University Of California, San Francisco

NIH Research Projects · FY 2025 · 2025-07

DGAT1 deficiency is a rare lipid metabolism disorder that results in congenital diarrhea with protein losing enteropathy and prolonged parenteral nutrition dependence. Despite its discovery over a decade ago, DGAT1 deficiency remains under-recognized and incompletely understood. The DGAT1 Deficiency Symposium, to be held virtually on Sunday, November 9, 2025, seeks to address the crucial question: how do we change the course of the disease? The conference will bring together patients and families, health care providers, investigators, and industry partners, to foster collaborative learning and innovation. The aims of the project are particularly relevant to the missions of NCATS, which focuses on translating research observations into practical solutions for patients, and of NICHD, which focuses on improving the health and well-being of children affected by genetic disorders: Establish a disease registry. Initiate a prospective longitudinal study of DGAT1 deficiency. Engage basic science and translational researchers in elucidating the mechanism of lipotoxicity induced enterocyte dysfunction in DGAT1 deficiency, providing further insight into genotype-phenotype correlations, and identifying therapeutic targets. Disseminate best practice guidelines to healthcare providers and share resources with patients and families. Facilitate early diagnosis. Identify and address the unmet needs of patients and families affected by this disorder.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY Homologous chromosome pairing during meiosis is a central process underlying Mendelian inheritance, in which homologous chromosomes move through the nuclear interior to locate and pair with their homologs, prior to synapsis via the assembly of the synaptonemal complex (SC). Failure of this pairing process can result in aneuploidy. Much of the effort to understand meiosis has focused on determining genes involved in homology recognition and recombination, but the physical process by which chromosomes come together inside the densely packed nucleus remains poorly understood. In order to pair successfully, chromosomes must carry out a huge number of homology tests while avoiding interlocks that would prevent complete pairing. Meiotic chromosome movement is facilitated by forces, generated by the cytoskeleton and motor proteins, that in turn are coupled to telomeres through the nuclear envelope (NE), leading to large-scale active telomere-led motions. But these telomere motions are randomly directed, raising the question of how exactly they facilitate pairing. In addition to random telomere movement, the meiotic nucleus can undergo an extensive re- organization, including clustering of telomeres to form a bouquet, coupling of centromeres between non- homologous chromosomes, and pre-meiotic homolog alignment, depending on the organism. Here we will investigate the cellular mechanism of meiotic pairing by testing a set of hypotheses regarding the role of telomere-led active motion and nuclear re-organization in increasing the fidelity of pairing, reducing the extent of homology searching, and avoiding or reducing interlocks between chromosomes. Our work uses live-cell imaging combined with genetic manipulation in budding yeast as a model system.

NIH Research Projects · FY 2026 · 2025-07

PROJECT SUMMARY Rheumatoid Arthritis (RA) is a chronic, destructive autoimmune disease that primarily targets joints. It affects millions globally and is associated with both disability and reduced lifespan. Antigen-dependent activation of CD4 T cells contributes to disease onset. However, the specific antigens that activate CD4 T cells and the mechanisms by which they evade tolerance remain undefined. Therefore, our long-term goal is to identify the earliest events that activate self-reactive T cells and define the mechanisms they employ to subvert tolerance. This knowledge may reveal vulnerabilities in disease-causing T cells that could lead to new diagnostic and therapeutic interventions. To identify the early mechanistic events that break T cell tolerance, we use the SKG mouse model in which a hypomorphic Zap70 kinase allele impairs TCR signaling; this deficit allows autoreactive thymocytes to escape negative selection, leading to mature autoreactive T cells that cause autoimmune arthritis resembling RA. Our group identified a subset of highly arthritogenic CD4 T cells in these mice. This subset of CD4 T cells is enriched for T cells that recognize superantigens (Sags) from an endogenous retrovirus (ERV) known as the mouse mammary tumor virus (MMTV). ERVs are implicated in human autoimmune disease, but determining their causal role has been exceptionally difficult. Our preliminary data indicate that these Sag-reactive T cells, which are normally deleted in the thymus of wild-type mice, escape deletion in SKG mice, accumulate in arthritic joints, and contribute to disease pathogenesis. These findings suggest that ERVs may play a crucial role in initiating and sustaining autoimmune responses. Furthermore, we find a similar process may be occurring in human RA synovial tissue. The central hypothesis of this proposal is that ERV Sags break immune tolerance and drive RA by persistently engaging and activating self-reactive T cells. To test this, we will use genetic engineering to create and study Sag-reactive CD4 T cells in both SKG and wild-type mice, assessing their role in arthritis initiation and analyzing their activation states and clonality through single-cell sequencing. Parallel human studies will explore the TCR repertoire and transcriptomes in RA synovial T cells. Additionally, we will eliminate MMTV Sags in SKG mice to examine their effects on T cell development, activation, tolerance mechanisms, and arthritis progression, providing insights into the influence of ERVs on central and peripheral tolerance. This research will elucidate how ERVs and their Sags influence T cell activation and tolerance, potentially leading to new therapeutic strategies for RA and related autoimmune diseases.

NIH Research Projects · FY 2025 · 2025-07

Project Summary Colorectal cancer (CRC) is a major health concern, ranking as the third most common cancer and the second leading cause of cancer-related deaths in the United States. Approximately 20% of patients present with metastatic disease at diagnosis, making early detection and accurate staging crucial. CRC often metastasizes to the liver, lymph nodes, bones, and peritoneum, with computed tomography (CT) serving as the primary imaging tool for initial staging, monitoring disease progression, and evaluating treatment efficacy. However, accurately interpreting CT images to detect metastatic disease early is challenging due to difficulties in distinguishing small liver metastases from benign lesions, identifying metastatic lymph nodes that do not meet size criteria, and detecting peritoneal metastases obscured by surrounding tissues. These tasks require radiologists to meticulously review images across multiple time points, perform measurements, and provide detailed reports to document changes, which are repetitive yet cognitively demanding and time-consuming. This paradoxically conflicts with the increasing workloads and time pressures faced by radiologists, leading to high burnout rates, increased diagnostic errors, and compromised patient care quality. Thus, there is a critical need for innovative solutions that can enhance both accuracy and efficiency in evaluating metastatic disease in CRC using CT. To address these challenges, we propose leveraging artificial intelligence (AI) to develop a comprehensive system capable of meticulously analyzing multiple time points of abdominal CT scans. This system could enable earlier detection of subtle changes in disease burden that might elude human experts while reducing the cognitive load on radiologists and allowing them to focus on more complex cases and comprehensive patient assessments. To overcome the limitations of current AI models, which are hindered by labor-intensive data curation and annotation processes, we propose creating a large-scale standardized imaging and report database. This will involve using large language models to automate data extraction from radiology reports, facilitating efficient data selection for AI training. We will also employ annotation-efficient methods, such as radiology-report supervision, data augmentation via realistic synthetic tumor generation, and active learning, to train our anatomy-aware vision–language AI systems on large datasets without extensive manual labeling. We will conduct prospective studies to evaluate the AI system's performance in real-world clinical settings, ensuring its robustness and generalizability across diverse environments, with the ultimate goal of enhancing the detection and tracking of metastatic disease in CRC, improving patient outcomes, and reducing radiologists' cognitive workload.

