Yale University

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY: The mammalian lung has the capacity to repair itself following various injuries. Alveolar repair is a dynamic and coordinated process whereby stem/progenitor cells in the lung undergo differentiation into specialized cells to repair the damaged epithelium. Recent studies have uncovered a distinct intermediate progenitor cell state that exists during the transition between stem/progenitor cells and these specialized cells; however, the dynamic cellular behaviors and molecular regulatory landscape that drives intermediate progenitor cell transitions toward repair is poorly understood. Here, we propose two aims to dissect the cellular and molecular mechanisms that control alveolar repair in vivo in the regenerating mammalian lung. First (Aim 1) we will utilize a permanent lung imaging window system to track the emergence, live behaviors and terminal differentiation of individual intermediate progenitor cells over time during alveolar repair. Second (Aim 2) we will utilize combined scRNA-seq and scATAC-seq together with advanced dynamical analysis and machine learning techniques to define the cellular state space (gene expression and chromatin accessibility), cellular trajectories and regulatory landscape of transitioning intermediate progenitor cells. We will perform both aims using complimentary in vivo lung injury models and fluorescent report mice in order to track the mechanisms that are unique to intermediate progenitor cells and potentially dependent on their cellular origin and/or injury context. This project will generate extensive, high quality datasets to enable quantitative and predictive models of the key regulatory mechanisms that mammalian drive alveolar repair in vivo. Given that many of the cellular and molecular mechanisms of lung biology are conserved between mouse and human, our findings have the potential to uncover putative targets for modulating alveolar repair in the context of human disease.

NIH Research Projects · FY 2025 · 2023-01

Project Summary. The project proposes a rodent preclinical stroke therapeutics testing site at Yale for the NINDS program Stroke Preclinical Assessment Network (SPAN) to Support Translational Studies for Acute Cerebroprotection. A team of investigators including basic scientists studying mechanisms of injury after stroke, translational researchers, biomedical imaging specialists, and clinical trialists form a highly collaborative and interactive team dedicated to testing candidate cerebroprotectants selected by NINDS under the oversight of a national coordinating center. The overall goal is to identify the most promising candidates for novel stroke therapies to then be tested in clinical trials, advancing the care for stroke patients. In Aim 1 of the project we will establish the SPAN standard operating protocols and timeline for testing of the interventions. In Aim 2, we will test the efficacy of candidate cerebroprotectants in multiple rodent models of transient middle cerebral artery occlusion, including models incorporating aging and comorbidities. In an exploratory Aim 3, we will test innovative methods to improve the predictive value of the SPAN testing protocols to identify candidate treatments that will be efficacious in patients and enhance the impact of SPAN.

NIH Research Projects · FY 2025 · 2023-01

Bridge2AI: a FAIR AI BRIDGE Center (FABRIC) Overall Summary We propose to develop a FAIR Bridge2AI Center (FABRIC) to organize a large Consortium of institutions that will be generating data and tools to enable the application of novel AI algorithms to solve biomedical or behavioral problems affecting human health. Our center’s approach is to develop an Administrative Core that, together with a Teaming Core, designs and manages structural components of the Bridge2AI Consortium with a focus on team science and inclusion. The products and activities of each of four Cores are led by experts in their respective fields, and who have an extensive track record of working together in large NIH-funded initiatives, such as BD2K, All of Us, etc. The Administrative and Teaming Cores are based at the University of California San Diego. The Ethics Core, led by Vanderbilt University, will oversee all ethical, legal, and social implications (ELSI), generate guidelines and assess fairness in data from Data Generation Project (DGP) and other Consortium products. The Standards Core, led by University of Texas Health, will ensure that all products and activities follow the FAIR (findable, accessible, interoperable, reusable) principles and are harmonized/standardized to agreed upon standards. The Tool Optimization Core, led by the Broad Institute, will use its experience developing large- scale software to build a platform for the whole Consortium, where Cores and DGPs can have their products available for other researchers. It will also optimize various tools that are deemed useful for Bridge2AI. The Skills and Workforce Development will develop and deliver educational materials and activities for the whole consortium and optimize software developed by others. A special emphasis for FABRIC is inclusion, as our vision is for a novel type of Coordinating Center where there is no single point of control and in which transactions (e.g., data access, data use) are immutably recorded in an easy-to-access ledger that is viewable by all. FABRIC is designed so that various threads in Cores and DGPs are interwoven into a durable, sustainable center for the Bridge2AI Consortium.

NIH Research Projects · FY 2026 · 2023-01

Project Summary The research proposed in the R00 phase will be conducted in the Department of Neuroscience at Yale University School of Medicine. The project description has been updated to reflect changes in the research from the original submission. The ability to remember past personal happenings to guide proper behavior and make correct decisions is essential to our everyday life. Memory transience observed in neurodegenerative disorders such as Alzheimer’s disease has severe afflicting implications. Hippocampal circuits have long been posited to mediate spatial information necessary for episodic memory processes. At the cellular level, a striking and behaviorally relevant form of spatial information can be found in the receptive fields of CA1 place cells which collectively produce a representation of the external world. However, it remains unknown how hippocampal circuits operate to give rise to these representations, coordinate their output at the population level and broadcast this information to the rest of the brain. Addressing these questions would serve as a major step towards a deeper mechanistic understanding of the hippocampus and allow for the development of targeted approaches to prevent the devastating consequences of hippocampal circuit dysfunctions in various brain disorders. In this proposal, I will leverage new electrophysiological, imaging, tracing and optogenetics methods to interrogate the functional organization of hippocampal circuits across multiple scales in awake behaving mice. The research plan is organized in three aims, split across a K99 training phase and a R00 independent phase. In the first aim (K99), I will initiate monosynaptic retrograde rabies tracing from a single CA1 neuron and record the activity of its inhibitory presynaptic partners with volumetric random access 2-photon calcium imaging during behavior. This will allow me to directly test the contribution of local inhibition on sculpting the input/output function of individual pyramidal cells. In the second aim (K99/R00), I will use patterned optogenetics manipulations with single-cell precision to produce network wide circuit maps between principal neurons and distinct inhibitory cell types. I will use it to identify core circuit motifs shaping the CA1 network structure and regulating the circuit’s specialized operations. In the R00 portion of the award, I will test whether the local organization of CA1 circuits helps dispatching and transferring spatial representations to brain regions downstream of the hippocampus. During the K99 phase, the work was conducted in the Zuckerman Mind Brain Behavior Institute at Columbia University under the supervision of Drs. Attila Losonczy, Stefano Fusi, Liam Paninski, Darcy Peterka and Ashok Litwin-Kumar.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT Stroke affects 800,000 Americans every year and remains a leading cause of long-term disability. Increased blood pressure variability (BPV) has consistently been associated with two to three times higher risk of disability or mortality after acute ischemic stroke (AIS) in retrospective analyses, independent of mean blood pressure. Our central hypothesis is that increased BPV is harmful after AIS and warrants reduction. However, prior BPV research in AIS patients has been retrospective and limited by non-standardized BP measurement and, therefore, BPV is not mentioned in current stroke guidelines. To address the limitations of prior BPV research, determine mechanisms of BPV's deleterious effect, and identify potentially effective methods to reduce BPV, the proposed study will: 1) prospectively validate that “short-term” and “long-term” BPV after AIS onset is associated with functional outcome and define the effect size of different levels of BPV, 2) utilize portable MRI to confirm that final infarct volume, infarct growth and hemorrhagic transformation between baseline (measured within 12 hours of hospital arrival) and 72 hours are mechanistically related to BPV, and 3) utilize bedside pupillometry to determine how the autonomic nervous system contributes to BPV after AIS and evaluate the class effect of antihypertensive medications on BPV. To achieve these goals, we will enroll 150 patients who have anterior circulation stroke and a baseline NIH Stroke Scale ≥6 within 12 hours of AIS onset at three study sites. With completion of the Aims, we will define the outcome for a future trial (disability at 90 days vs. infarct volume or HT vs. post-stroke cognitive impairment at 12 months vs. composites), the effect size of BPV on individual outcomes and composites, the duration for lowering BPV (24-72 hours vs. weeks or months), and potential interventions to reduce BPV. Pharmacologic BPV reduction would be an inexpensive and widely available intervention, able to be administered in a range of healthcare settings. By completing the proposed Aims, we will be ideally positioned to test accessible targeted interventions to diminish the morbidity and mortality of AIS.

