctoral slots. Various areas of research in immune dysregulation are displayed amongst our participating faculty trainers, ranging from immunity against infection, autoimmunity, molecular and cellular immunology in various organ systems. Each faculty trainer (except for the 3 junior trainers) has extensive experience in mentoring post-doctoral fellows into science-oriented careers. As a catalyst for developing the next generation of principal investigators, this training program will impact directly on improving treatment, decreasing costs, and most importantly, improving the lives of patients with immune dysregulation, all of which are directly relevant to public health.

NIH Research Projects · FY 2025 · 2024-07

PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a leading cause of cancer mortality and is significantly affected by multifactorial influences including host genetic and epigenetic factors, diet, and microbial composition. One of the most intriguing interventions for mitigating cancer risk and progression is modifying dietary behavior, a powerful approach that has been increasingly investigated. While many studies focus on individual dietary components, it is unclear how distinct metabolic inputs from the dietary milieu integrate to influence cancer progression. Two major nutrients that have been extensively linked to CRC are fructose and fiber. Fructose is highly enriched in Western diets and promotes intestinal tumorigenesis by accelerating de novo lipogenesis (DNL) and glycolysis. Fiber is metabolized by the gut microbiota to produce short-chain fatty acids (SCFAs), which exert anticarcinogenic activity. Fructose and acetate, the most abundant SCFA, converge at a common downstream metabolite, acetyl-CoA, which can be used for DNL or histone modification, making it a central metabolite critical to metabolism and epigenetic regulation. This makes fructose and acetate prime candidates for evaluating crosstalk between multiple dietary inputs in CRC. ACSS2 is the enzyme responsible for converting acetate to acetyl-CoA and is a gene target of several transcription factors which are activated in response to fructose consumption. Thus, ACSS2 is important due to its position at the nexus of catabolic and anabolic metabolism. A key focus of this proposal is on the metabolic and epigenetic effects of dietary fiber and fructose and the role of ACSS2 in mediating these effects. My preliminary data suggest that loss of ACSS2 expression is associated with greater CRC tumor grade and progression. This is potentially due to the downregulation of cell differentiation genes and upregulation of genes relevant to CRC tumor metastasis, such as epithelial-mesenchymal transition. Using a mouse model, we found that manipulating dietary fiber and fructose led to changes to host metabolism in opposing directions, highlighting the need for understanding the integrated effects of these particular nutrients in the cancer context. I hypothesize that fructose manipulates acetyl-CoA pool utilization to prioritize biosynthetic pathways that are advantageous for tumor growth and that acetate exerts epigenetic effects on colonic differentiation gene targets, which are mediated by ACSS2-directed histone acetylation. Aim 1 will determine how acetate and fructose interact to affect CRC growth and acetyl-CoA metabolism through in vitro organoid and cell culture models and in vivo genetic mouse models. Aim 2 will identify the mechanism by which acetate, fructose, and ACSS2 regulate CRC epigenetic modifications and differentiation status through histone proteomics, RNA sequencing, and chromatin immunoprecipitation sequencing. This project will provide novel insights into the combinatorial effects of fiber and fructose and the influence of host gene-diet interactions on susceptibility to dietary impacts on CRC. Our work has significant implications for dietary interventions that can profoundly impact cancer patient care.