NIH Research Projects · FY 2026 · 2025-06

Defining and treating genetic abnormalities in psoriasis/atopic dermatitis-overlap dermatoses Project Summary/Narrative Immunomodulatory therapies now robustly improve cases of the inflammatory skin diseases psoriasis and atopic dermatitis, based on selective targeting of cytokine pathways. However, no biomarkers are available to diagnose or predict treatment regimens for the frequent, ambiguous rashes harboring overlapping clinicopathologic features of both diseases. The objective of this proposal is to use high-resolution, quantitative biomarkers specific for psoriasis and atopic dermatitis to understand the etiology of these overlap cases, which we term “PSO-AD” rashes. We will also use observational studies to determine if PSO-AD rashes predictably respond to targeted drugs based on their biomarker profile. Our long-term objective is to develop a biomarker that can predict the medication most likely to elicit clinical improvement in a given patient's PSO-AD rash. Our central hypothesis is that PSO-AD rashes arise from a spectrum of genetic abnormalities and that cases closely resembling psoriasis or atopic dermatitis will respond to the corresponding targeted medication. The rationale underlying this proposal is our preliminary data defining highly specific biomarkers for psoriasis and atopic dermatitis and evidence that based on these markers, therapies can be rationally chosen for PSO-AD overlap cases. We will pursue these aims using innovative technical approaches that include spatial transcriptomics (to validate that epidermal pathology arises from T cell pathology) and targeted bulk RNA-seq methods that can assess PSO-AD cost effectively on the patient level. Our proposal is significant because it advances the first companion diagnostic to targeted biologic medications.The expected outcome of this proposal is a biomarker set that predicts drug response in PSO-AD rashes. These data will have a positive impact on treatment because they will reduce the substantial clinical delays and economic impact stemming from failed ad hoc treatment of PSO-AD rashes.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract The proposed Enamel 11 conference is designed to: 1) critically address significant advances in enamel research that have taken place since the Enamel 10 meeting in Pittsburgh, PA in 2022; 2) promote the further understanding of the biological, biochemical and physicochemical mechanisms of enamel formation, its malformation and destruction in vivo and in vitro; 3) encourage communication and collaboration among attendees from clinical, basic and translational science disciplines; 4) stimulate new ideas that will serve as the basis for the development of novel biomimetic and therapeutic approaches for the repair and/or regeneration of the tissue, as well as strategies for the prevention of enamel decay; and 5) disseminate the presented new knowledge through publication of a meeting proceedings in a peer-reviewed journal, thus broadening national and global accessibility of critical findings and discussions at the meeting. Furthermore, we plan to use this meeting to 6) encourage the involvement of new or early-stage investigators, underrepresented minorities (URM), women, and persons with disabilities in this field of research by providing financial support through awards for travel to the meeting and career mentorship opportunities at the meeting. The planned symposium will emphasize new developments in enamel research in particular around environmental pathologies, ion transport and novel strategies for enamel repair. Enamel 11 will have a profound impact on enhancing research activities, promoting international and interdisciplinary collaboration among scientists, providing networking and training to early-stage investigators leading to improvements in oral health in line with the basic mission of the NIDCR.

NIH Research Projects · FY 2026 · 2025-06

ABSTRACT/PROJECT SUMMARY This application presents a five-year mentored research and training plan that will prepare Dr. Gabriel Loeb to lead a successful independent academic research program focused on the mechanisms underlying ciliary kidney diseases. Dr. Loeb completed his MD and PhD at the Tri-Institutional MD-PhD Program at Weill Cornell Medical College/Rockefeller University/Memorial Sloan Kettering where he studied the function of microRNAs in the lab of Dr. Alexander Rudensky. Dr. Loeb completed his fellowship in Nephrology at UCSF and his long- term career goal is to advance our understanding of the mechanisms underlying genetic forms of kidney disease, particularly those caused by mutations in proteins that localize to primary cilia including Autosomal Dominant Polycystic Kidney Disease and Nephronophthisis. This project will lay the foundation for his independent research program, by dissecting ciliary pathways involved in kidney cystogenesis. Mutation in either of two ciliary membrane proteins, PKD1 and PKD2, is responsible for most Autosomal Dominant Polycystic Kidney Disease. However, the effectors of PKD1/PKD2 signaling remain unknown. GLIS3 is a transcription factor that localizes to primary cilia and the cell nucleus. Loss of GLIS3 phenocopies the severe cystogenesis caused by loss of PKD1 or PKD2. These data suggest the hypothesis that GLIS3 is a long-sought effector of ciliary PKD1/PKD2 signaling. This hypothesis is tested in two independent aims by 1) testing whether GLIS3 is regulated by Polycystins and acts downstream of Polycystins to prevent cystogenesis and 2) testing whether GLIS3 traffics from primary cilia to the nucleus and is regulated by ciliary signaling mediators. This work will employing innovative approaches to study ciliary signaling to generate fundamental knowledge about the pathways underlying cystic kidney disease. The proposed career development plan includes training to help establish Dr. Loeb’s ability to dissect pathways involved in genetic forms of kidney disease using mouse models, organoid models, and advanced imaging. In addition, the training plan will help Dr. Loeb develop all the non-experimental skills necessary for a career as a successful independent academic investigator. These additional skills include laboratory leadership, trainee mentoring and supervision, and grant writing. Dr. Loeb has assembled a world-class mentorship team with complementary expertise in mouse models and ciliary signaling (Primary mentor, Dr. Jeremy Reiter), imaging and ciliary ion channels (Co-mentor Dr. Markus Delling), organoids and kidney disease models (Advisory Committee Member Dr. Andrew McMahon), Ciliary biochemistry and protein analysis (Advisory Committee Member Dr. Maxence Nachury), Polycystic Kidney Disease (Advisory Committee Member Dr. Meyeon Park), and human genetics and single-cell genomics (Dr. David Erle). Dr. Loeb, his mentors, and the Department of Medicine at UCSF are fully committed to this proposal and to his goal of becoming an independent scientist-nephrologist by the completion of this training period.

