University Of Pennsylvania

NIH Research Projects · FY 2024 · 2024-09

There is a public health need for veterinary laboratories to have significant capacity to test animal diagnostic specimens and food/feed samples rapidly and accurately for the presence of important pathogens including Salmonella, Campylobacter, Listeria and SARS-CoV-2. The FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) is a group of veterinary laboratories tasked with protecting the health of humans by being able to rapidly and robustly respond to outbreaks associated with animals and animal products. This equipment-only grant requests support for the purchase of a QuantStudio 3 real-time polymerase chain reaction (PCR) system to maintain the capacity of a Vet-LIRN member laboratory (Ryan Small Animal Hospital Clinical Microbiology Laboratory at the University of Pennsylvania) to perform testing for diagnostic, monitoring and surveillance purposes. It will also allow the laboratory to more fully engage with proficiency testing and interlaboratory comparison exercises developed by Vet-LIRN.

NIH Research Projects · FY 2025 · 2024-09

Ischemia reperfusion injury (IRI) is a major source of morbidity in renal transplantation (Tx), as well as after cardiac arrest, cardiopulmonary bypass, and trauma. IRI and related early allograft dysfunction occur in ~30% of renal Tx recipients and are associated with poorer long-term outcomes. Kidney scarcity has also led to acceptance of grafts with greater degrees of baseline ischemic insult. These factors help explain why short-term graft survival has steadily improved in conjunction with the development of better immunosuppression but why long-term graft survival has been unchanged for nearly 2 decades. Our central goal is to develop ways to limit renal IRI and preserve renal function following renal Tx and other clinical scenarios. This proposal builds on our work in murine models showing that estrogen therapy preserves renal function and mitigates development of fibrosis after warm or cold renal IRI. We found that estrogen’s actions via estrogen receptor-alpha (ER) are renal-extrinsic, whereas actions of estrogen receptor-beta (ER) may diminish the benefits of estrogen therapy on renal IRI and are renal-intrinsic. We plan to further explore these actions, with an eye to improving clinical outcomes. Aim 1. Assess the cells/tissues responsible for the differential effects of ER and ER on renal IRI. Whole animal ER deletion leads to decreased renal IRI tolerance, and in Tx experiments, renal ER deletion does not impact IRI tolerance whereas deletion in the recipient dramatically worsens IRI. In contrast, whole animal ER deletion leads to increased renal IRI tolerance, and in Tx experiments, ER deletion in the renal Tx promotes IRI tolerance whereas deletion in the recipient has no benefit on IRI. Using cellular, biochemical, and transcriptomic studies, we will assess various cell lineages for their contribution to the opposite effects of 1.1) ER and 1.2) ER deletion on renal IRI. Aim 2. Probe the utility of SERMs and related compounds to modulate outcomes of renal IRI. We have tested various SERMs for their impact on IRI in mice and shown that raloxifene, a clinically approved osteoporosis drug, is superior to estrogen in promoting renal protection, while several other SERMs have no impact on renal IRI. Using murine models of renal IRI we will test whether: 2.1) ER antagonists, or 2.2) a SERM, such as raloxifene, in combination with an ER antagonist can maximize beneficial effects against renal IRI. Our studies will identify pathogenetic mechanisms and test new approaches to prevent acute and chronic kidney injury with significant potential for translational intervention.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The placenta is a transient, vascular organ necessary for in utero gas, nutrient, waste exchange in the majority of mammals. Placental dysfunction may lead to hypertensive disorders of pregnancy, fetal growth restriction, or intrauterine fetal demise in humans. In mice and humans, congenital cardiac defects are associated with placental insufficiency. In mice, endothelial cells from the allantois generate a highly branched exchange interface with the maternal circulation in a region termed the labyrinth. The molecular pathways that pattern the labyrinth remain poorly characterized. As the embryo develops, its metabolic demands change, and the number of placental vessels appears to increase accordingly. However, signals from the embryo proper that may communicate its oxygen and nutritional requirements remain obscure. I have deleted Endoglin (Eng), an endothelial BMP10 co-receptor, in the labyrinth using Hoxa13-Cre and found it to be lethal in midgestation. BMP10, secreted by the developing heart and liver, acts on ENG and the TGF-β/BMP type I receptor ALK1 on ECs to promote activation of SMAD1/5/8. Histologically, Eng mutant placentas have fewer, narrower fetal vessels than controls, and these vessels appear poorly perfused. Therefore, I hypothesize that ENG and ALK1 in placental ECs function to promote angiogenesis in response to BMP10 secreted by cardiomyocytes. Through this project, I will use in vivo mouse genetic tools to understand the function of ENG in the labyrinth, as well as assess the requirement for a BMP10 signal from the embryo to instruct placental vascularization. In aim 1, I will identify the primary deficit in Hoxa13-Cre; Eng fl/- placentas through morphometric, histologic, and transcriptomic analyses. In aim 2, I will determine whether ENG, along with ALK1, promotes activation of SMAD1/5/8 in placental ECs in response to cardiac-derived BMP10. Together, these experiments will enhance our understanding of how placental development is regulated and informed by the embryo proper, which may have implications for placental insufficiency and congenital heart disease in humans.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract In-hospital mortality prediction models (MPMs) are widely used in clinical research and practice; but existing MPMs suffer from algorithmic bias, or systematic differences in performance by group. We and others showed that MPMs for hospitalized patients – the Sequential Organ Failure Assessment score (SOFA) and Laboratory- based Acute Physiology Score, version 2 (LAPS2) – overestimate mortality for Black patients with acute respiratory failure (ARF) or sepsis, and underestimate mortality for white patients. Biased MPMs may thus produce healthcare inequities and flawed inferences about contributions of sociodemographics to clinical outcomes. Therefore, we seek to develop, validate, and demonstrate the impact of a novel MPM that optimizes fairness (i.e., defined by ‘groupwise optimality,’ optimizing subgroup performance without sacrificing predictive accuracy) across key subgroups defined by race, ethnicity, sex, age, primary language, insurance status, and social vulnerability without sacrificing accuracy. We will address key causes of bias in model development: differential missing data and calibration biases. We will study hospitalized ARF and sepsis patients because they face high risks of biased predictions due to diagnostic uncertainty and high mortality risk, and these syndromes pose increased mortality risks for racial and ethnic minorities. In Aim 1, we will develop a fairness- informed, in-hospital MPM. We will identify predictive features using those in common MPMs and structured data within 24 hours of presentation. We will assess missing data bias by comparing feature proportions by subgroup, excluding biased features, using a 2018-2023 cohort of ~220,000 encounters across 28 hospitals in the University of Pennsylvania and Kaiser Permanente Northern California health systems. We will select features using elastic net regression, and develop and internally validate a set of novel MPMs for use at admission, building logistic and elastic net regression, and machine learning models. We will implement model bias audits and mitigation strategies (i.e., multicalibration, optimizing calibration across subgroups without sacrificing predictive accuracy) to develop a set of optimized MPMs. We will evaluate performance overall and by subgroup, and compare performance to SOFA, LAPS2, and the Epic Deterioration Index. In Aim 2, we will conduct focus groups among key stakeholders to present blinded results of the novel MPMs, varying subgroup performance tradeoffs and decision thresholds, to select the model and thresholds that best promote equity and accuracy. In Aim 3, we will test the external validity of this MPM among patients admitted to MedStar Health, a health system serving primarily racial and ethnic minority patients, using a different electronic health record. In Aim 4, we will quantify the impact of the novel MPM on key use cases, by (1) re-analyzing our team’s pragmatic trials to assess the impact of risk adjustment on effect estimates overall and by subgroup; and (2) performing microsimulation informed by intensive care unit (ICU) demand in our health systems during peak COVID surges to compare ICU bed allocation across subgroups, compared to SOFA and LAPS2.