NIH Research Projects · FY 2025 · 2024-07

Project Summary Placental vasculature is critical for the exchange of oxygen, nutrients, and waste products between maternal and fetal circulation. Failure to establish this vascular network is associated with fetal growth restriction and maternal cardiovascular pathologies. However, the signaling pathways that regulate placental vascular growth and function remain largely unknown. My long-term goal is to elucidate the cellular and molecular basis underlying placental vascular network formation and function at the blood- placental barrier, as well as how the placental endothelium affects fetal growth and maternal cardiovascular health. By utilizing a new genetic tool, the Hoxa13Cre line, that specifically targets placental vasculature but not vasculature in the embryo proper or yolk sac, I have found that placental endothelial-specific deletion of transcription regulators YAP/TAZ results in embryonic lethality and compromised placental vasculature. I propose to investigate the molecular basis for placental vascular growth and function by leveraging unique genetic tools, advanced imaging, and single-cell genomics (Aim 1). In addition, I will explore the roles of the placental endothelium in maternal-fetal exchange by combining mouse genetics and proteomic analysis (Aim 2). These lines of investigation will reveal transcriptional and epigenetic mechanisms by which placental vasculature grows and offer insights into the roles of the endothelium in the blood-placental barrier. The proposed studies complement my prior skills while acquiring new training in placental biology, single-cell technologies, and proteomics analysis to establish a strong foundation to build an independent research career. With a world-class team of mentors and collaborators with expertise in placental biology, cardiovascular metabolism, and bioinformatics to ensure exceptional guidance and a supportive, stimulating training environment at the University of Pennsylvania, I am ideally positioned to fully develop my technical skills and knowledge in placental vascular biology. Together, the proposed research and career development activities will be critical for me to develop an independent research program centered around understanding endothelial regulation in the blood-placental barrier.

NSF Awards · FY 2024 · 2024-07

Addressing the questions of how the two-meter-long human DNA fits into the space of a cell's nucleus (~20 um) and how it is organized within this space has been among the major mysteries of cell biology. DNA is packaged into the nucleus in the form of chromatin, consisting of a complex between DNA and histone proteins. DNA wrapped in compacted histones is thought of as “repressed” and “inaccessible”, and thus chromatin compaction plays a critical role in regulating gene activity. Current chromatin modeling is based on polymer simulations at different levels of resolution. However, given the slow time scales of these processes of the order of minutes to hours, the size scales of the order of 5--10 um (typical size of nucleus) are not accessible using methods such as molecular or dissipative dynamics approaches. The objective of this project is to decode the quantitative relationship between the physical microenvironment, multiscale 3D genome organization, and transcriptional output. The project will employ a convergent research strategy that integrates super-resolution microscopy, genomics, biophysical modeling and simulation, and machine learning. The new tools developed in this project will impact many areas in biology, including normal and abnormal tissue development, tissue degeneration in disease, as well as tissue regeneration. The research team will educate future scientists and a diverse workforce with a collaborative expertise in interdisciplinary training. Additional outreach activities will include research experiences for undergraduate students and high school students. Tissue-resident cells continuously sense changes in their chemo-physical environment and use this information to maintain their phenotype and tissue homeostasis. The project will develop a predictive framework of emergent epigenetic and transcriptional features of cells in response to changes in their physical environment. The project will develop new quantitative models for the distribution of heterochromatin domains in the interior of the nucleus as well as along the nuclear periphery. Specifically, a mathematical model will be developed to study the effect of rates of histone tail acetylation, methylation, and transcription on determining the distribution of heterochromatin domains in the interior of the nucleus. The project will further extend this model to include the formation of lamina-associated domains (LADs) by incorporating the energetic interactions between chromatin and the nuclear lamina via chromatin anchoring proteins. To verify the theoretical model, cells of fixed fate and fluid fate will be grown under varying micro-environments, and their whole genome organization at the nano- and micro-scale will be visualized and quantified through super-resolution microscopy. In addition, a machine learning framework leveraging novel deep neural operators will be developed for nonlinear inverse problems to extract the high-dimensional parameter fields implicitly and explicitly from noisy experimental images. To enhance the robustness, accuracy, and efficiency of neural operators with small data, the project will endow neural operators with prior knowledge, physics, multifidelity data, and active learning. 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 · 2024-07