NIH Research Projects · FY 2026 · 2025-06

Project Summary/Abstract In the past decade, genome-wide characterization of gene expression in cells dissociated from biological tissues has transformed the understanding of cell types that build organs in a variety of organisms. However, to define the precise arrangement of cell types within a tissue or organ, analysis of large-scale spatial transcriptomics and integration with other spatial datasets such as neural connectivity patterns is needed. To achieve these integrative analyses of multiple highly dimensional datasets (e.g., hundreds of genes in cellular resolution, measured across the whole organ), all datasets need to be brought together in the same common coordinate system and new computational algorithms that can handle the complexity of data need to be developed. Studies from the Allen Institute for Brain Science and the Broad Institute are now providing the first whole-brain spatially resolved transcriptomics datasets and providing an integrative view of the cells that make up the brain and their spatial location. One opportunity that these new datasets provide is to define a completely data-driven anatomic parcellation/atlas of the mouse brain. Such a parcellation will be an enormous resource for the systems and molecular neuroscience communities both in formulating new hypotheses for the mechanisms of brain function and investigating existing results. However, current methods are unable to accommodate the scale, complexity, and inherently multimodal nature of integrating these spatial cellular and molecular taxonomies with the wealth of other data (such as connectomics, proteomics, and functional). In this proposal, we aim to utilize new developments in the field of machine learning to address this need for the development of an unsupervised computational algorithm that can synthesize disparate and large datasets of the mouse brain into the next generation of reference anatomical parcellation/atlas. Specifically, we propose a novel deep learning framework called DeepGene to predict spatial cell-type clusters in whole-brain spatial transcriptomics datasets (Aim 1). Our proposed method takes advantage of the unique flexibility of the transformer neural network architectures to scalably model groups of observations with minimal structure. We will then train a more comprehensive version of DeepGene using a combination of eight whole-brain MERFISH datasets. We will use this model to develop a novel parcellation/atlas of the adult mouse brain shared across eight mouse brains. We hypothesize (and provide evidence through preliminary results) that our model delineates cellularly distinct subregions in the mouse brain with differentially abundant cell types (Aim 2). Finally, we will establish a two-stage sequential version of DeepGene to integrate spatial transcriptomics datasets with axonal projection datasets and discover finer brain subregions (Aim 3). The proposed computational frameworks are generalizable to spatial transcriptomics datasets in any tissue, organ, or organism and from any species.

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY Adverse Childhood Experiences (ACEs) are among the strongest predictors of adolescent depression and are estimated to account for 44% of depression cases. The pervasive impact of ACEs on health has galvanized policy makers, resulting in federal and state-wide efforts to screen for ACEs with the goal of providing preventive services to those with high levels of ACEs. However, with 15% percent of children experiencing 4 or more ACEs, meeting the needs of all these individuals is simply not possible given mental health care shortages. The primary rationale for this study is that the precision and efficacy of ACEs-focused programs and policies could be dramatically improved with better insight into what factors increase risk for, and resilience to, depression symptoms following ACEs. The overarching hypothesis for this study is that there is significant heterogeneity in the association between ACEs and adolescent depression symptoms that is due to interactions between a broad range of factors. Prior investigations into such heterogeneity, however, have faced a conceptual gap between the theories that inform adolescent depression research, which highlight interacting, developmentally specific sources of vulnerability and resilience, and the predominant statistical approach of testing one or two risk or promotive factors at a time. We advance ACEs and depression science in two foundational ways: 1) we draw on empirical work and theories across disciplines to identify an inclusive yet concise list of factors mostly likely to promote resilience or increase vulnerability to depression following ACEs, and 2) we identify the combination of these a priori defined characteristics that most affect the ACEs- adolescent depression symptoms association using a robust, well-powered, data driven approach (Aim 1a). Our innovative analytic pipeline includes 10-fold replication of our primary findings, as well as replication using alternative algorithms (Aim 1b). We also explore novel, understudied effect modifiers to generate new hypotheses (Exploratory Aim 2). We use the longitudinal Adolescent Brain and Cognitive Development (ABCD) study (release 6.0, available Spring 2025), which will include data on approximately 10,000 socioeconomically and racially diverse children from 21 sites across the country with yearly data collection from age 9-10 through age 14-15. We model effect modifiers of the association between ACEs (parent and child report) and change in depression symptoms (assessed using the Child Behavior Checklist) between ages 9-10 and 14-15, thereby accounting for baseline depression symptoms. A priori identified effect modifiers include sociodemographic characteristics, family and peer relationships, media use, and pubertal timing. The proposed work will yield robust insights about adolescents in the United States right now with implications for ACEs focused programs and policies. Future research will replicate results in other pediatric studies, expand inquiry to adult depression outcomes, and inform new collaborations with interventionists to design clinical trials powered to detect heterogeneity in the effectiveness of ACEs focused treatment.

NIH Research Projects · FY 2026 · 2025-06

ABSTRACT Determining the molecular function of myriad unannotated pathogen genes remains extremely challenging, yet such knowledge is critical for understanding pathogen biology and developing new drugs. Genome-wide profiling in model yeasts has shown that genes that function in the same pathway show correlated patterns of genetic interactions (double mutant phenotypes) or phenotypes in response to small molecule challenges (chemical- genetic phenotypes). Such studies of gene-gene co-fitness have produced numerous important functional insights by identifying new components of pathways or connecting pathways. However, its application to fungal pathogens at a genomic scale has yet to be achieved. Cryptococcus neoformans, the focus of our work, is the most common cause of fungal meningitis, causing >100,000 fatalities annually. We constructed a gene deletion collection for C. neoformans corresponding to ~4400 knockout strains. To understand gene function, we developed a very high-precision method (replicate r2 ~ 0.98) for quantifying the abundance of gene deletions in pools (KO-seq). We profiled the entire knockout collection under >130 diverse in vitro challenges, including small molecule/drug treatments. We also quantified the fitness of the knockout collection in pools in mice. The high precision of the in vitro measurements enabled the clustering of gene deletion strains based on their phenotypic signatures, revealing numerous high-confidence clusters, most of which correspond to conserved pathways/complexes, supporting the exquisite quality of the data. Importantly, we also discovered many new clusters containing unstudied genes. Below, we focus on three clusters related to fungal sensing of the environment, which is critical for pathogenesis and offers targets for antifungal drug development. Focusing on this theme, we hypothesize that the phenotypic map of C. neoformans will enable the identification and study of new components of key pathways relevant to pathogenesis and drug development. To accomplish this goal we will 1) Dissect function of a new component of a conserved pH sensing pathway, 2) Dissect function of new components of the calcineurin pathway, and 3) Test the model that negative regulation of a putative cell wall amylase is essential for survival under host-like conditions: Successful accomplishment of these aims will illuminate important new aspects of C. neoformans environmental sensing mechanisms highly relevant to drug development and pathogenesis while demonstrating the utility of high-precision phenotypic measurements under diverse environmental conditions to decipher a deadly human fungal pathogen.