NIH Research Projects · FY 2025 · 2023-01

Project Summary Large data sets are important in the development and evaluation of artificial intelligence (AI) and statistical learning models to predict morbidity, mortality, and other important health outcomes. Healthcare institutions are stewards of their patients’ data, and want to contribute to the development, evaluation, and utilization of predictive analytics tools. However, they also know that simple “de-identification” per HIPAA rules is not sufficient to protect patient privacy. Additionally, other factors such as protection of market share, lack of control about who uses shared data for what purposes, and concerns about patients’ reactions to having their data shared without explicit consent make initiatives such as certain registries and centralized repositories difficult to implement. We have shown that it is possible to decompose algorithms so that they can run on data that stays at each healthcare center, thus mitigating the concerns about control and potential misuse. In the first phase of this project, we concentrated on demonstrating the accuracy and performance of these algorithms for the study of chronic diseases in which (1) acquisition of new knowledge about the condition is slow (i.e., the disease is well understood, so scientific discoveries are not being published at a rapid pace); and (2) the incidence and presentation of the disease do not vary dramatically from place to place, and from person to person. In this competitive renewal, we propose to develop decentralized predictive models that meet all requirements for chronic diseases, but the methods are also applicable to rapidly evolving acute conditions such as COVID-19. We propose new approaches to deal with sites that may be missing certain patient profiles or certain variables but can still participate in model learning, evaluation and implementation. These new AI algorithms will permit supervised and unsupervised learning across institutions, using data from multiple modalities (e.g., imaging, genomes, laboratory tests), and will allow privacy-protecting record linkage. We will test these algorithms and approaches in data from three highly diverse medical centers across the US: Emory University in Atlanta, University of Texas Health Science Center at Houston, and University of California, San Diego.

NIH Research Projects · FY 2026 · 2022-12

Patients with diabetes mellitus (DM) remain at high risk for the development of significant co-morbidities, specifically those associated with autonomic dysfunction, peripheral artery disease (PAD), and cardiovascular complications with a 2 to 10-fold higher mortality rate. PAD may manifest as stress induced leg pain (claudication) due to vascular insufficiency or critical limb ischemia (CLI) as the most severe manifestation of lower extremity PAD characterized by lower limb ischemic rest pain and/or the presence of tissue loss. The sympathetic nervous system plays a critical role in the normal autoregulation of the vasculature, and loss of vasomotor control is responsible for postural hypotension but also for the remarkable increase of peripheral blood flow and arteriovenous shunting in the neuropathic diabetic foot. Therefore, sympathetic denervation increases blood flow, as it results in vasodilation and non-nutritive, arterio-venous shunting. Sympathetic denervation may cause structural damage to peripheral arteries resulting in degeneration of arterial medial smooth muscle with subsequent medial artery calcification (MAC) a feature of diabetic neuropathy. Positron emission tomography (PET) imaging with 82Rb provides high-sensitivity and high-resolution images for quantification of absolute flow, while a 18F-labeled norepinephrine analog (18F-LMI1195) provides information about sympathetic activity. This project will optimize and apply dual isotope (82Rb/18F-LMI1195) hybrid PET/CT imaging for evaluation of lower extremity flow and denervation and vascular calcification in pre-clinical models of PAD and in patients with DM and PAD. We propose a clinical imaging sub-studies of two active multi-center observational registry called SCOPE-CLI and PORTRAIT, which were designed to phenotype patients with CLI and claudication, respectively. These registries collect observational data on treatment patterns, and other outcomes that are relevant to patients with DM and PAD. In Aim 1, our hybrid PET/CT imaging approach will be optimized using a porcine model of hindlimb ischemia in the presence and absence of an acute peripheral nerve block. These studies will establish methods for evaluation of skeletal muscle rest and stress flow and flow heterogeneity. Aim 2 will apply dual isotope PET/CT imaging for quantification of acute and chronic changes in regional flow and flow reserve before and after regional denervation of the lower extremities in relation to development of MAC in a chronic rabbit model of peripheral denervation. In Aim 3 we will translate this approach to patients with DM and a spectrum of PAD disease severity to evaluate the prognostic value of hybrid PET/CT imaging of lower extremity for predicting progression of MAC, rates of amputation, and major adverse cardiovascular events. The proposed multi-isotope PET/CT imaging of lower extremity flow and innervation developed and applied in this project in conjunction with imaging of vascular calcifications will characterize the pathophysiology of autonomic dysfunction in critical limb ischemia and this information may lead to a paradigm change in the evaluation and long-term management of patients with DM and PAD.