Project Summary/Abstract Candidate: Jasleen Minhas, MD is a pulmonary and critical care physician-scientist passionate about developing personalized interventions promoting physical activity to improve patient outcomes in PAH. She seeks didactic and experiential training to develop expertise in advanced statistical methods for multidimensional data, longitudinal data analysis, and qualitative methods to advance her career goals towards research independence. Research Context: Activity limitation is an early manifestation of PAH and often persists despite treatment. Patients with PAH prioritize the impact of disease on their daily physical activity over its effect on exercise capacity. However, PAH care focuses on improving exercise capacity as measured by the 6-minute walk test. Clinic-based tests do not capture the daily variability in functionality. This proposal uses a non-invasive biosensor device that continuously captures multiple data streams (including ECG, respiratory rate, and activity) to define the clinical implications of continuously measured cardiopulmonary measures and physical activity in the patient’s home environment. Additionally, we will assess patient’s attitudes and preferences toward behavioral interventions targeting physical activity. Specific Aims: 1) Identify determinants of patterns of heart rate and daily physical activity in PAH. 2) Phenotype patients by integrating cardiopulmonary and physical activity measures and determine the association of phenotypes with clinical outcomes. 3) Assess patient attitudes and preferences for activity-based interventions. Research Plan: Dr. Minhas will utilize data from the ACTiPH-VP study to accomplish these aims. She will identify predominant patterns of heart rate and physical activity using a multilevel functional principal component analysis and identify their determinants. She will then integrate cardiopulmonary measures with physical activity and utilize the Joint and Individual Variance explained method to identify novel phenotypes and investigate their association with clinical outcomes. For Aim 3, Dr. Minhas will use semi-structured interviews to assess patient attitudes towards physical activity, and their preferences for activity-based interventions. Career Development Plan: Working closely with her mentors and advisors, Dr. Minhas’s goals are to 1) expand expertise in advanced statistical methods and dimensionality reduction techniques for multidimensional data gathered by biosensor devices; 2) obtain skills to design, launch, monitor and troubleshoot a multisite, prospective study; and 3) gain proficiency in qualitative methods to elicit patient preferences. Additionally, she will remain heavily involved with the parent ACTiPH study, gaining skills in startup, oversight, and completion of a multisite, prospective cohort study of patients with PAH across the US. Environment: The University of Pennsylvania offers an ideal environment to pursue this training, with well- established mentors. Her division is heavily invested in Dr. Minhas’s success.

NIH Research Projects · FY 2025 · 2024-07

(NOTE THAT margins are .75 in for abstract—original abstract was .50 inch margins) We propose a flexible interdisciplinary Training Program in Cell and Molecular Biology – the Cell and Molecular Biology Training Grant (CMBTG) – at the University of Pennsylvania (Penn). The CMBTG is a University-wide, interdepartmental and interschool program whose mission is to provide a multifaceted doctoral program to prepare students for research-focused and/or research-related careers in cell and molecular biology in academia, industry, or government. The goals of the CMBTG are (1) to provide Trainees with both in-depth and broad-based training in cell and molecular biology research and modern methodology, while at the same time matching the Trainees’ specific interests, and (2) endow students with an appreciation for life-long learning in which they can adapt to shifting research needs within the cell and molecular biology fields. These goals are achieved through general and specialized courses, literature survey courses, laboratory rotations, a qualifying examination, thesis research with oversight from an advising committee and annual IDP submission, training in responsible conduct of research and scientific rigor and reproducibility, and training grant-specific activities. Trainee-specific activities include: an annual oral presentation of ongoing thesis research; attending the annual Cell and Molecular Biology Graduate Group Retreat; attending the Annual Trainee Organized Invited Lectures and meeting with the speakers; participating in Alumni Day designed to expose Trainees to a broad range of PhD-dependent careers; Current and Former Trainee Lunch in which former Trainees present a talk on their thesis research; Senior student mentored preparation of individual fellowships; Interactions with Pennovation Works, a business incubator and laboratory that aligns and integrates researchers, innovators, and entrepreneurs for the commercialization of research discoveries; and other activities. Trainees also work on presentation skills, including elevator talks, and learn how to write, defend, and review grants productively. Students completing their first year of graduate studies are appointed for two years and are selected annually by the Executive Committee. Trainers come from multiple Departments within the Penn Schools of Medicine, Veterinary Medicine, and Arts and Science, and affiliated Children’s Hospital of Philadelphia and the Wistar Institute. Trainers have active research programs in cell and molecular biology and a strong commitment to graduate education. The CMBTG has formal mechanisms to monitor Trainees both during and after CMBTG support and to train Trainers in mentorship and monitor them. Direct management of the CMBTG is done by an Executive Committee that sets and reviews policy and selects Trainees. Based on the number of potential trainees, we request support for 10 predoctoral trainees/year for the next 5 years.