NIH Research Projects · FY 2025 · 2025-06

Abstract After birth, the developing human immune system encounters a multitude of new environmental antigens. Balancing protection against infection with immune tolerance to limit detrimental and excessive tissue inflammation is thus a critical function of the neonatal immune system, regulated in part by the capacity of naïve CD4+ T cells to preferentially differentiate into regulatory T cells (Tregs). Immune cell metabolic state is closely tied to function, and emerging research has revealed a distinct metabolic signature for neonatal versus adult CD8+ T cells. Yet, how the metabolic states of early life T cells, particularly CD4+, relate to their distinct functional capacities remains an understudied area of high biomedical relevance to pediatric health and the design of early life therapeutics. I have shown that human neonatal naïve CD4+ T cells preferentially use glycolysis for ATP generation and that this is closely tied to their heightened expression of CD38, a metabolic enzyme with many cellular functions including NAD+ metabolism and intracellular calcium regulation. When treated with a pharmacologic CD38 inhibitor, I observe that neonatal naïve CD4+ T cells demonstrate diminished capacity for Treg differentiation despite preserved cell proliferation and activation. Through the proposed experiments, I aim to test the hypothesis that the distinct metabolic state of human early life naïve CD4+ T cells, driven by their high CD38 expression, facilitates their unique potential for Treg differentiation. To do so, I will define how the metabolic state of neonatal naïve CD4+ T cells impacts their functional potential (Aim 1) and the mechanism by which CD38 shapes the glycolytic dependency and tolerogenic potential of neonatal naïve CD4+ T cells (Aim 2). This work will utilize cutting edge metabolic techniques and immunology assays, as well as pharmacological and genetic approaches, to elucidate the connection between metabolism, CD38, and tolerance in early life. Completion of these aims will shed light on fundamental mechanisms behind a critical time in immune development and build a foundation for precision therapies that aim to augment or limit early life tolerance via modulation of CD38 or other metabolic pathways.

NIH Research Projects · FY 2026 · 2025-06

Project Summary/Abstract: This research proposal aims to develop advanced in vivo magnetic resonance imaging (MRI) technologies for studying infant brain development. The objective is to achieve motion-robust, high-resolution (600µm isotropic) whole-brain quantitative MRI (qMRI) and diffusion MRI (dMRI) within a clinically feasible scan time. By improving image resolution and quality, we can better understand the microstructural changes in cortex and superficial white matter during infancy. Specifically, the development of myelin, a crucial indicator of brain development, will be assessed using qMRI and dMRI techniques such as myelin water fraction mapping (MWF), longitudinal relaxation time T1, transverse relaxation time T2, mean diffusivity (MD) and fractional anisotropy (FA). These techniques provide valuable information about tissue properties, contributing to a comprehensive understanding of early brain development. Increasing the spatial resolution of qMRI and dMRI is crucial for accurately studying myelination and tracking developmental trajectories. However, current image resolutions in large-scale clinical and research studies typically range from 1.0 to 2.0mm, leading to partial volume effects and potential inaccuracies in assessing myelin content. Overcoming challenges such as long scan times, motion sensitivity, and decreased signal-to-noise ratio is essential for increasing image resolution in infant brain development studies. To address these challenges, this grant proposes three specific aims. Aim 1 focuses on developing acquisition and reconstruction strategies for 600µm whole-brain multi-parametric qMRI. This includes the use of our proposed ViSTa-MRF sequence for simultaneous MWF, T1, and T2 mapping, as well as motion-robust subspace reconstruction for improved identification of myelin components. Aim 2 aims to develop acquisition and reconstruction strategies for high-fidelity whole-brain mesoscale dMRI, incorporating techniques such as simultaneous multi-slab gSlider encoding and blipped-up/-down circular-EPI acquisition. Motion-robust dMRI approaches will also be developed to mitigate head motion and correct image distortions. Aim 3 involves translating the developed qMRI and dMRI techniques for infant brain development by establishing a protocol using a home-built baby coil and validating the techniques through ex-vivo and in-vivo measurements. Longitudinal assessment of the visual cortex and superficial white matter will be conducted to validate developmental variations, explore the association between microstructural development and tissue proliferation, and investigate myelination and U-fibers. Overall, this proposal leverages expertise in MRI technology development, data acquisition, reconstruction, and analysis to advance our understanding of infant brain development. By enhancing the capabilities of qMRI and dMRI, we can gain valuable insights into the intricate processes of early brain development.