NIH Research Projects · FY 2025 · 2022-12

PROJECT SUMMARY The thirteen-lined ground squirrel is an obligate hibernator that seasonally drops its body temperature from 37° in the active state to 2-4°C during torpor. While such temperatures typically inhibit peripheral signaling in mammals, torpid squirrels remain sensitive to tactile cues and can be awakened by the sense of touch (mechanosensation). However, the neurobiological mechanisms involved remain unknown. The long-term goal of this study is to identify the cellular and molecular signatures that afford cold-resistant mechanosensation to support extreme thermal tolerance in mammals. Pressure stimuli are sensed by a specialized subset of neurons in the dorsal root ganglion (DRG) known as mechanoreceptors. Mechanically gated ion channels such as Piezo2 convert pressure into ionic current that activates voltage-gated ion channels, which relay the signal downstream through action potentials (APs). Piezo2 and voltage-gated ion channels have temperature-dependent dynamics ripe for evolutionary manipulations permitting thermal resistance. This study, which will be conducted at the laboratories of Drs. Elena Gracheva and Slav Bagriantsev at Yale University, assesses the central hypothesis that ion channel modifications in mechanoreceptors underlie cold-adapted mechanosensation in the thirteen-lined ground squirrel. Past work from the Gracheva and Bagriantsev labs has shown that this species shows constitutively low thermosensitivity and decreased function of nociceptive AP machinery during torpor. Our preliminary work also shows that mechanosensitive currents are preserved during torpor. Accordingly, we use a combination of in situ hybridization, RNA sequencing and electrophysiology to assess whether squirrel DRG neurons have selectively potentiated mechanosensitivity at the cost of other sensory modalities, relative to mouse DRG neurons (Aim 1). Moreover, cold exposure widens APs and disrupts voltage gradients in typical mammalian neurons due to slowed kinetics of the Na+/K+ ATPase pump. In contrast, our preliminary work shows that cold-exposed torpid neurons show decreased AP widening and intact resting membrane potential. In light of these data, we use whole-cell and ex vivo electrophysiology to assess whether squirrel mechanoreceptors have cold-resistant electrogenic machinery (Aim 2). To the best of our knowledge, this study represents the first attempt to dissect cellular mechanisms of cold- adapted mechanosensation in mammals. Elucidating neural function in extreme cold has the potential to provide novel targets for combatting neuronal damage following clinical hypothermic procedures.

NIH Research Projects · FY 2025 · 2022-12

Project Summary/Abstract PTEN is highly associated with autism, macrocephaly, and congenital hydrocephalus, which are increasingly prevalent neurodevelopmental disorders that present in early childhood. Currently, there are no treatments that address the cause of these conditions, despite mounting genetic evidence through large scale whole exome sequencing studies that mutations in specific genes, including PTEN, confer increased risk for these disorders. While these studies produce compelling targets for investigation, the role of PTEN in neurodevelopmental disorders remains poorly understood. We seek to understand how PTEN, through its role as a negative regulator of the greater mTOR pathway, contributes to early neurodevelopment using CRISPR-Cas9 loss-of-function models in zebrafish. We have generated zebrafish mutants with frameshift mutations in exon 5 of both pten alleles present in the teleost duplicated genome: ptena (Δ10) and ptenb (Δ2). We first aim to characterize neurodevelopmental abnormalities in these fish, which preliminary data indicates have significant differences in brain volume, brain activity, brain ventricle size, and startle response. We will further explore the function of PTEN in regulating cell proliferation, differentiation, and establishment of the excitatory and inhibitory circuits in the brain. Secondly, we aim to use this model to provide a new and accessible tool for screening the PTEN variants identified in children with neurodevelopmental disorders, by injecting embryos with human mRNA constructs at the 1-cell stage and evaluating neurodevelopmental changes. Lastly, we will perform a high-throughput drug screen of mTOR pathway inhibitors and additional compounds which oppose or match the behavioral changes observed in our mutant larvae. This will identify new potential pharmacological candidates. Our preliminary data already shows partial phenotype rescue upon treatment with mTORC1-inhibitor sirolimus. In summary, these loss of function lines demonstrate PTEN plays a critical role in early vertebrate neurodevelopment. Furthermore, these models provide a new and accessible tool for screening the PTEN variants identified in neurodevelopmental disorders as well as potential pharmacological candidates.

NIH Research Projects · FY 2026 · 2022-12

The WHO listed air pollution as one of the top ten threats in 2019, and earlier research indicates links between weather exposures and brain health. Further, the burden of older persons with Alzheimer’s disease (AD) and related dementias (ADRD) is expected to double by 2060. Simultaneously, wildfires are a substantial contributor to extremely high levels of air pollution . To date, little is known regarding impacts of heat or air pollution, including wildfire smoke, on the elderly with AD/ADRD. Most studies on environmental exposures and related vulnerabilities investigated a single factor at a time rather than the real-world settings characterized by multiple factors (co-occurring air pollution and heat, frailty, chronic conditions). Further, previous studies have not leveraged recent developments in satellite imagery, machine learning, and causal inference methods, which can increase the rigor and validity of statistical analysis. We propose to address these scientific gaps using a large, validated cohort of US Medicare beneficiaries (>65y) with AD/ADRD (approx. 10 million for the period 2000-2019) and spatially resolved weather data combined with state-of-the-science machine learning for estimates of air pollution exposure, which leverages satellite imagery, land use data, and monitors. Our long-term goals are to characterize the vulnerability and health impacts of weather-related exposures within a large cohort of older adults with AD/ADRD. First, we will estimate the impacts of short-term exposure to heat and heatwaves on cause-specific hospital admissions, readmissions, mortality, and a novel patient-centered outcomes of days-at-home, and develop machine learning algorithms to identify which subpopulations with AD/ADRD are most vulnerable with respect to several individual- and community-level factors (e.g., sex, chronic conditions, frailty). Next, we will estimate vulnerability of older persons with AD/ADRD to air pollution including wildfire smoke using our state-of-the-science approach to estimate air pollution and wildfire smoke exposure. We then estimate the impacts and vulnerabilities from co-occurring heat and air pollution (including heat waves and wildfire smoke) by developing Bayesian hierarchical spatio-temporal models to quantify synergistic effects. Finally, we will disseminate all methods, exposure data, and statistical software, making them publicly available free of charge. Characterizing the factors that increase vulnerability for older persons with AD/ADRD will allow decisionmakers to design effective interventions. Findings will inform impact assessments of the health burden of environmental exposures, specifically heat and air pollution including wildfires, and for understanding environmental health. Our results will have implications for management of health during co-occurring heat and air pollution events, including wildfires, and increase knowledge on weather and air pollution preparedness, response, and recovery.