NIH Research Projects · FY 2025 · 2024-09

Abstract CAR T cells can mediate deep, durable cancer remissions but their activity is not controllable once infused, they are not easily tracked, their risk for toxicity remains an issue, and they are seldom designed to combat the immunosuppressive tumor microenvironment (TME). To provide a means for quantitative control of CAR T cell activity, our team first created universal immune receptors (UniCARs), a versatile CAR-like platform for the de novo generation and quantitative control of tumor antigen-specific T cells where human T cells are genetically engineered with adaptable docking immune receptors and can be conferred with highly personalized tumor specificity via pre-targeting with “tagged” antigen-specific small molecules, antibodies, scFvs or receptor ligands. Building upon these principles, and with a multidisciplinary team of physicians and researchers with scientific expertise in advanced T cell gene-engineering with molecular imaging and chemistry, we propose clinical development of an orthogonal imaging-enabled, adaptable CAR T cell (ImAC) platform where localization of infused UniCAR T cells can monitored via noninvasive PET imaging and UniCAR activity can be controlled through the administration of “tagged” biologics in order to facilitate safe and effective targeted therapy for cancer. This theranostic ImAC method utilizes a single UniCAR construct with two novel and distinct agents that share the same clinically-validated CAR-binding tag (DOTA); an imaging small molecule, [18F]-DOTA-Y, that permits tracking of the cellular product, and a new targeting biologic, folate-DOTA-Y, that redirects the specificity and activity of UniCAR T cells against folate receptor expressing cells. Folate receptor isoforms are expressed by the majority of ovarian cancers and by most immunosuppressive tumor-associated macrophages (TAMs) in the TME, allowing simultaneous targeting of cancer and TAMs via dosing with a single agent. An additional benefit, beyond the immediate scope of this study, is that targeting biologics can also be applied for diagnostic imaging prior to CAR T cell delivery to assess localization of the agent to the tumor, to predict response to therapy, and to test for potential on-target off-tumor toxicity. In the UG3 phase, the goal of our multidisciplinary team is to refine and optimize the ImAC method to validated optimal dosing schedules, routes and concentrations that confer a strong tumor response, and to confirm small molecule-based imaging of the administered CAR T cells in mouse xenograft models of ovarian cancer. With this data in hand, and with the small molecule targeting biologic being generated by the Immune Cell Network Core (ICN), we will seek to conduct a 3+3 dose-escalation phase I clinical trial of administration of autologous UniCAR T cells with folate-DOTA-Y for recurrent high grade serous ovarian cancer with PET-based imaging of the infused cell product in the UH3 phase of the study. Successful clinical development of this ImAC platform will significantly advance CAR gene therapy by allowing for the therapeutic coupling of adaptable antigen-targeted T cells with PET-guided monitoring for therapeutic activity, drug delivery and safety in vivo, which can be readily re-deployed for the treatment of other malignancies.

NIH Research Projects · FY 2025 · 2024-09

Project Summary The long-term goal of this proposal is to understand the mechanisms that regulate sub-cellular organization and the significance of this organization to organelle and cell function. Cells are highly compartmentalized at multiple length scales. At the micron scale, organelles are organized within the cytoplasm and occupy specific sub- cellular zones. Proteins and nucleic acids organize into assemblies at the nanometer scale in the cytosol, nucleus and on cellular membranes to ensure high fidelity of biochemical reactions. This highly orchestrated spatiotemporal organization maintains cellular homeostasis. Not surprisingly, disruptions to the spatiotemporal organization of organelles, proteins and nucleic acids are hallmarks of diseases. In this context, we seek to address three key biological questions: 1. How do molecular assemblies of motor and adapter proteins regulate the transport and spatial positioning of organelles and how are these assemblies disrupted in diseases. 2. How is the molecular identity of organelles such as lysosomes, in turn, linked to organelle positioning and dictate organelle function in health and disease states? 3. What is the cause/consequence relationship between the spatial organization and physical compaction of the genome within the nucleus, the epigenetic modifications, and gene activity? To address these questions, we will take an innovative approach of combining cell biological tools with new, advanced, and quantitative microscopy methods that enable us to visualize the spatial organization of cells in situ and with near molecular spatial resolution. This proposal builds on major advances made by my group in the past 12 years in developing quantitative advanced microscopy tools including quantitative, multiplexed super-resolution microscopy. These methods make it possible to address the molecular scale questions that we are asking in the cell context and with unprecedented quantitative detail. We have used these tools to visualize organelles moving along individual microtubule filaments inside cells, protein nanoplatforms forming on microtubules, inside the cytosol and on organelle membranes, and the folding of the chromatin fiber within the nucleus. These approaches provided new insights into how the microtubule cytoskeleton and motor proteins collectively regulate organelle transport and how the folding of chromatin relates to cell identity under physiological and pathological states. This proposal will build on our advances to elucidate how multiple molecular parts assemble into functional transport units to regulate the positioning and ultimately the function of organelles. We will further map the spatial proteome of these organelles to determine how their molecular identity is linked to organelle positioning and function. Finally, we will seek to address the causal relationship between chromatin structure and function. These areas and our method development integrate synergistically to advance our understanding of how sub-cellular organization emerges, and the importance of this organization for cell physiology and pathology.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Somatic mosaicism results from the accumulation of mutations over time, leading to a unique genotype in every cell. These mutations can be as small as a single base pair or as large as an entire chromosome – affecting hundreds or even thousands of genes. Mosaic chromosomal alterations (mCA) are one type of somatic mosaicism characterized by large chromosomal gains or losses and are associated with aging, heart disease, kidney disease and cancer. The role of mCA has been best characterized in blood, but recently we used single- cell sequencing to show that mCA are also common in the kidney and are associated with disease progression. Kidney and blood are hotspots for mCA, but these data raise the possibility that mCA are present in other organs where they exert tissue-specific effects. Existing algorithms for mCA detection are tailored to cancer and rely on the assumption that cancer cells are clonal and carry the same variants. This is a significant limitation because mCA in non-neoplastic tissue are not clonal and may only be present in a small fraction of cells. Moreover, mCA that do not affect copy number or transcript abundance and do not reside in open chromatin regions are all undetectable with current methods. This proposal aims to develop methods to improve detection of mCA that will enable novel lines of inquiry in kidney, blood and other non-neoplastic tissues. Our single-cell methods will be complemented by algorithms to localize mCA in spatial transcriptomics datasets, which may provide insight into how mCA affect disease progression via interaction with neighboring cells. In addition, we will design high- throughput assays that can be used to evaluate mCA burden at low cost in a wide variety of sample types. All methods will be released as open-source protocols and software packages that researchers can tailor to their tissue of interest and may lead to novel therapies that aim to eliminate cells with mCA.