NIH Research Projects · FY 2025 · 2025-06

Neurodegenerative disorders such as Alzheimer’s Disease (AD), frontotemporal dementia (FTD) and limbic-predominant, age-associated TDP-43 encephalopathy (LATE), are age-associated diseases characterized by aberrant protein homeostasis. Lysosomes and the lysosome-dependent autophagic processes degrade and recycle macromolecules, thereby promoting cellular protein homeostasis and preventing inclusion formation. Proteolysis in the lysosome is carried out by a suite of proteases, known as cathepsins, that each recognize specific linear amino acid sequences. Declines in lysosomal function with age have been shown to contribute to neurodegenerative disease pathogenesis and genetic evidence also strongly supports the importance of lysosomes in AD, FTD and LATE. For example, loss-of-function mutations in the Pgrn gene lead to protein haploinsufficiency and FTD with TDP-43 aggregates. PGRN is a lysosome-resident protein that can be cleaved first into bioactive multi- granulin fragments (MGFs) and then individual granulins (henceforth collectively referred to as PGRN species). We have demonstrated that 1) relative levels of PGRN species change with age, 2) PGRN and MGFs promote the activity of a key lysosomal protease, CTSD, while 3) granulins inhibit cathepsin activity and TDP-43 clearance. Still unclear are precisely how PGRN species alter TDP-43 cleavage and why these processes change with age and Pgrn haploinsufficiency. The major focus of my research group is to utilize multi-disciplinary approaches to understand how lysosomes contribute to aberrant proteostasis in aging and neurodegeneration. Our objective here is to determine how PGRN species contribute to TDP-43 accumulation with age and Pgrn haploinsufficiency. Our central hypothesis is that age and Pgrn haploinsufficiency alter the relative levels of intact vs cleaved PGRN species, thereby impacting the ability of lysosomal proteases to degrade TDP-43. The rationale for this work is that if TDP-43 is not efficiently degraded, steady-state levels will increase, promoting TDP-43 aggregation and potentially contributing to selective vulnerability to TDP-based diseases. To address our hypothesis, we propose the following specific aims. Aim 1: Test the ability of PGRN species to modulate TDP-43 proteolysis in vitro. Aim 2: Define the capacity of induced neurons, astrocytes and microglia with and without Pgrn deficiency to degrade and clear TDP-43. Aim 3: Determine how PGRN species and proteolysis of TDP-43 changes with age and PGRN deficiency in murine models. Successful completion of the proposed aims will enhance understanding of the factors governing TDP-43 proteolysis with age and disease. This information could reveal new therapeutic avenues for treating TDP-43 diseases and has implications for precision medicine in other forms of neurodegeneration.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract The exceptional strength of enamel can be attributed to its high mineral content within the matured matrix. This biomineralization process relies heavily on maturation ameloblasts (MABs), responsible for transporting approximately 86% of the required minerals. Among the key players in this intricate process is the K+- dependent Ca2+/Na+-exchanger SLC24A4. Disruptions of SLC24A4 contribute to the development of an enamel disorder called amelogenesis imperfecta. SLC24A4 is highly upregulated in those post-secretory stages of ameloblasts, where it shuttles between the apical plasma membrane (PM) of the ruffle-ended and the cytosol of the smooth-ended MAB subpopulations. The mechanisms governing SLC24A4’s temporal and spatial expression in MABs remain largely unexplored. During development, secretory ameloblasts (SABs) are succeeded by the transition stage of ameloblasts (TABs). TABs play a crucial role in modifying the protein composition and mechanical stress within the underlying enamel matrix by introducing the potent proteinase KLK4. Our preliminary data indicated that factors present in the transition stage of enamel, including amelogenin hydrolysis peptides (Amelx20, C-domain, and C-P1), along with gradually intensified mechanical stiffness, enhance the transcription of Slc24a4. Additional data indicated a correlation between the transcription of Slc24a4 and heightened levels of H3K4 methylation. Simultaneously, we discovered that exposure to BSA-saturated palmitic acid (PA) elevated the levels of protein palmitoylation and enriched SLC24A4 along the PM of ameloblast lineage cells (ALCs). Furthermore, our findings revealed a significant concentration of palmitoyltransferase ZDHHC21 within PM of the ruffle-ended MABs, implying a potential role of protein palmitoylation in the cellular trafficking of SLC24A4. We hypothesize that the combined influence of developmental cues and protein palmitoylation regulates the calcium transport capacity of SLC24A4 at both the transcriptional and activity levels. To investigate this hypothesis, we have formulated two specific aims. Specific aim 1: To determine the molecular mechanisms underlying the upregulation of SLC24A4 transcription in MABs. To assess the impact of development cues, we will conduct a comparative analysis of H3K4me3 marks in ALCs treated with and without these cues using ChIP-seq. To validate the intensity of H3K4me3 marks in the regulatory region of the SLC24A4 gene within rare cell populations of wt mouse SABs and MABs, we will employ the recently developed CUT&Run-seq analysis. Specific aim 2: To define the significance of protein palmitoylation in governing the activity of SLC24A4. We propose to quantitate the protein palmitoylation levels of SLC24A-transduced ALCs treated with or without BSA-PA using click chemistry and proximity ligation assay. Next, we aim to determine the palmitoylation sites of SLC24A4 using immunoprecipitation and mass spectrometry. Understanding the factors and mechanisms that regulate SLC24A4 transcription and activity is important for both enamel biology and regenerating ameloblast/enamel.

NIH Research Projects · FY 2025 · 2025-06

Project Summary/Abstract In most countries approaching elimination, Plasmodium vivax (Pv) represents an increasing proportion relative to P. falciparum (Pf). Mass drug administration (MDA), as a way target subpatent, asymptomatic infections, is recommended for P. falciparum elimination, but the recommendation does not extend to P. vivax given limited evidence, tools, and safety concerns. The objective of our study is to evaluate the long-term impact, safety, and cost-effectiveness of focal MDA (fMDA) for Pv transmission reduction. To test our hypothesis that fMDA, in addition to standard aggressive interventions, will safely reduce transmission, we propose a 3-year open-label CRCT in the low endemic setting of Loreto Region, Peru. Villages or clusters will be randomized to control or fMDA. The control arm will receive standard interventions (vector control, symptomatic case management, and active case detection of asymptomatic cases). The treatment arm will receive standard interventions plus fMDA, which will utilize a new drug for radical cure of P. vivax, tafenoquine, and a new quantitative glucose 6 phosphate dehydrogenase (G6PD) deficiency rapid test to support safe administration of tafenoquine. fMDA will be targeted to consenting and eligible high-risk villagers, defined as household members and neighbors of recent Pv index cases. fMDA will be conducted in 2 rounds per year, two months apart during the low malaria season, and over 3 years. Eligibility will be re-assessed each year, and prior to each fMDA round. Specific aims are to: 1) Determine the effectiveness of fMDA to reduce Pv transmission as measured in a primary outcome of incidence and secondary outcomes of infection prevalence, seroprevalence, and genetic diversity, 2) Evaluate the safety and tolerability of fMDA, and 3) Measure the cost-effectiveness of fMDA. To maximize