NIH Research Projects · FY 2026 · 2022-12

Project Summary: Nephrotic syndrome (NS) is characterized by proteinuria and is associated with podocyte actin cytoskeletal disorganization termed foot process effacement (FPE). Podocytes are incapable of self- renewal, and podocyte loss above~40% per glomerulus associates with glomerulosclerosis (FSGS) and kidney failure. Distinct from FSGS, Minimal Change Disease (MCD) also shows diffuse FPE, but has preserved podocyte numbers, and is highly treatment responsive with a low rate of progression to ESRD (5-20% in 20 years). FSGS has been associated with glomerulomegaly and podocyte hypertrophy in later stages. However, early FSGS can be morphologically indistinguishable from MCD and a debate exists whether some MCD cases transition to FSGS, representing a “switch” between diseases. Hence, understanding signals specific to MCD will reveal mechanisms facilitating podocyte survival and preventing a phenotype “switch”. Interestingly, Fyn kinase inactivation was specifically identified in human MCD. In mice, Fyn inactivation (by Shroom3 silencing) also associated with FPE without podocytopenia - an “MCD-like” pathology. Hence Fyn inactivation was a candidate MCD-unique signal. Downstream of Fyn-inactivation, investigation of anti-hypertrophy and pro- survival pathways in podocytes revealed enhanced activation of AMP-kinase, explaining these effects. Fyn inactivation activated AMPK by increasing cytoplasmic efflux of LKB1. Moreover, inhibition of Ampk in MCD-like mice induced podocyte loss, glomerulomegaly and FSGS, while AMPK activation prevented podocyte loss after glomerular injury induced by hypertrophy and direct toxins. Invitro data show increased autophagy as the central pro-survival mechanism in podocytes after AMPK-activation. We hypothesize that in the context of injury causing podocyte FPE, AMPK signaling regulates the “switch” between MCD and FSGS by enhancing autophagy and preventing podocytopenia. In this proposal, we will test the role of podocyte AMPK signaling in MCD vs FSGS, and establish downstream mechanisms regulating podocyte survival. In Aim I, we will use genetic and pharmacologic model systems to specifically inactivate or activate AMPK to induce phenotype changes from MCD-to-FSGS and vice versa. In Aim-II, we will modulate autophagy in podocytes while activating AMPK to show the central role of AMPK-mediated autophagy in podocyte survival. We will also specifically examine the role of autophagy in restricting glomerulomegaly during injury. Finally, in Aim-III, applying state-of-the-art and multidimensional technologies to the largest NS cohort in the US, we will investigate the specific role of AMPK signaling in human MCD vs FSGS. Our work will provide novel MCD-FSGS diagnostics, and develop novel AMPK therapeutics as well as help repurpose FDA-approved AMP-activators.

NIH Research Projects · FY 2026 · 2022-12

Project summary Thermoregulation is a fundamental process that affects virtually all aspects of animal physiology. In mammals, core body temperature is monitored by neurons in the preoptic area of the hypothalamus. Activity of these neurons determines the neuronal output that drives thermoregulation and controls body temperature. Despite its fundamental physiological significance, the molecular basis of temperature homeostasis under normal, adaptive and pathological conditions remains obscure. Mammalian hibernation is a cyclical reprograming of a thermoregulatory phenotype. This process is associated with dramatic physiological perturbations: heart and respiration rate decrease, core body temperature drops from 37°C to 2- 4°C. Upon arousal from hibernation, all physiological parameters return to normal within hours. This remarkable plasticity suggests the presence of specific adaptations in the thermoregulatory system of hibernators, but the underlying cellular and molecular mechanisms remain enigmatic. In this basic scientific proposal, we will employ a comparative multidisciplinary approach to understand cellular and molecular principles of reversible hypothermia at the level of POA, using a novel hypothermia-tolerant animal model, hibernating thirteen-lined ground squirrel, and laboratory mice, a non-hibernating species. Understanding how hibernators achieve reversible hypothermia will facilitate the development of pharmacology to induce and regulate hypothermia in mammals.

NIH Research Projects · FY 2026 · 2022-12

PROJECT SUMMARY Psychiatric and neurodegenerative disorders are highly heritable and debilitating brain diseases that together affect nearly fifty million Americans, caused by the complex interaction of genetic and environmental risk factors. Although genomic studies indicate that much of disease risk reflects the aggregate impact of hundreds of genetic variants, to date, a substantial proportion of both the heritable and environmental components remain unexplained. A major challenge in the field has been illuminating the pathways connecting genetic variants (the vast majority of which fall in non-coding sequences) to target genes and causal cellular phenotypes, particularly in a cell-type-specific and context-dependent manner. We previously uncovered an unexpected combinatorial effect between risk genes that was not predicted from single gene perturbations, one that concentrated on synaptic function and linked the rare and common variant genes implicated in psychiatric disease risk. Based on our preliminary analyses and the work of others, we hypothesize that impact of genetic variants and stress converge and interact to impact critical neuronal and glia functions. Here our objective is to evaluate psychiatric and neurodegenerative risk variants, investigating convergent relationships between risk variants and stress across the major cell types of the brain. To do this, we will functionally dissect the impact of genetic variants significantly associated with brain disease, exploring their regulatory impact across cell types (neurons, astrocytes, glia) and contexts (physiological and environmental stressors) (Aim 1). To extend these insights, we will explore additive effects between risk variants and stressors at the level of network expression and cellular function (Aim 2). Finally, to test the extent that these insights might result in clinically actionably information, we will the clinical consequences of gene-environment interactions across two large healthcare and population- based biobanks (Aim 3). The translational impact of our work includes potential improvements to additive polygenic risk scores, prioritization of convergent genes for mechanistic follow-up, and identification of pathways that might serve as potential therapeutic targets. Our overarching goal is to advance the field towards an era of precision medicine, whereby not just each patient’s genetic variants, but also the expected interactions between them, can be used to predict disease trajectory and potential therapeutic interventions.