NIH Research Projects · FY 2024 · 2024-09

Project Summary Aging related disorders, such as Parkinson's Disease with dementia (PDD), Lewy Body with Dementia, and other Alzheimer Disease and related dementias (ADRDs), are debilitating neurological disorders that affect more than 8 million people in the US alone. Amyloid deposits, characterized by their insolubility and disruptive protein clumps, pose a distinct challenge in structural biology and medicine due to their atypical structure compared to soluble proteins. In PDD, the protein alpha-synuclein (αS) misfolds and aggregates into self-templating fibrils. These fibrils consist of repeating beta-sheet units, capable of adopting various shapes and surface structures, known as fibril morphologies (FMs). Ensuring consistent FM for in vitro assays remains a formidable challenge across literature given the variability introduced by factors such as salt concentration, buffer selection, and additive choices to name a few. These assays are vital for the assessment of compound binding and cellular toxicity, yet no expedient assay has been created to rigorously determine FM. The University of Pennsylvania (UPenn), sponsor, and principal investigator of this research proposal are uniquely positioned to accomplish the goals outlined below. UPenn provides a robust infrastructure and extensive resources for groundbreaking research, the sponsor contributes deep expertise in chemistry and biophysics, vital for the project's scientific rigor, and the principal investigator, with extensive experience in the relevant areas of this research, is well- equipped to lead and ensure the successful execution of the project. The project proposed relies on the hypothesis that proteolytic cleavage rates of a fibril are determined by stability of the fibril fold and enzyme steric clashes resulting in a specific kinetic proteolytic profile (KPP) which can distinguish FMs. Current methods require laborious assays to pinpoint FM, and with FM often varying unpredictably under identical preparations, the need is clear: a streamlined and efficient assay to guarantee uniformity across samples and literature. Therefore, in Aim 1 introduces an assay that is designed to determine FM by KPP. Pinpointing the FM is foundational to reproducibility and enables Aim 2: a statistically rigorous high- throughput screen (HTS) for exploration into the diversity of possible FMs. The envisioned HTS utilizes site selective chemistry via a cysteine to introduce mutations to the monomer structure of αS which is envisioned to induce conformational change when fibrils are formed. It is anticipated that a large FM library will be generated within the first few iterations which will not only expand the collective knowledge of the FM manifold, or coverage of all possible FMs, but also enable the creation of consistent preparations with specific features. Due to the number of potential combinations, Machine learning (ML) will be employed to guide future iterations of this HTS. By addressing these challenges, the proposed comprehensive MORFS assay, HTS, and ML will revolutionize the analysis of amyloids, paving the way for reproducible in vitro assays for advancements in our collective understanding ADRDs.

NIH Research Projects · FY 2025 · 2024-09

Under-diagnosis occurs when an individual living with a disease condition has not received a diagnosis. Reasons for under-diagnosis are often complex and context specific, and the extent may vary across sensible population subgroups leading to disparity in care. Electronic Health Records (EHRs) contain a wealth of health information for patients, and the diagnosed and under-diagnosed patients may bear similarity in their EHR profiles, which differ from those condition-free. Therefore, EHRs provide a unique opportunity to address under-diagnosis in the standard healthcare setting. Full exploitation of such opportunity is challenging, however, because of the very fact that under-diagnosed patients are embedded in the large number of condition-free patients. Noting that patients who have been diagnosed with the condition can be identified from EHRs, we propose that EHR data, when enriched with additional disease labels from a small scale targeted screening, allows development of data-driven approaches to identifying under-diagnosed patients and assessing disparity in under-diagnosis. To this end, we will develop an arsenal of statistical and machine learning methods and accompanying software tools to address under-diagnosis. Our methods enable (1) a risk-based approach to identifying patients in EHRs who most possibly miss the diagnosis (Aim 1); (2) unbiased comparison between diagnosed and under-diagnosed patients to understand disparity in under-diagnosis (Aim 2); and (3) leveraging of existing models and targeted screening data to address under-diagnosis in a new clinical setting. We will apply the developed methods to address under-diagnosis in Primary Aldosteronism and Familial Hypercholesterolemia using data from Penn Medicine and VA EHRs.