NIH Research Projects · FY 2025 · 2025-06

PROJECT SUMMARY AND RELEVANCE Ocular anterior segment dysgenesis (ASD) is a genetically heterogeneous group of developmental disorders affecting the front of the eye. Individuals with ASD have a greatly elevated risk of developing severe and early onset glaucoma that is refractory to treatment. Between 50-75% of patients with ASD develop glaucoma, which occurs at significantly younger ages than in the general population, leading to disproportionately diminished quality of life for these individuals and their families. Despite decades of study, the cellular and molecular details of normal ocular development are still poorly understood, and disease mechanism remain elusive. Mutations in the genes encoding collagen IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause a multisystem disorder that includes ASD and glaucoma. Importantly, we recently discovered that Col4a1 mutant mice have elevated TGFb signaling and that reducing TGFb signaling partially rescued ASD. We hypothesize that Col4a1 mutation and elevated TGFb signaling affect differentiation of the periocular mesenchyme (POM) – a transient embryonic tissue derived from neural crest cells and paraxial mesoderm. The POM gives rise to structures of the ocular anterior segment including corneal endothelium and stroma, iris stroma, trabecular meshwork, and ciliary muscle. Despite its importance, the small size and transitory nature of the POM has made it difficult to study. This project will apply recent advances in single cell and spatial transcriptomics to overcome this obstacle and enable unprecedented investigation of the cellular and molecular mechanisms of anterior segment development and dysgenesis. The proposed project uses both unbiased and hypothesis driven approaches to answer central questions about where, when, and how pathogenesis arises, reveal other potential disease mechanism, and understand how molecular mechanism(s) might converge with other known genetic causes of ASD in humans. The successful completion of this project will provide foundational knowledge of normal ocular development and novel insights into molecular mechanisms underlying ASD and developmental glaucoma.

NIH Research Projects · FY 2026 · 2025-06

ABSTRACT Our research aims to uncover the ways in which evolutionary forces shape patterns of genetic variation and drive phenotypic variation across human populations. We focus on three broad areas of investigation. Firstly, we ex- plore the effects of recessive deleterious alleles and associative overdominance (AOD). While previous studies have mainly focused on additive fitness effects, we investigate the unique patterns of genetic variation caused by recessive deleterious alleles. We analyze additional summary statistics such as the site frequency spectrum (SFS), ancestral recombination graphs (ARGs), and haplotype structure to better understand the impact of AOD. We also examine the dynamics of genetic diversity in the context of demographic changes and investigate the likelihood of AOD in the MHC locus of the human genome. Secondly, we move beyond traditional racial and ethnic categorizations and develop a more refined approach based on genetic relatedness groups. We utilize ancestral recombination graphs (ARGs) to trace genetic lineages over time and identify relatedness clusters without relying on predefined reference populations. Our analysis focuses on different timescales, including con- tinental and sub-continental population structure, allowing us to capture the complexities and variabilities within human populations. Lastly, we build simulation tools that incorporate genetic and non-genetic interactions to study complex traits. By integrating statistical methods, population genetics theory, and flexible simulation tools, we aim to simulate multivariate traits with varying genetic architectures. We focus on both common and rare variants, developing power simulators for rare variant association tests (RVATs) to address the challenges of studying rare variants in complex populations. Overall, our research aims to provide novel insights into the evo- lutionary mechanisms driving phenotypic variation and enhance the realism and accuracy of simulations for studying complex traits in diverse human populations.

NIH Research Projects · FY 2026 · 2025-06

SUMMARY Tauopathies including Alzheimer’s disease (AD) and frontotemporal dementia (FTD) are a group of devastating neurodegenerative diseases that currently affect over 30 million people worldwide. Tau pathology correlates with neuronal loss in AD and several mutations that increase tau aggregation cause familial forms of FTD, suggesting tau aggregation plays an important role in the pathogenesis of neurodegeneration in these diseases. Indeed, tau-lowering therapies are being evaluated in clinical trials for AD. Despite this central importance of tau in the pathogenesis of AD and related tauopathies, the molecular and cellular mechanisms by which pathological tau drives neurodegeneration are poorly understood. Thus, a systematic elucidation of these mechanisms would transform our understanding of AD and related dementias, and also open up new avenues for therapeutic approaches that could be beneficial in patients in which tau pathology has already been established and downstream effects have already affected neuronal function. Here, we propose to combine our expertise in mass spectrometry-based proteomics (Swaney) and functional genomics (Kampmann) to comprehensively map the impact of tau pathology on the proteome, which we expect to be mediated in part through disruptions in mRNA splicing, and measure the functional consequences of this pathology with cell type, subcellular, and molecular resolution. This work leverages AD mouse models and human iPSC tauopathy models for discovery of protein drivers of tauopathy, as well as validates such drivers in human post-mortem brains. Importantly, our approach will measure multiple modalities of protein function to provide a holistic view of the diversity of functional consequences incurred in neurons and brains with tau pathology. We anticipate these results will deepen our understanding of molecular mechanisms of AD and ADRD and have the potential to pinpoint novel therapeutic targets.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY We are developing novel minimally invasive catheter-based devices placed under real-time x-ray angiography guidance: functional endovascular filters that remove chemotherapy from the blood in order to reduce systemic toxicity during locoregional infusion therapy. The fundamental challenge of eliminating a specific agent from the blood in veins draining an organ undergoing intraarterial chemotherapy (IAC)—after the agent has its therapeutic effect in the treated organ and before it causes a systemic toxic effect—can be met by capture strategies based on physicochemical properties of the target agent. Preclinical ChemoFilter devices containing ion exchange resins (IER) that reduce doxorubicin (Dox) deposition in the heart of a hepatic IAC swine model by 46% are now being commercialized through startup company Filtro. We will use advanced imaging to quantify the degree to which venous drug filtration changes the biodistribution of target drugs infused into the hepatic artery and the carotid artery (with and without blood brain barrier disruption), simulating the treatment of liver tumors and brain tumors, respectively. Prototype ChemoFilters will be modeled, built, validated in vitro for efficacy, and tested in vivo for efficacy and safety. Experienced teams from UCSF, Penn State, and Purdue will undertake the following specific aims: (SA1) mathematically model drug capture by ChemoFilters, (SA2) synthesize radiolabeled chemotherapeutic analog tracers, (SA3) validate ChemoFilter designs in vitro for capacity to capture drugs, and (SA4) assess safety and efficacy of optimized filters in vivo in small and large animal models. Achievement of these aims will create a family of minimally invasive medical devices that could markedly increase the efficacy of image-guided locoregional intraarterial chemotherapy by lowering systemic drug concentrations and reducing systemic toxicities, thus permitting dose escalation in any given IAC procedure and better local tumor control in fewer IAC sessions. The imaging techniques developed herein will enable superior drug distribution monitoring, providing insight into modifying biodistribution clinically.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY / ABSTRACT This application for the Mentored Clinical Scientist Research Career Development Award (K08), sponsored by the National Institute of Allergy and Infectious Diseases, describes the five-year career development plan of Dr. Matthew Kan, a pediatric immunologist and early career physician-scientist in the Division of Pediatric Allergy, Immunology, and Bone Marrow Transplant at University of California, San Francisco. Dr. Kan's long-term career goal is to develop genome editing therapies for patients with inborn errors of immunity (IEIs), and in this proposal he specifically focuses on developing a preclinical therapy for Artemis- deficient severe combined immunodeficiency (ART-SCID). The career development goals outlined in this application include developing expertise in non-viral genome editing of human hematopoietic stem cells, preclinical modeling and genetic repair of ART-SCID, in vivo delivery of genome editors, and developing potency assays for genome-edited cell products. The primary mentor for accomplishing these career training goals is Dr. Jennifer Doudna, Li Ka Shing Chancellor's Chair in Biomedical Sciences at UC Berkeley, who is a co-discoverer of CRISPR genome editing. Dr. Jennifer Puck, Professor of Pediatrics, will be co-mentor, and she is a renowned clinical immunologist who is an expert in ART- SCID and has advanced multiple lentiviral gene therapies through clinical trials. The career development plan for Dr. Kan includes individualized mentorship, a career development team that includes other leaders in the field of genome editing and immune diseases, formal coursework, and a research program that builds upon Dr. Kan's strong experience in basic and clinical immunology with training in biochemistry, RNA expression, xenograft mouse models, and lipid delivery of genome editors. The overall objective of the research plan is to develop a non-viral platform technology for genome editing of hematopoietic stem cells that comprehensively addresses all pathogenic mutations in each gene. As over 1/3 of patients with ART-SCID have megabase deletions in DCLRE1C, the affected gene, the native locus cannot be used for “universal” gene repair. The central hypothesis is that targeted integration of a DCLRE1C transgene in the AAVS1 safe harbor locus will restore Artemis in ART-SCID, and the safe harbor will serve as a platform for other IEIs. The specific aims are to 1) develop non-viral techniques of targeted integration to restore Artemis expression in human hematopoietic stem and progenitor cells, initially ex vivo and then in vivo, and 2) develop a novel assay for Artemis activity for both patient diagnosis and potency of gene-edited cell products. This application is relevant to the NIH and the NIAID because Dr. Kan's goal is to develop methods for genome editing that will improve outcomes for a devastating inherited immune disease and the technologies developed will translate to other inborn errors of immunity.