NIH Research Projects · FY 2025 · 2022-11

The goals of this R01 Renewal are driven by the results of our successfully conducted randomized controlled clinical trial comparing endoscopic third ventriculostomy combined with choroid plexus cauterization (ETV/CPC) to shunt treatment for post-infectious hydrocephalus (PIH, the most common hydrocephalus etiology in Uganda) in infants < 6 months of age (ClinicalTrials.gov NCT01936272, R01HD085853, R21HD068213) at the CURE Children’s Hospital of Uganda (CCHU). The interim outcomes of this study are becoming a landmark in children’s medicine, finding that: a) at up to 24 months, there was no significant difference between ETV/CPC and shunt in regard to failure rate, developmental outcome, and brain growth; b) brain volume, not CSF volume, correlated strongly with developmental outcome; c) successful treatment of the hydrocephalus led to improved brain growth in the first year, which stagnated during the second year. Because almost all of the failures of endoscopic treatment occurred early in the first year of life, we anticipate that the longer term outcomes of hydrocephalus treatment may favor endoscopy if we follow these children through primary school age from 5 to 10 years. Furthermore, our ability to perform much more detailed neurocognitive assessments during this further followup will be much more accurate than during our pre-school assessments. Lastly, we can gather all of our data and perform a much more definitive cost-effectiveness analysis if we can follow this unique cohort for a full 10 years. We have engaged in substantial capacity building during the previous 5 years of support during this clinical trial. We have developed a thriving research unit at the CURE Children’s Hospital of Uganda, enabling high-quality research to be conducted in multiple other NIH funded studies unrelated to this clinical trial (DP1HD086071, R01AI145057, and R01HD096693). Furthermore, since all hydrocephalus treatment requires brain imaging, we have developed a thriving center for sustainable low-field MRI engineering at the Mbarara University of Science and Technology. This present project seeks to follow this unique cohort to 10 years of age, seeking to definitively establish whether endoscopic treatment is more sustainable for infants in the developing world, establish low field MRI as a sustainable imaging technology, and establish the capacity of both the medical and engineering sites in Uganda as independent research and technology development centers for Africa.

NIH Research Projects · FY 2025 · 2022-10

Project Summary INTRODUCTION. Face perception is central to social functioning and commonly disrupted in psychosis. Prior research found that face processing in the dorsal and lateral visual streams is modulated by visual behavior, pointing to the existence of a distributed face visual processing neural network which is an extension of visual acquisition and is a candidate for dysfunction in mental illness. Building upon the Bayesian Brain hypothesis which posits that the brain is an inference machine which models the environment to make predictions and drive behavior, we theorized that a lateral-dorsal network emerges during dynamic face viewing as a function of fixation behavior, enabling emotional expression recognition. Further, this network is a candidate driver of the emotion recognition deficits common in psychosis. AIM 1 is thus to test hypotheses that a fully connected lateral-dorsal network emerges during dynamic face viewing as a function of fixation behavior, using dynamic causal modelling (DCM). AIM 2 is to test hypotheses that this network is related to expression perception and social functioning, with disruptions emerging in those with psychosis and attenuated in those who are clinical high risk for psychosis(CHR). HYPOTHESES. A fully connected lateral-dorsal network is hypothesized to be the model of best fit for face viewing electrocortical data. Mean fixation duration is posited to modulate the intra-lateral and lateral to dorsal connections as mean fixation is theorized to determine precision of bottom-up stimuli. Significant differences in behavior and parameter estimates are hypothesized to occur between those with and without psychosis, with the CHR group as an intermediate phenotype. A mixed effects model including parameter estimates for lateral edges and mean fixation duration is hypothesized to predict expression accuracy and social functioning across pooled subjects. APPROACH. Participants (n=30 each of those with and without psychosis, and those who are CHR) will view dynamic RADIATE faces11 and control videos normalized for brightness and movement, reporting perceived emotions per event. 128-channel scalp EEG and 1000hz binocular eye tracking will be acquired, supplemented by the Global Assessment of Functioning (GAF)12. Data will be simulated using biophysically realistic head and cortex DCMs13,14 with various connectivity patterns between lateral and dorsal streams, and compared to the recorded data. The best fit model will be identified with Bayesian model selection and single subject parameters will be estimated with Variational Bayes15. Average parameters and behavioral measures per group will be compared to identify significant differences. Mixed effects models will be generated to relate parameter estimates and mean fixation duration to emotion perception accuracy and GAF score. SIGNIFICANCE. These hypotheses may shed light on a core deficit in psychosis—social functioning—through identification of a mechanism of action—atypical acquisition of dynamic facial information leading to disrupted connectivity in a lateral-dorsal neural network and disrupting emotional expression perception. If true, it will enable new avenues for targeted treatment and symptom amelioration.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Glioma make up 80% of all primary malignant brain tumors. The current standard treatment for newly diagnosed gliomas includes maximal surgical resection, radiation therapy (RT), and chemotherapy. A key technical challenge in RT treatment planning is accurate target volume delineation of gliomas. The current clinical guidelines for target volume delineation rely primarily on structural Magnetic Resonance Imaging (MRI) images. Gross Tumor Volume (GTV) is defined based on contrast-enhanced T1-weighted MRI and T2-weighted MRI. However, structural MRI alone lacks specificity for delineation of true tumor boundaries. Accordingly, Clinical Target Volume (CTV) is often defined as the GTV plus a large margin (e.g., 20-25 mm) to account for possible microscopic infiltration. The lack of specificity of structural MRI is a critical factor limiting the investigation and clinical application of new RT techniques for better clinical outcome. MR spectroscopic imaging (MRSI) has long been recognized as a potentially powerful tool for label-free molecular imaging of brain tumor. In a recent Phase I clinical trial, MRSI is used to guide dose escalation in RT for Glioblastoma multiforme patients, showing very promising preliminary results. Although general clinical applications of MRSI have been impeded by its limited spatial resolution and long scan time, significant progresses have been made in addressing these technical challenges over the past decade using advanced data acquisition and processing methods. Our group have successfully developed a powerful MRSI technology, known as SPICE (SPectroscopic Imaging by exploiting spatiospectral CorrElation). SPICE effectively integrates rapid scanning, sparse sampling, quantum simulation of molecule resonance structures, and machine learning to enable rapid high-resolution MRSI. Preliminary results by our and other groups have shown an exciting potential of SPICE to achieve an unprecedented combination of resolution, speed, and SNR for metabolic imaging. We have also demonstrated, for the first time, the feasibility of mapping T1, T2 and proton-density parameters of brain tissues using the unsuppressed water signals from the SPICE scans. The primary goal of this project is to leverage this significant advance in MRSI technology and investigate the use of high-resolution metabolic and structural information to achieve more accurate target volume delineation for RT treatment planning of gliomas. We will: 1) further develop and optimize SPICE for MRI/MRSI-guided RT of gliomas in clinical settings, 2) perform systematic performance evaluation of the proposed method on phantoms, healthy subjects, and glioma patients, and 3) investigate the use of metabolic and structural biomarkers for delineation of biological target volume to improve image-guided RT of gliomas. The proposed research is innovative in developing a novel molecular imaging technology and a timely effort on improving RT treatment planning of gliomas with quantitative metabolic and structural biomarkers. Successful completion of the project will have a significant impact on image-guided RT for gliomas, opening up new opportunities for better control of recurrence in glioma patients using dose escalated RT.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death and soon to become the second in the next few years. Numerous epidemiologic studies have shown that obesity increases the risk of developing and dying of PDAC. Given the rise in worldwide obesity rates, a better understanding of the mechanisms by which obesity promotes PDAC progression is necessary. To study how obesity drives PDAC, our lab recently combined a well-established genetic model of obesity with an oncogenic Kras-driven pancreatic cancer model and showed increased tumor burden and decreased survival compared to non-obese controls. Obese mice exhibited aberrant expression of the neuropeptide hormone cholecystokinin (CCK) in pancreatic islet beta (b) cells, the latter of which was sufficient to enhance Kras-driven pancreatic tumorigenesis. These results uncovered a novel mechanism of obesity-driven PDAC by local hormonal signaling between endocrine islets and exocrine acinar cells. Therefore, my overall goal is to elucidate the cellular and molecular mechanisms by which islets adapt in response to obesity and in turn promote PDAC progression through endocrine-exocrine hormonal signaling. In Aim I, I will perform lineage tracing studies in vivo and in silico to identify the cell-of-origin that gives rise to b cells mis-expressing hormones, such as CCK. In Aim II, I will determine whether loss of transcription factors required for b cell identify lead to aberrant hormone expression in mouse insulinoma (insulin-producing) cells and primary human b cells using genetic knockdown experiments and chromatin immunoprecipitation. Lastly, in Aim III, I will perform in vivo gain-of-function and loss-of-function experiments using islet specific gene manipulation by adeno-associated viruses to evaluate the pro-tumorigenic potential of hormones beyond CCK that are overexpressed in b cells in obesity. Together, these studies will reveal novel endocrine adaptations that could be targeted to halt obesity-driven pancreatic exocrine tumorigenesis. In addition, through the acquisition of new technical skills in this project, extensive mentorship (from her sponsor, co-sponsor, and collaborators), interactions within an outstanding scientific environment, participating in advanced classes and workshops, and attendance and presentation at conferences and seminars, the comprehensive training plan will markedly broaden the applicant’s skillset in preparation to be a successful independent research scientist.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY/ ABSTRACT In multicellular organisms, germ cells provide all the material necessary to generate offspring, including both genetic instructions encoded in DNA and regulatory information that guides developmental gene expression. Importantly, germ cells must retain the potential to establish totipotency while also functioning as terminally differentiated cells. Epigenetic modifications are one mechanism that encodes information about germ cell- specific regulatory programs while also permitting retention of developmental plasticity. A specialized epigenetic state called bivalency exists in germ cells and embryonic stem cells (ESCs), and may help to balance the competing requirements for cell fate restriction and plasticity. At bivalent domains, two contradictory histone modifications occupy the same nucleosome in promoters of transcriptionally silent genes: trimethylation of lysine 4 on histone 3 (H3K4me3), which promotes transcriptional activation, and H3K27me3, which promotes transcriptional repression. Bivalency is established in promoter regions of developmental genes and is thought to ‘poise’ these genes for conditional expression during somatic lineage specification. However, despite its potential importance in regulating early development, there is currently a gap in our understanding of the molecular machinery that regulates bivalency and its functional contributions to germ cell biology, embryo plasticity, and development. The goal of this project is to discover cis- and trans- regulatory mechanisms that contribute to bivalency. Specifically, we will utilize transgenic mouse embryonic stem cells to test the hypothesis that distinct sequence elements are responsible for establishing bivalency and that there are proteins maintaining histone modifications specifically in a bivalent context. Experiments in Aim 1 will test the contribution of specific sequence elements to establishment and maintenance of bivalency using both candidate and unbiased approaches. First, we will evaluate the role of a putative CCCTC-binding factor (CTCF) binding site in regulating bivalency at a specific test locus, Traf6. Second, we will systematically interrogate sequence elements in the Traf6 promoter using clustered regularly interspaced short palindromic repeats (CRISPR) technology to systematically ablate short pieces of the promoter and determine which sequence motifs are necessary to establish bivalency. Aim 2 will identify trans-acting novel regulators of bivalent chromatin by using a genome- wide CRISPR screen in three mouse ESC reporter lines. Together, these experiments will identify both locus- specific and global mechanisms important for defining and maintaining bivalent promoters. These data will advance our understanding of the cis- and trans- regulatory control of bivalency and provide insight into the function of this chromatin state in development. Our results will have implications in germ cell function and fertility, epigenetic inheritance, and embryonic development and differentiation.