NIH Research Projects · FY 2024 · 2024-09

Summary: Periodontitis is a chronic inflammatory disease in which a highly orchestrated host-microbial interaction leads to the destruction of the tooth-supporting structures including periodontal tissue attachment and alveolar bone. Mast cells are found in the gingiva; their numbers are increased in chronic periodontitis and the degree of their activation correlates well with disease severity. Not surprisingly, it has recently been shown that mast cells contribute to Porphyromonas gingivalis-induced periodontitis in mice, but the mechanisms involved in their activation and regulation remain unknown. Mas-related G protein-coupled receptor X2 (MRGPRX2, mouse counterpart MrgprB2) is a newly described cell surface receptor that is expressed in a subtype of mast cells found predominantly in the skin and the gingiva. We recently demonstrated that MRGPRX2-expressing mast cells are present in normal gingiva and that their numbers are increased in patients with chronic periodontitis. In addition, our unpublished preliminary data demonstrated that compared to wild-type mice, MrgprB2-/- mice are protected from mast cell recruitment, gingival inflammation, and bone loss in a ligature-induced model of periodontitis. Based on these findings, we hypothesize that recruitment and activation of mast cells through MRGPRX2/B2 contribute to periodontitis. In aim 1, we will develop two models of humanized mice. The first involves retroviral transduction of MRGPRX2 into MrgprB2-/- mouse bone marrow stem cells, their differentiation into bone marrow-derived MCs (BMMCs) ex vivo and their engraftment into mast cell-deficient Wsh/Wsh mice. The second involves CRISPR/Cas9-mediated replacement of MrgprB2 with MRGPRX2. These humanized mice will be used to study Porphyromonas gingivalis and ligature-induced periodontitis. In aim 2, we will modulate periodontitis by targeting MRGPRX2/MrgprB2-mediated cofilin and NF-κB signaling in mast cells. Successful completion of this study will lead to the development of new preclinical models to modulate periodontitis through specific small molecule receptor antagonists and by targeting signaling in mast cells.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Lewy body disorders, a subset of Alzheimer’s disease and related disorders (ADRDs), are the second most common neurodegenerative disorders worldwide and the 14th leading cause of death in the United States. Lewy body ADRDs are progressive, incurable, and mortality rates are rising, making end-of-life (EoL) care a significant public health concern. The majority of persons with Lewy body ADRDs are hospitalized in their last 6 months of life with high rates of intensive care unit admission, in-hospital death, and low rates of discharge to hospice care. Because these markers of poor care quality are often incongruent with patient care preferences, urgent interventions are needed to improve EoL care quality in acute care hospitals. Despite this critical need, there are two fundamental knowledge gaps in developing inpatient EoL interventions for persons with Lewy body ADRDs: 1) lack of a prognostic model to determine risk of 6-month mortality and 2) lack of a validated tool to measure EoL care quality. This proposal requests support for a mentored career development award for Dr. Whitley Aamodt, a movement disorders specialist, neurodegenerative neurologist, and clinical researcher at the University of Pennsylvania. The overarching goal of this project is to improve prognostication and EoL care quality for hospitalized persons with Lewy body ADRDs by identifying patients at the greatest risk of death and ensuring that hospital-based care is appropriate and aligned with care preferences. In Aim 1, Dr. Aamodt will use comprehensive Medicare data to develop a risk-prediction model for 6-month all-cause mortality in a nationally representative sample of hospitalized patients with Lewy body ADRDs using advanced predictive modeling. This model will be externally validated in a second cohort of Medicare beneficiaries from the post- pandemic period. In Aim 2, Dr. Aamodt will create, test, and externally validate a patient-centered EoL care quality instrument based on data gathered from diverse Lewy body ADRD patients, care partners, and practitioners using qualitative research methods and factor analysis. In executing these aims, Dr. Aamodt will obtain additional training in neuroepidemiology, biostatistics, and qualitative research under the guidance of mentors and advisors in epidemiologic research methods (John Farrar, MD, PhD), neurodegenerative disease epidemiology and health services research (Allison Willis, MD, MS), predictive modeling (Warren Bilker, PhD), qualitative research methods (Katharine Rendle, PhD, MPH), and palliative care research (Scott Halpern, MD, PhD). The results of this project will provide fundamental knowledge about hospitalized patients with Lewy body ADRDs nearing EoL and will guide the development of future interventions to improve EoL care quality. This work aligns with the strategic goal of the National Institute on Aging to address EoL care needs in ADRDs. Through the research training and mentorship gained during this career development award, Dr. Aamodt will establish herself as an independent investigator in the field of applied epidemiology, outcomes research, and palliative and EoL care for aging Americans with neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2024-09

Project Summary Stillbirths and neonatal deaths are two adverse birth outcomes of critical global health relevance. In 2021, an estimated 1.9 million babies were stillborn, and 2.3 million liveborn babies died before reaching 28 days of age. India stands out globally as the country having the largest number of stillbirths and neonatal deaths (respectively 290,000 and 440,000 in 2021). These large numbers not only reflect the size of India’s population but also its high levels of stillbirth and neonatal mortality. Moreover, not only are India’s neonatal mortality rates high relative to other countries, but they are high relative to India’s own levels of postneonatal mortality. This indicates the existence in India of a distorted age pattern of mortality at early ages with excess mortality at neonatal ages, impeding the country’s ability to meet Sustainable Development Goals targets. In spite of the significance of these patterns, measurement and understanding of stillbirth rates and neonatal mortality in India are hindered by major gaps in data availability and quality. Undercount of stillbirths and neonatal deaths as well as misclassification of neonatal deaths vs. stillbirths in existing, mostly retrospective sources remain major concerns. Issues with availability and quality of information on small for gestational age, preterm and low birthweight – three major risk factors that can play a large role in both levels and age patterns of early-age mortality – are additional gaps that further impede proper monitoring of India’s patterns of mortality during the late fetal and neonatal periods. The goal of this project is to improve our understanding of why India stands out globally in terms of both its levels and age patterns of mortality during the late fetal and neonatal periods by collecting new prospective data in 4 surveillance sites located in different regions of India, covering a variety of contexts. While the focus of this project is on India due to the outsize role it plays in global rates of stillbirth and neonatal mortality, results will have methodological and substantive implications for other low-income countries, including other South Asian countries and countries in Sub-Saharan Africa which, like India, are characterized by a high burden of stillbirths and neonatal mortality as well as large data gaps in their stillbirth and neonatal mortality information.

NSF Awards · FY 2024 · 2024-09

Silicon quantum dot devices hold significant promise for scalable quantum computing. However, tuning these devices into the desirable states for quantum applications is highly challenging, creating substantial barriers to entry. Traditionally, tuning has been a manual process that is time-consuming, heavily reliant on experimental intuition, and inherently unscalable. This situation underscores the need for automated tuning (autotuning) approaches. The development of autotuning algorithms has been impeded by the lack of experimental training data and the limitations of existing quantum dot simulators, which only capture the physics of already-tuned devices. To this end, this project aims to provide full-stack support for quantum dot device autotuning research by delivering new quantum dot device simulation infrastructures for cold start and exploring corresponding autotuning algorithms. This initiative will democratize autotuning research, offering researchers without access to experimental facilities both training data and a low-cost autotuning testbench. These advancements will promote the progress of science by facilitating broader access to quantum computing research and enhancing the efficiency and scalability of quantum dot device tuning. This project will provide training opportunities for the next-generation quantum computing workforce, and the research outcomes will be integrated into undergraduate and graduate education efforts. The proposed research will significantly advance our understanding of quantum device modeling and tuning, providing innovative tools, data, and methods that can shape the tuning process of quantum dot devices. Specifically, this project will develop the QDREAM (Quantum Dot Real-Time Emulation and Autotuning Model) framework. QDREAM consists of 1) device-physics-based cold start simulations that focus on combining a finite element electrostatic simulation with a constant interaction quantum dot model to simulate devices in a completely untuned regime; 2) an FPGA-based quantum dot device emulator that will take in real voltages and output a charge sensor signal in real-time; and 3) a series of autotuning algorithms targeting various stages of the device tune-up process from cold start. QDREAM will be validated using real quadruple quantum dot devices routinely fabricated and measured in our lab. These comprehensive advancements will serve as a foundational step towards realizing larger-scale, more advanced quantum-dot-based quantum computers. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NSF Awards · FY 2024 · 2024-09