NIH Research Projects · FY 2026 · 2025-05

Research Project Summary The skin is a vital barrier tissue, which manages to maintain immune homeostasis amidst ongoing microbial stimulation. Cutaneous immune cells continually sense skin commensal microbes, yet a healthy symbiosis is preserved through mechanisms that actively regulate this commensal-specific immune response. Disruption of this tolerance contributes to chronic inflammatory diseases such as acne vulgaris, atopic dermatitis and hidradenitis suppurativa. Prior work from my lab has highlighted that early exposure to skin bacteria promotes immune tolerance through the generation of commensal-specific regulatory T cells (Tregs). Recent studies specifically identified CD301b+ type 2 dendritic cells (DC2) as the primary population sampling commensal bacteria in neonatal skin and critically supporting generation of commensal-specific Tregs. The Treg-promoting function of CD301b+ DC2 is facilitated by their retinoic acid production, which is further augmented upon uptake of commensal bacteria. However, many questions remained from this work, most notably, what are the commensal signals that reinforce tolerogenic function in CD301b+ DCs and how are these sensed? Preliminary data I’ve generated since starting my PhD have started to address this knowledge gap. I have shown that in vivo deletion of toll like receptor 2 (TLR2) leads to greatly reduced commensal-specific Tregs in neonatal mice. Additionally, through in vivo and ex vivo studies using mutant Staphylococcus epidermidis strains, I have seen that fewer commensal-specific Tregs are generated by CD301b+ DC2 in response to bacteria lacking D-alanine modification of their surface teichoic acids, known ligands for TLR2. Collectively, these results shed new light on the role of conserved microbial ligands not only as stimulators of cutaneous immune defense against pathogens but also as fundamentally supporting commensal-specific immune tolerance. Building on these solid lines of evidence, this project will investigate how TLR2 sensing of modified ligands on commensal Staphylococcus spp. supports Treg induction by CD301b+ DC2s. Aimed at dissecting these mechanisms and furthering my training goals, I will employ a combination of approaches, including transgenic mouse models, genetic manipulation of bacterial strains, and human skin explants. Collectively, these studies will deepen our understanding of skin immune tolerance, potentially inform therapeutic targets for inflammatory diseases and advance me towards my long-term career goal of becoming an independent investigator and academic educator studying host-microbe interactions.

NIH Research Projects · FY 2026 · 2025-05

Project Summary/Abstract The current paradigm of asthma attributes epithelial cells in recruiting immune cells that promote pathologies seen in asthmatic airways, including infiltration of immune cells and mucous metaplasia. What is less clear is how stromal cells surrounding the airway alter the inflammatory response to allergic challenge, and whether targeting these cells represents a viable strategy for asthma therapeutics. This proposal aims to characterize a targetable stromal factor that alters immune cell accumulation in the lung and define the downstream effects of these immune cells on airway epithelial cells. Our preliminary data demonstrate that fibroblast-specific deletion of Hhip promotes the accumulation of T cells with tissue residency features (tissue resident lymphocytes, or TRLs) within the adventitial space surrounding the airways, suggesting that host factors in the lung can alter the inflammatory response after allergen challenge. Utilizing a combination of novel genetic tools to trace and delete Hhip+ fibroblasts, human organoid platform, and a novel pharmacologic reagent made in our lab to target TRL accumulation, this proposal will determine the mechanism by which stromal factors in the lung modifies TRL accumulation in response allergen challenge. Furthermore, we will determine whether host factors that modify TRLs can be leveraged as pharmacologic therapy to attenuate the inflammation and airway metaplasia seen in asthmatic airways. Finally, we will develop an ex vivo organoid model of human asthmatic airways that preserves the stromal-epithelial-immune architecture for drug screening. Successful completion of this proposal will highlight an unrecognized axis whereby a dysregulated lung stromal niche can drive the maladaptive expansion of TRLs that promote mucous metaplasia in asthmatic airways, and provide preclinical studies of novel therapeutic agents to target the stroma in asthma.