NIH Research Projects · FY 2024 · 2022-09

Summary/Abstract This application proposes a program of research, development, and evaluation of the use of consumers’ assessments of health care for informing health care choices and quality improvement. The goals of the program are organized in five areas: (1) Survey and supplemental item set development, revision of surveys, and trademarking; (2) Survey methods research aimed at achieving equity in patients’ experience of care; (3) Analysis and reporting of patient experience data, including assessments of how well different subgroups in the population are represented in survey responses; (4) Patient experience quality improvement studies based on CAHPS data; and (5) Developing internal and external program communication strategies to critical stakeholders. The proposed methods include focus groups, cognitive interviewing, interviewing of expert informants, establishing technical expert panels with nationally recognized experts, and field tests of draft surveys. The team will use standard psychometric statistical techniques to analyze survey data. The team will conduct experiments to assess reporting strategies and quality improve initiatives. The applicants are a consortium of experts in consumer survey design, report development, experimental assessment of consumer perceptions and medical care choice behavior, patient experience quality improvement, evaluation research, and communication and dissemination from the Yale School of Public Health, Mass General Brigham healthcare system and Harvard Medical School, the Center for Survey Research at the University of Massachusetts, the University of Pennsylvania, Columbia University, and several nationally known independent researchers and consultants. This consortium proposes maintaining continuity in the CAHPS development process by continuing critical activities and well-established collaborations from previous CAHPS projects and Centers for Medicare & Medicaid Services (CMS) projects, while strengthening the team to bring new skills, perspectives, and expertise to the project. The team has close partnerships with collaborating demonstration sites and has established relationships with public and private organizations that share an interest in refining and assessing CAHPS products for improved consumer decision making and quality improvement.