This project aims to serve the national interest by closing the academic completion gap for Latina/o STEM students through the implementation of a "Circle of Champions." The Circle of Champions framework seeks to organize individuals around students, actively supporting them throughout their academic journeys. Under this approach, students nominate parents, other family members, friends, former high school teachers, professors, and similar individuals as their champions. With a Circle of Champions around each student, the project tracks their journey, informs champions of progress, and facilitates their learning on how to provide support effectively. The goal is to leverage students' assets and community wealth into traditional forms of social, cultural, and academic capital. This project employs a cultural assets approach to student learning combined with an intentional focus on harnessing the considerable resources students possess within their families, communities, and themselves. By addressing this oversight, the project will set the stage for an equity-oriented approach to supporting student success. This project seeks to accomplish four goals: 1) advance the understanding of converting social capital into academic capital; 2) investigate conditions under which the Circle of Champions model can be optimized to impact student success in STEM; 3) narrow or close the equity gap in STEM at Gavilan College; and 4) develop a replicable model for other Hispanic Serving Institutions. Project activities aimed at achieving these goals include supporting the Circle of Champions model for all Gavilan College students enrolled in STEM courses, expanding and developing an AI assistant platform, studying the effectiveness of research and program evaluation to fully understand variables influencing success, and disseminating findings. The researchers aim to explore existing assets in the lives of Latina/o students, particularly their social capital, and how these assets can contribute to academic success. To investigate the impact of social networks on students' lives, the project utilizes Community Cultural Wealth (CCW) and Funds of Knowledge (FK) models as guiding frameworks and employs a mixed method of analysis, including qualitative analysis of user opinions, quantitative analysis of user activity using machine learning, and quantitative assessment of student academic outcomes. The NSF IUSE: Innovation in Two-Year College STEM Education (ITYC) Program seeks to accelerate the impact of and advance knowledge about emerging and evidence-based practices in undergraduate STEM education at two-year colleges. This project is partially funded by the HSI Program, which aims to enhance undergraduate STEM education, broaden participation in STEM, and build capacity at HSIs. 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 · 2024-09

The long-term care industry is one of the fastest growing sectors of the economy, while also creating some of the lowest-paid and lowest-quality jobs. These precarious working conditions are costly for workers – who are disproportionately women and women with various racial and ethnic backgrounds – and the patients they care for. On the worker side, the conditions of long-term care jobs keep families in poverty and contribute to sex and racial economic disparities in the workforce. Worker turnover is high, and worker shortages are pervasive. These precarious working conditions may deteriorate the quality of care workers can provide and have a detrimental impact on patient outcomes, potentially contributing to or exacerbating health disparities among patients. This could be particularly an issue for people living with Alzheimer's disease and Alzheimer's disease and related dementias (AD/ADRD), where continuity of care and communication between the care team is paramount. The working conditions of this workforce, both wages and workload, have important implications for turnover and overall well-being. In Aim 1, we will study the impact of wage changes, instrumented through minimum wage laws and pandemic-related wage protections, on home health care worker outcomes, including wages, turnover, and poverty. In Aims 2 and 3, we will study the impact of staffing regulations and unionization, respectively, in nursing home settings, on the welfare of the workers, measured by wages, turnover, and reliance on contract staff. We will look for heterogeneity in the effects of workplace policies on workers, based on geography (urban vs. rural), and agency/facility metrics (star rating, percent Medicaid, percent patients who are of different racial or ethnic group). In each aim, we will take the next important step, which is to estimate how these workforce changes are related to patient outcomes, including functional status, physical and mental health, and health care utilization and mortality. We will focus our analysis on people living with dementia; over one-third of home health care recipients and one-half of residents in nursing homes have AD/ADRD.10 This group is likely the most vulnerable to staffing changes, training, and disruptions. We will look for heterogeneity in the effects of workplace policies on patients by both facility (e.g.,profit status, rurality) and patient-level characteristics (e.g., race, ethnicity, sex). This project will build the evidence base on which we can make informed policy recommendations that could improve the lives of the dementia care workforce and their patients.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Dysbiosis, an alteration of the gut microbiome associated with chronic diseases, has been previously described in patients with cirrhosis. Common features of dysbiosis include reduced bacterial diversity and the outgrowth of the family of human pathogenic bacteria Enterobacteriaceae, including Escherichia coli. Infectious complications, namely spontaneous bacterial peritonitis (SBP) and bacteremia, are deadly for patients with cirrhosis with up to 50% mortality. E. coli is one of the most common causes of these infections, thought to derive from bacterial translocation across the intestinal epithelium from reduced barrier function. While this suggests a connection between dysbiosis and disseminated infection, the underlying risk factors of disseminated infection are not well understood in this population. Lactulose, a simple carbohydrate which is the first line treatment for the cirrhosis complication hepatic encephalopathy, has been associated with higher abundance of E. coli in the gut microbiota. Our work demonstrates that lactulose also increases colonization of Enterobacteriaceae, by overcoming carbon limitation in the colon for these pathogens. My preliminary data show that E. coli acquire mutations allowing it to utilize lactulose as a carbohydrate source both in culture and in the mouse gut, thus increasing its fitness. This phenotype is dependent upon constitutive expression of the lactose (lac) operon via deactivation of the transcriptional regulatory protein LacI, a repressor of the lac operon, which encodes the lactulose metabolizing enzyme β-galactosidase. I therefore hypothesize that lactulose treatment selects for mutant Enterobacteriaceae capable of metabolizing lactulose, thereby increasing colonization and the risk of disseminated infection in patients with liver disease. My objectives in this proposal are to characterize the gain of function mutations that enable lactulose metabolism by E. coli, determine the competitive advantage imparted by these mutations, and assess the impact of lactulose on disseminated infection in mice and humans with liver disease. My hypothesis will be tested through two inter-related Specific Aims that will evaluate the adaptive mutations and competitive fitness imparted by lactulose treatment to E. coli (Aim 1) and test the impact of lactulose on disseminated infection (Aim 2). This proposal takes advantage of several innovative techniques and unique resources including a novel mouse model of disseminated infection in liver disease and creation of a human disseminated infection strain library. The University of Pennsylvania is an ideal research environment for these studies given the local expertise in gut microbiome, pathogen biology, and comparative bacterial genomics. The candidate will acquire critical skills for his career development as an independent investigator, including in bioinformatics, bacterial genomics, and mouse disease modeling. Successful completion of this proposal will enable the investigator to reach their career goal to become an independently funded tenure-track faculty member, identifying the dietary and bacterial factors involved in gastrointestinal diseases.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT As large-scale genome-wide association studies (GWAS) continue to yield now thousands of genomic loci robustly associated with neurodevelopmental and psychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia (SCZ), the major defining challenge of the post-GWAS era is to characterize the concrete biological mechanisms through which this polygenic variation confers disease risk, at scale. To this end, we and others have recently developed methods and resources for systematic integration of GWAS results with population-level functional genomic reference panels -- identifying isoform-regulation during the second trimester of human brain development as mediating the greatest proportion of heritability across multiple neuropsychiatric GWAS studies compared with earlier or postnatal timepoints. Yet, no studies have characterized genetic regulation of alternative polyadenylation (APA) in the developing brain, a critical yet understudied tissue-specific gene-regulatory mechanism with established roles in neuronal mRNA metabolism, subcellular trafficking, and cellular differentiation. Our preliminary data indicates widespread dysregulation of APA in stem-cell-based models and postmortem brain tissues from subjects with ASD and SCZ, as well an outsized enrichment of psychiatric GWAS signal with APA quantitative trait loci (QTL) in the developing human brain. This proposal seeks to integrate large-scale functional genomics, single-cell and long-read sequencing, deep learning, and genome-editing in human neuronal stem-cell models to develop a detailed, mechanistic understanding of APA regulation during human brain development and its contribution to neuropsychiatric disorder pathophysiology. Specifically, we will generate a comprehensive atlas of APA regulation across neurodevelopment, leveraging data from >3650 bulk tissue samples as well as single-nucleus RNAseq data across >700 unique donors, including 170 with SCZ/ASD. We will train and validate a deep learning model predicting APA changes from primary sequence. Through integration with psychiatric GWAS, we hypothesize that APA regulation will provide substantially greater resolution to detect candidate biological mechanisms underlying psychiatric GWAS loci. Finally, predicted SNP-UTR-disease mechanisms will be experimentally tested via high-throughput screens and genome-engineering in iPSC-derived neurons. Together, these studies will systematically characterize a critical, yet underexplored area of genomic regulation in the human brain across development, thereby providing novel insights into psychiatric disease mechanisms and identifying potential neurobiological targets for therapeutic development and intervention.