NIH Research Projects · FY 2025 · 2022-09

Neovascular age-related macular degeneration (AMD) is a neuroinflammatory disease that is a leading cause of blindness in the elderly. While the involvement of photoreceptors in AMD has been well established, our research will study the less understood role that inflammation plays in the development of AMD. Our proposal will address this knowledge gap and yield a detailed understanding of inflammation-associated AMD pathogenesis in humans, in addition to the identification of potential therapeutic targets and treatments for AMD. Our access to human tissue with advanced neovascular AMD from the Yale Rapid Autopsy Service and single-cell RNA sequencing expertise will allow us to perform studies that profile the transcriptome in activated innate immune cells. Our preliminary data indicate that the critical inflammatory pathways reside in microglia and monocyte-derived macrophages. Our overarching hypothesis is that functional changes in the innate system influence neovascularization in AMD, and these changes may be targeted to halt disease progression. To explore this hypothesis, we propose the following two specific aims. In Aim 1, we will perform highly parallel single-nucleus transcriptional profiling with a novel enrichment technique for glia from human eyes with exudative AMD. The primary goal is to define and interrogate the molecular signature of microglia and macrophages. Our preliminary data revealed activated microglia in AMD with secretion of the proinflammatory cytokine interleukin-1b. We hypothesize that there is as upregulation of inflammatory molecules, which is associated with choroidal neovascularization in AMD. In Aim 2, we will target the pro- inflammatory cytokines that regulate activation of reactive Müller glia in AMD to identify targetable pathways to reverse the chronic inflammation in disease. Based on preliminary data, our targets will include the IL-1b, IL-10, and IL-17 pathways as well as additional ones identified in Aim 1. We hypothesize that inflammatory molecules are critical for the transformation of homeostatic Müller glia to a reactive, pro-angiogenic state in AMD. The proposed research plan will provide unprecedented insight into the molecular mechanisms of AMD progression and has significant potential to identify novel therapeutic targets for drug discovery. We anticipate that our work will lead to the development of the first effective therapeutic approaches targeting inflammation, thereby improving the quality of life for individuals suffering from neovascular AMD.

NIH Research Projects · FY 2026 · 2022-09

Project Summary Since 1999, there has been a 400% increase in the rate of drug overdose (OD) deaths in the U.S., with over 70% of the deaths in 2019 related to opioids.The opioid crisis continues to worsen in the State of Connecticut (CT) for all racial/ethnic, gender, and age groups, with the number of overdose deaths increasing by 285% from 2012 to 2020. While the deployment of first responders in the field for overdose, including police, fire, and emergency medical services, provides life-saving resuscitation and naloxone, it is unknown whether other evidence-based interventions are available and being utilized. To date, we lack critical and actionable real-time data from first responders and emergency departments (EDs), including whether a treatment referral was offered to those who have overdosed, and from individuals who overdosed, such as time from overdose to treatment engagement. This real-time data could assist local authorities in predicting rates, timing, and location of overdoses, as well as the types of services needed. In response, our research team has partnered with the CT Department of Public Health (DPH) to develop a system dynamics (SD) model that allows us to assess the impact of key interventions, including the implementation of Good Samaritan Laws (GSLs) and the widespread distribution of naloxone, on important clinical outcomes, such the number of OD deaths. This model has been carefully calibrated for CT and has already been used to identify where data gaps are limiting the development of evidence-based interventions (e.g., absence of information about bystander use of naloxone during OD event, etc.) and to predict the clinical outcomes that can anticipated if specific policy changes or interventions are pursued. Our team has also developed a comprehensive telehealth platform that can be deployed in the field where the overdose occurred or in the ED with minimal time or effort by existing staff. This platform will provide real-time access to providers who prescribe medication for opioid use disorder (MOUD) and other harm reduction services for high-risk individuals, and we hypothesize that it will remove many of the barriers to follow up that these individuals face. Thus, the main objectives of this proposal are twofold: (1) To implement a novel, scalable, evidence-based, intervention (i.e., our telehealth platform) at the time of an opioid overdose that links people who have overdosed with access to medication for opioid use disorder (MOUD), harm reduction services, and recovery supports, and (2) to collect high-quality data about the processes and outcomes associated with deployment of this platform that can be integrated with our existing SD model to determine if, where, when, and what interventions should be implemented in the future. There is a great need to expedite and facilitate MOUD access and respond effectively to witnessed overdoses. Our long-term goal is to implement these novel SD modeling and telehealth strategies in CT, with subsequent dissemination nationally, ultimately improving access to MOUD and reducing OD events and fatalities.

NIH Research Projects · FY 2025 · 2022-09

ABSTRACT Bioinformatics skills and tools are critical for the analyses of vast amount of data generated by Next-Generation Sequencing (NGS) and have become an integral field for gaining insight into cancer biology. While laboratory protocols to generate NGS data have become more standardized, researchers find it difficult to bring expertise in bioinformatic data analysis into their practice. The Yale Cancer Center (YCC), one of the 51 NCI-Designated Comprehensive Cancer Centers in the nation, aims to understand and prevent cancer, detect cancer early, and manage cancer treatments more accurately and effectively. YCC is committed to fulfilling these goals by supporting collaborative research and education activities aimed at achieving breakthrough discoveries and training future leaders in cancer science and medicine. The YCC supports and manages Shared Resources that provide unique expertise and enabling technologies that enrich the scope and expedite progress for each of the YCC research programs. As Associate Director of Bioinformatics at the Yale Center for Genome Analysis (YCGA), one of the YCC Shared Resources, I established the bioinformatics analysis services for the YCC with the goal of bringing bioinformatics and NGS expertise closer to the cancer investigators. My bioinformatics work with them for the last 9 years has focused on three areas: 1) providing full “design-to-publication” bioinformatics support; 2) facilitating access to new genomic technologies and develop/build new analytical pipelines to enable cutting edge genomics; and 3) providing training and education in cancer bioinformatics through mentoring, seminars, and workshops. I have brought such expertise to over 300 projects for 78 laboratories, have developed new data analysis pipelines and enabled new NGS applications, and have trained many students and researchers to become proficient in their own bioinformatics analyses. During my work for the YCC, I have co-authored over 28 publications in leading scientific journals with YCC investigators, involving a diverse set of analysis for different techniques, cancer types, and organisms. The support of the R50 award will allow me to continue to develop and use bioinformatics tools to increase the progress and scope of cancer research and provide broader access and training to YCC researchers.