NIH Research Projects · FY 2025 · 2024-09

Project Abstract The mammalian genome is organized into various regions at different scales as one mechanism to regulate gene expression and mediate cellular identity. One type of well-characterized region is the lamina-associated domain (LAD), which contains areas of chromatin that directly interact with the nuclear lamina (NL) at the nuclear periphery. Found across all chromosomes, LADs dynamically interact with the NL to release or attach genes and regulatory elements in accordance with cell-type and differentiation state-specific gene expression programs. Patients with mutations in LMNA, encoding the A and C type lamins in the NL, develop a heterogenous group of diseases, known as laminopathies. Laminopathies preferentially affect striated muscle, and patients often develop dilated cardiomyopathy (DCM), which can be fatal. Evidence from mouse models and human genetic studies of laminopathies have suggested a potential role for the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in mediating the disease phenotype. Abrogation of the LINC complex in a laminopathy mouse model resulted in a phenotypic rescue. Additionally, characterization of LMNA mutations in various cell types demonstrated a disruption to chromatin-lamina interactions in a cardiomyocyte-specific manner. This suggests a mechanism where the nuclear lamina and LINC complex are each playing a role in the pathogenesis of laminopathy phenotypes, and potentially affecting genome organization. However, while a LINC-LMNA-gene positioning axis has been previously suggested, the mechanism of how this may occur remains elusive. Using a combination of population-based genomics analyses, single-cell microscopy, and cellular functional assays, I will test the hypothesis that cytoskeletal-nuclear interactions in lamin variant cardiomyocytes destabilize LADs and contribute to abnormal cellular function. I aim to define the role of the LINC complex in mediating chromatin organization in LMNA mutant cardiomyocytes and will determine if disruption of LINC complex components can preserve the changes to genome organization observed. In addition, I aim to determine how cardiomyocyte function is affected by disruption of the LINC complex in the presence and absence of LMNA mutations. These studies will provide mechanistic insights into how the nuclear lamina and LINC complex are contributing to both LAD organization and cardiomyocyte function, which will begin to provide novel understanding of the molecular basis of laminopathy phenotypes.

NIH Research Projects · FY 2025 · 2024-09

Cytokines govern key cellular processes of pregnancy with both pro- and anti-inflammatory activities. To prevent immune dysregulation at the maternal-fetal interface, cytokines are thought to dictate these seemingly contradictory immune responses through meticulous immune regulation. Yet, there is a gap in knowledge regarding the mechanistic roles of many cytokines in the context of pregnancy, including the versatile cytokine Interleukin 27 (IL-27). Our proposal will address this gap in knowledge, as we have recently established that the IL-27 signaling circuit is active at the maternal-fetal interface and discovered that IL-27 is a protective cytokine during congenital infection. IL-27 serves as a potent regulatory of inflammation, where it can be pro- or anti- inflammatory dependent upon cellular context. Although initially recognized for its proinflammatory activities in promoting immunity, IL-27 was later acknowledged for its profound ability to serve as an anti-inflammatory cytokine during various infections where IL-27 has been found to act on various T subsets cells to limit inflammation during infection. Moreover, IL-27 can directly influence viral infection through induction of antiviral genes in IL-27 responsive cells. Based on literature and our extensive preliminary data, we hypothesize that IL- 27 is both an immunoregulatory and antiviral cytokine during pregnancy. This proposal describes targeted objectives to define the mechanistic features of IL-27 signaling at the maternal-fetal interface that underlie its protective capacity during congenital infection. In Aim 1 we will focus our investigations on defining the immune regulatory functions of IL-27 at the maternal-fetal interface. We will uncover the cellular targets and impacts of IL-27 signaling in dictating activation state of localized T cells. In Aim 2 we will focus our studies on the antiviral functions of IL-27 in the placenta. We will evaluate the transcriptional dynamics of IL-27 signaling and antiviral capacity of IL-27 signaling in mouse and human placental trophoblast organoids. Altogether, outcomes from this proposal will embody new fundamental insights into regulatory cytokine communication at the maternal-fetal interface. Specifically, this proposal will define the mechanisms underlying IL-27 protective immunity during congenital infection, which could ultimately be leveraged to improve maternal and neonatal outcomes.