NIH Research Projects · FY 2026 · 2022-09

Modified Project Summary/Abstract Section Chronic diseases and infectious diseases have had a disproportionate impact on those who are incarcerated and work in our nation’s prisons and jails. High rates of disease in correctional systems spill over into surrounding communities, particularly in jails with rapid turnover and in neighborhoods where correctional officers reside. Improving respiratory preventive healthcare interventions in correctional systems is therefore an important community-wide health improvement strategy for all Americans. Yet acceptance of preventive healthcare interventions in correctional settings varies considerably, shaped by distrust, stigma, and unique institutional policies. Prior work to enhance uptake among incarcerated populations has rarely focused on strategies suited for correctional environments, leaving a key knowledge gap that hinders efforts to reduce the burden of chronic and infectious disease in both facilities and the communities to which people return. The overall objective of this proposal, Advancing PreVentive Healthcare AcceptaNce in Carceral Settings through Community Engagement (ADVANCE), is to identify feasible and effective interventions to improve preventive healthcare uptake in prisons. Building on the P3 health prevention acceptance model, we will create a novel Patient, Provider, Practice, Prison-level (P4) framework tailored to correctional systems, guided by the central hypothesis that strategies developed in partnership with directly impacted individuals will lead to greater acceptance. To test this, we will pursue three aims: (1) identify promising correctional system-based strategies; (2) adapt these strategies into an updated P4 framework through a community-based participatory research approach; and (3) evaluate their effectiveness through rapid-cycle, cluster-randomized trials in the Pennsylvania correctional system. Throughout, currently and formerly incarcerated people and correctional staff will contribute critical input to ensure interventions are relevant, feasible, and sustainable. ADVANCE represents a substantial departure from prior work by building an evidence base for preventive healthcare intervention acceptance specifically in correctional settings, with strategies designed for scalability and adaptation to incarcerated populations nationwide. By filling this gap, the proposed research directly strengthens health in correctional systems, improves outcomes in surrounding communities, and contributes to the broader goal to make America healthy.

NIH Research Projects · FY 2025 · 2022-09

Project Summary People who experience incarceration and have mental health challenges are disproportionately Black and Latinx, and low-income. This group experiences disproportionate financial hardship regardless of incarceration due to racism and other forms of discrimination, with associated negative health outcomes. If incarcerated, financial hardship worsens, creating barriers to community reentry. Support is provided to help people with income from employment and/or disability benefits, but less attention is paid to aspects of finances including problem debt, poor credit, and barriers accessing banking institutions. These financial issues have a direct impact on health, create barriers to employment and housing, stress social networks, contribute to feelings of exclusion and contribute to recidivism, all of which are health determinants. Financial capability programs including one-on-one coaching and access to safe and affordable financial products can improve low-income people's financial well-being and mental health, especially when they integrate into existing services, and partner to work towards reform of laws and policies to address upstream causes of financial difficulties. This project's goal is to intervene at the community level to reduce financial difficulties of individuals with incarceration histories and mental health challenges, who are predominantly Black and Latinx. We will use Community Based Participatory Research (CBPR) methods to achieve the following specific aim: 1) Change community level determinants that impact financial well-being and health of the target group by training existing service provider including: i) community-based financial capability providers to be able to address financial difficulties of the target group; ii) service providers along the criminal justice pathway to be able to provide basic financial guidance to target group; iii) financial institution staff to reduce discrimination related to financial consequences of justice-involvement and mental illness. We will also support community collaborations working for legal/policy reform that impacts finances of target group. 2) Use mixed methods to assess impact on community determinants, measuring integration of financial capability support into existing services, ability of financial coaches to support target group, access to financial products, attitudes, knowledge and behavior of bank staff, strength of community collaborations, and progress towards changes in laws and policies. We will assess impact on individuals by measuring target mechanisms (financial skills, self-efficacy and behavior) hypothesized to mediate the relationship between financial capability support and primary outcomes including financial well-being and other health determinants (employment, housing, social support, mental health supports, and belonging), secondary outcomes (health and recidivism) and mediators between primary and secondary outcomes (hope, empowerment, and mastery). 3) Assess the value of integrating peer support into community-based financial capability support for the target group by randomizing participants into two groups, financial capability support only, or financial capability support plus peer support.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Chromosome segregation is driven by a spindle machinery that distributes copies of the genome between daughter cells. In eggs and their progenitor oocytes, chromosomes are segregated in a specialized meiotic division program. Oocytes and eggs are remarkably vulnerable to chromosome segregation errors that give rise to aneuploidy in embryos, a leading cause of spontaneous miscarriages and developmental disorders. Embryo aneuploidy can nonetheless arise even after error-free completion of meiotic chromosome segregation. However, our understanding of the underlying causes of embryo-specific aneuploidies has been restricted by a tendency to focus only on meiosis-derived aneuploidies. Until recently, it was believed that microtubules are the only cytoskeletal components required for chromosome segregation. This view was successfully challenged by our discovery of spindle F-actin in oocytes and eggs that boost chromosome-spindle attachments and prevent aneuploidy. How is spindle F-actin assembled and how does it exert its function at the chromosome-microtubule interface? These are among outstanding questions raised by this paradigm shift in our understanding of cell division. A major goal of my lab is to understand the mechanisms that safeguard accurate chromosome segregation in mammalian oocytes and embryos. Driven by our discovery that spindle F-actin constitutes one such protection mechanism, we are combining advanced microscopy assays with rapid protein degradation tools to identify proteins required for spindle F-actin assembly and function in oocytes. This approach has revealed key actin- and microtubule-binding proteins that govern oocyte chromosome segregation, some of which were independently implicated in sporadic miscarriages and developmental disorders in recent genetic studies of infertility patients. We propose to build on this progress and study the origins of embryo-specific aneuploidy by 1) expanding our candidate-based rapid protein degradation screens to a larger subset of actin-microtubule crosstalk proteins, and 2) developing a new biochemical and proteomics-coupled experimental pipeline for unbiased identification of novel spindle F-actin assembly proteins in oocytes and embryos. Furthermore, we will take direct experimental approaches of adding or removing centrosomes using microinjection and laser microsurgery tools to address why spindle-shaped F-actin structures are unique to acentrosomal spindles. Early mouse embryo mitotic divisions, which are executed without canonical centrosomes, will provide us with an attractive experimental model in which to answer this long-standing question in cell biology. When this research is completed, we will have discovered and functionally characterized spindle F-actin assembly proteins in oocytes and embryos. Overall, this study will reveal how distinct cytoskeletal systems cooperate to drive accurate chromosome segregation in early development.