NIH Research Projects · FY 2026 · 2024-09

PROJECT SUMMARY Visual decision-making in the brain is thought to depend on two behaviorally distinguishable computational components: one that converts uncertain visual inputs into a decision variable, and a second that applies a rule to the decision variable to commit to a choice. Our long-term goal is to understand the neural mechanisms that implement these computational components of high-order visual processing, which represent key building blocks of cognition. Here we propose to examine where and how visual decision rules are implemented in the brain. We build on three primary innovations: 1) a novel theoretical framework predicting that normative decision rules tend not to be static, as prescribed in many commonly used decision models, but rather dynamic with flexible adjustments both within and across decisions; 2) a novel task design that allows us to control the decision variable and measure decision commitment directly for each decision; and 3) measurements and manipulations of neural activity at multiple cortical and subcortical components of a key oculomotor pathway to assess their relative contributions to implementing and updating flexible decision rules. We have three Specific Aims. Aim 1 is to characterize flexible decision rule use by monkeys. Aim 2 is to identify correlative relationships between neural activity in the oculomotor pathway and decision rules on single trials. We targe the frontal eye field and lateral intraparietal area of the cortex; the substantia nigra pars reticulata, which is a major output structure of the oculomotor basal ganglia; and the superior colliculus, which receives input from the other three regions. Aim 3 is to identify causal relationships between neural activity in these brain regions and decision rules on single trials. Results from the proposed project will provide new, theoretically motivated, and empirically grounded insights into circuit mechanisms that control a major building block of deliberative information processing in the brain: the rules that govern when and how to end the deliberations and commit to an action. These results will help to provide a solid foundation for investigating cognitive impairments associated with dysfunction of the cortico-basal ganglia pathway.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY My long-term goal is to become an independent investigator focusing on how pollutant exposures may adversely affect respiratory health and identify measures to effectively mitigate such exposures. My primary project objective is to investigate the exposure and toxicity of chemical constituents of concern (CCOC), namely metal and aldehydes, as well as the pulmonary health effects, including inflammation, of using new and emerging electronic cigarette (e-cig) devices among young adults. E-cig devices work by heating a mixture of chemicals to generate an aerosol that is inhaled by the user. Use of e-cigs has increased and, among adults, remains the highest among those aged 18 to 24 years. More recent e-cig devices such as disposable PODs have grown in popularity, yet it is currently unknown whether these new devices’ design characteristics in conjunction with user vaping regimen impact CCOC exposure and influence respiratory health. Thus, my specific aims are to 1) evaluate the relationship between e-cig use and CCOC exposure and effect, 2) assess the association of e-cig use with respiratory outcomes and inflammatory markers, and 3) assess CCOC exposure as a mixture and potential mediator in e-cig related respiratory health outcomes. In this cross-sectional study, to achieve Aim 1 (K99 phase), 150 participants (75 e-cig users, 75 non-users) will be recruited to assess biomarkers of exposure (aldehydes, metals) and effect (metallothionein) from e-cig use. This will leverage the ongoing EMIT study which looks at metal exposure and collects e-cig user regimen via questionnaire, aerosol samples, biospecimens (blood, urine), and spirometry measures. After receiving training in chemical analysis, respiratory clinical outcomes, and inflammatory markers, including gene expression changes, a new cohort of 150 participants (75 e-cig users, 75 non-users) will be recruited for Aim 2 (R00 phase). This phase will not only collect the same data as in Aim 1 but also biomarkers of effect and inflammation (blood, urine, FeNO) and gene expression profiles (in nasal epithelial cells). Whether e-cig users have increased respiratory symptoms, inflammation and altered gene expression profiles compared to non-users will be evaluated. Combining Aims 1 and 2 cohorts (n= 300), Aim 3 will employ the use of Bayesian and causal mediation methods to assess if CCOC exposure is positively associated with and explains, at least in part, the respiratory effects from e-cig use. With the proliferation of newer e-cig devices, there is an urgent need to characterize exposure and respiratory health effects resulting from their use. This study has the potential to generate critical data to inform FDA regulation to limit adverse exposures and health outcomes and curb the increasing prevalence of use among young adults. Through this research, my didactic coursework, and the guidance of my mentoring team consisting of a pulmonologist, exposure scientist, immunologist, analytical chemist, and environmental epidemiologist, I will acquire critical skills needed to be a successful independent researcher in environmental health and tobacco control.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Emotion dysregulation, defined as the inability to regulate the intensity and quality of emotions, is a common transdiagnostic symptom of mood disorders in early adolescence and a major risk factor for youth suicide. Evidence indicates that sleep disruption may be a driver of emotion dysregulation, and that sleep indices such as poor sleep efficiency – a greater time spent awake during attempted sleep – both precede and predict the onset of mood disorders. It is therefore critical to understand the neurodevelopmental mechanisms through which sleep disruption relates to emotion dysregulation during adolescence. Prior studies have linked alterations in the Default Mode Network (DMN) to both sleep disruption and emotion dysregulation. In parallel, findings indicate that sleep significantly impacts synaptic plasticity mechanisms crucial for brain maturation. Importantly, the protracted maturational program of the DMN may render this network particularly vulnerable to sleep disruption during adolescence; however, this has not been directly tested. Recent methodological advances provide a strong premise for the relevance of low frequency fMRI fluctuation amplitude as a non-invasive marker of developmental plasticity. Using this measure, our lab recently demonstrated a period of increased plasticity in the DMN during early to mid-adolescence, indicating that the DMN is highly malleable during this period. However, no work has comprehensively characterized the impact of sleep disruption on DMN developmental plasticity and emotion dysregulation in humans. In response to this gap, the current proposal will test the overarching hypothesis that poor sleep efficiency in early to mid-adolescence blunts developmental plasticity in the DMN and is associated with emotion dysregulation. To test this, the current proposal will apply advanced neuroimaging approaches to two complementary datasets. In Aim 1, cross-sectional relationships between sleep efficiency, emotion dysregulation, and DMN plasticity will be defined in a prospectively collected sample (ages 11-16) of 100 individuals with mood disorders and 50 typically developing comparators. This sample will include high-precision phenotyping not available in large-scale data, including digital phenotyping of emotion regulation and next generation multi-echo fMRI sequences that enhance sensitivity. Aim 2 will delineate how sleep efficiency impacts longitudinal development of plasticity in the DMN and emotion dysregulation by leveraging the large-scale longitudinal Adolescent Brain Cognitive Development (ABCD) Study (ages 11-16). Both datasets will include wristwatch-recorded sleep, providing objective measures of sleep efficiency. Taken together, findings will provide critical new insights into the neurodevelopmental underpinnings of sleep disruption and emotion dysregulation. Supported by a strong mentorship team with complementary expertise (Drs. Satterthwaite, Mackey, Gehrman, Shou, and Barch) and the world-class resources of the University of Pennsylvania, this proposal aligns with a cohesive training plan that will form the ideal foundation for an independent career at the intersection of neurodevelopment, sleep, and computational psychiatry. 1

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY Cells are exquisitely sensitive to the mechanical properties of their environment, altering fundamental processes like adhesion, migration, cell division, and cell fate specification in response to how the polymers comprising the extracellular matrix (ECM) and surrounding cells react to applied forces. Thus, environmental mechanical cues serve as crucial drivers of development, homeostasis, tissue regeneration, and disease progression. To understand the molecular mechanisms underpinning cellular mechanosensing, engineered systems are required that can decouple the influence of multiple confounding parameters, such as matrix stiffness (elasticity), viscous force dissipation, plastic deformation, microstructure, and adhesive cues. While there is an extensive body of literature exploring how cells sense and respond to stiffness and, increasingly, viscous force dissipation, present materials systems used in these studies are only capable of independently controlling one or two mechanical parameters at a time and make use of chemistries that can react with biologically relevant molecules, leading to altered material properties over time and potential off-target effects on cells. My lab leverages interdisciplinary expertise in bioorthogonal chemistries, protein engineered biomaterials, and stem cell biology to develop new platforms to study fundamental mechanisms of cellular mechanosensing under physiologically relevant conditions. This includes developing new chemistries and hydrogel materials to enable simultaneous, independent, and dynamic tuning of matrix stiffness, viscous force dissipation, and presentation of cell adhesive cues within 3D organotypic ensembles of cells. Recent efforts have focused on the development of highly- selective, stimuli-responsive chemistries to alter the stiffness and force dissipation rate of hydrogel materials on demand to model changes that occur in various diseases and during aging. We have also developed new protein engineered materials with genetically encoded viscoelasticity and are applying these materials to develop chemically-defined and highly tunable 3D organotypic culture platforms. In this proposal, we extend our work by developing new bioorthogonal chemistries that will enable tuning of viscoelastic force dissipation without off- target chemical reactivity, permitting casual relationships to be identified in complex systems over long culture durations without deterioration of material properties. We will also introduce new mechanically-labile crosslinking chemistries to provide additional modes of plastic deformation induced by cells. Finally, we will address a limitation of cellular force generation measurement techniques in native-like viscoelastic materials by developing new force sensors through protein engineering and chemoenzymatic modifications. The platforms developed in this proposal will be broadly useful to elucidate the molecular mechanisms by which cells sense and respond to changes in their mechanical microenvironment and to study how these changes drive desired phenotypes during development and tissue regeneration and undesired phenotypes during aging and disease progression.

NIH Research Projects · FY 2024 · 2024-09

Project Summary Chromatin architecture plays important regulatory roles in gene expression and is critical to maintain cellular identity. However, there are many unanswered questions regarding the interplay between chromatin architecture and transcriptional gene regulation, specifically regarding how one influences the other and what factors are involved in orchestrating long-range regulatory chromatin contacts. My proposal attempts to address these questions by focusing on an understudied architectural factor called Ldb1. Ldb1 has been studied at select loci and can facilitate long-range chromatin interactions between regulatory elements and target genes. The direct role of Ldb1 in facilitating chromatin architecture genome- wide to drive lineage-specific gene expression profiles during hematopoiesis has not been clearly defined. We will characterize Ldb1’s role in these processes using a well-studied cellular model system for erythroid differentiation. We will measure genome-wide features of chromatin architecture and Ldb1 genomic occupancy during the dynamic process of erythroid maturation. Additionally, we will acutely deplete Ldb1 to test how and to what extent it is required to maintain chromatin architectural features and gene expression profiles before and after terminal erythroid differentiation. Additionally, we will exploit the natural and highly dynamic processes that occur during cell cycle progression to test Ldb1’s role in establishing chromatin architectural features genome-wide. Mitosis is marked by the eviction of transcription factors, dissolution of most chromatin structure and a global cessation of transcription. During exit from mitosis, daughter cells must re-establish 3D chromatin architecture and transcriptomes that reflect the cell identity of the mother cell. Many features of chromatin architecture arise during mitotic exit through unknown mechanisms and are independent of well-studied architectural factors such as CTCF and Cohesin. We will profile Ldb1 genomic occupancy at closely spaced timepoints during the mitosis-G1 phase transition to determine if its binding dynamics are correlated with the formation of chromatin structures. Furthermore, we will test the requirement of Ldb1 to establish features of chromatin architecture by selectively depleting it in mitosis and measuring cell cycle progression and chromatin architecture reformation genome-wide. By using two natural cell state transitions (erythroid differentiation and cell cycle progression) in addition to acute degradation, we will glean new insights into the underlying mechanisms of chromatin architecture and how they drive lineage-specific gene expression.

NSF Awards · FY 2024 · 2024-09

NON-TECHNICAL SUMMARY: Enzymes are important molecules in biology. Enzymes are catalysts that convert substrates to products and are essential for driving processes that allow cells to function and respond rapidly to environmental conditions. There are a wide variety of enzymes in Nature corresponding to the diverse array of chemical reactions that a cell must execute for proper physiological function. It has been found that enzymes can exert forces when they convert substrates, and in this project, these forces will be harnessed to drive the motion of cell-sized capsules in response to substrates. The capsules will be made from biocompatible polymers, so they can be used to deliver drugs and communicate with cells by secreting bioactive compounds. Microfluidics will be used to assemble biocompatible capsules of defined chemistry, size, and composition. It has been found that asymmetry – either in the chemistry or the geometry of the capsule, or both – is required for robust motion. Asymmetry can be systematically built into the capsules using capsule chemistry and microfluidic design. A wide variety of enzymes will be tested to understand how the mechanism of action of each enzyme relates to its ability to support the propulsion of capsules. Capsules of tailored asymmetry with two faces – Janus capsules – will be used to understand how geometrical asymmetry can drive capsule motion via enzyme turnover. Systems in which capsules can communicate by secreting substrates to activate the motion of nearby enzyme-laden capsules or real biological cells will be developed, and furthermore, the motion of capsules in gradients of substrate will be measured. The project will train two graduate research assistants and two undergraduates, and the investigators will communicate their ideas to the broader research community through demonstration lectures to middle school and high school students and faculty. TECHNICAL SUMMARY: Enzymes are a diverse set of molecules that catalyze a host of biochemical reactions throughout biology. Enzymes are known exert forces during enzymatic turnover, and previously catalase and urease were used to drive the motion of biocompatible cell-sized capsules. Based on the hypothesis that propulsion is due to osmotic influx at the catalytic binding site (osmophoresis), a wide array of enzymes will be tested to relate fundamental features of enzyme activity (reaction rates, Michaelis constants, and reaction schemes) to capsule propulsion. Of particular interest are cleaving enzymes, such as amylase and nucleases, which are hypothesized to generate augmented osmotic forces and hence avid propulsion. Furthermore, higher order cell behavior, such as chemotaxis and multicellular organization, will be recapitulated with enzyme-functionalized microcapsules. The microcapsules are made by microfluidics using biocompatible polymers (poly-lactic-co-glycolic acid), allowing the control the size, composition, porosity and asymmetry of the capsules. Asymmetry in capsules chemistry and geometry has been demonstrated to enhance capsule motion. The aims of this project will be to measure the motility of single capsules across a range of enzyme-substrate systems and measure the dynamics of motion of asymmetric (Janus) capsules; to quantify the directional motion of capsules in gradients of substrates, analogous to the chemotactic motion of cells; and to determine the ensemble motion of capsules, both with different volume fractions of active particles, as well as mixtures of active and passive particles, to understand how motility can be used to separate and organize multi-particle assemblies. Another goal is to build communication systems in which one capsule can secrete a substrate and drive the motion of a neighboring capsule. Finally, we will understand how motile, synthetic capsules can communicate chemically and physically with biological cells. The capsules can be thought of as synthetic cells (protocells) inspired by and designed to mimic biology. Since the capsules are biocompatible, a host of applications, such as targeted drug delivery and tissue assembly, can be envisioned in which these motile protocells can interface with real biological cells and tissues. 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.