NIH Research Projects · FY 2026 · 2025-05

Proposed Approach: We will use data science and data integration across 13 different data sources to study the intersection of climate change, children, and emergency care. The project will provide a comprehensive evaluation of climate and health among children, with a focus on heatwaves. We will use existing research infrastructure, weather data with detailed spatial resolution, geospatial analyses, advanced analytics, modeling, and an interdisciplinary team to address this critical public health issue. Importance: Children are vulnerable to climate change, yet the science of climate and health in children is underdeveloped. We have recently shown that high emergency department (ED) pediatric readiness is independently associated with improved survival, but the function of high-readiness EDs during heatwaves is unknown. Addressing children in a changing climate is critical to their welfare, including methods to mitigate adverse outcomes and ways the U.S. healthcare system can remain resilient. This project is designed to inform EDs, hospitals, healthcare systems, and national health policy for children. Objectives: There are three specific aims: Aim 1: Using the national assessment of ED pediatric readiness, detailed weather data, pediatric census data, and geospatial analysis, we will identify the most geographically vulnerable areas for children due to heatwaves that also lack access to high-readiness EDs. Aim 2: We will create three national samples of children receiving emergency services (prehospital, EDs, and trauma centers) to evaluate the association of heatwaves with excess mortality and healthcare utilization across millions of children. Aim 3: Using two longitudinal pediatric cohorts, we will assess whether high-readiness EDs mitigate excess mortality during heatwaves and are resilient in their known survival benefit. Study Design & Setting: We will build three large samples of children using emergency services from 1/1/2012 to 12/31/2023 during the “warm season” (May to September), including 9-1-1 responses from over 14,000 emergency medical services (EMS) agencies (the Prehospital Sample), ED visits to 1,856 EDs across 16 states (the ED Cohort), and admissions to 999 trauma centers across the U.S. (the Trauma cohort). To assess access to high-quality emergency care, we will estimate the driving time to 4,840 EDs across the U.S., combined with their level of pediatric readiness. For weather data, we will use a 1 x 1 kilometer spatial resolution for heatwaves (defined as average daily temperature ≥ 95%ile for ≥ 2 days), air quality (fine particulate matter), humidity, and greenspace (Normalized Difference Vegetation Index). Participants: Children 0–17 years using emergency services, including 4.9 million 9-1-1 EMS responses, 53 million ED visits, 1.8 million hospitalizations, 29,527 deaths after ED presentation, plus 311,490 injured children admitted to US trauma centers and 4,517 deaths after injury. Outcome measures: We will evaluate mortality (primary) and healthcare utilization among children.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY Vascular contributions to cognitive impairment and dementia (VCID) describes any level of cognitive alteration attributable to cerebrovascular pathologies. After Alzheimer disease, VCID is the second leading cause of dementia and accounts for ~15-30% of all dementia cases. Cerebral small vessel disease (cSVD) accounts for up to 20% of all strokes and is the most common pathology underlying VCID. Importantly, the pathogenesis of cSVD is poorly understood which represents a major barrier for developing therapies. COL4A1 and COL4A2 mutations cause multisystem disorder for which cSVD disease is the major consequence. Cerebrovascular disease in individuals with COL4A1 or COL4A2 mutations have hallmarks of cSVD – subcortical microbleeds, enlarged perivascular spaces, and lacunar infarcts and Col4a1+/Mut mice faithfully model patient phenotypes. Moreover, Col4a1+/Mut mice have age-related cerebrovascular dysfunction including loss of myogenic tone and impaired hyperemic responses that are thought to be critical to VCID progression. Importantly, strong genetic evidence indicates that COL4A1 and COL4A2 contribute to general cerebrovascular health and idiopathic cSVD suggesting that understand pathogenic mechanisms contributing this form of monogenic cSVD may also provide important insight into idiopathic cSVD and VCID. We discovered that TGFβ signaling is elevated in Col4a1+/Mut mice, genetically decreasing TGFβ signaling ameliorates cSVD severity in mice, and that acutely inhibiting TGFβ signaling restores myogenic tone to ex vivo cerebral arteries from Col4a1 mutant mice. Here, we seek to understand the molecular mechanisms by which collagen IV a1a1a2(IV) regulates TGFβ signaling. We will also use mouse models to perform intravital imaging of the cerebral vasculature and cerebral hemodynamics, evaluate behavioral assays of cognitive impairment, and test hypothesis that dysregulation of matrix metalloproteinases contribute to pathogenesis. The successful completion of this project could provide significantly greater understanding of idiopathic cSVD and establish clinically relevant in vivo outcomes for testing future disease modifying therapies for an important monogenetic form of cSVD.

NIH Research Projects · FY 2026 · 2025-05

Project Summary/Abstract At cell division, the cell must accurately segregate its chromosomes to its two daughter cells. The kinetochore connects spindle microtubules to chromosomes and plays both physical and biochemical roles: it must resist and transmit microtubule forces to move chromosomes, and process microtubule signals to control cell cycle progression. Errors in these processes can lead to disease and birth defects. The mammalian kinetochore consists of ~100 protein species, with innermost kinetochore proteins binding DNA and outermost ones binding microtubules ~100nm away. Parallel protein linkages connect DNA and microtubules. While much is known about the mammalian kinetochore’s architecture, molecular composition and biochemistry, much less is known about its mechanics. Yet, the kinetochore has a key mechanical function – segregating chromosomes – and spends much of its life under force. To what extent and how the mammalian kinetochore maintains its structural integrity under force remain unclear. A major challenge has been in measuring kinetochore structure in live cells, under different cellular forces, given the kinetochore’s small size and rapid movements. Our recent approach development measuring inner and outer kinetochore shape changes with high spatiotemporal resolution in vivo during normal mitosis brings this question within reach. Here, I propose to test physical and molecular models for how mammalian kinetochores maintain their structure under spindle forces at metaphase. I will do so using super resolution live imaging and computational shape analysis of inner (CENP-A) and outer (Hec1) kinetochore proteins in PtK2 cells. In Aim 1, I will test the hypothesis that centromere stability contributes to kinetochore structural maintenance. I will do so by decreasing centromere stiffness genetically using an inducible Condensin II knock out cell line and measuring the impact on resulting kinetochore deformations. In Aim 2, I will test the hypothesis that lateral reinforcement between parallel kinetochore protein linkages contributes to kinetochore structural integrity. I will do so by replacing an inner kinetochore protein with a mutant which disrupts crosslinking between parallel linkages, and also by inducing an acute, partial loss of this candidate protein. In Aim 3, I will test the hypothesis that incorrect, merotelic attachments give rise to the most dramatic kinetochore deformations at metaphase. I will do so by enriching for merotelic attachments and asking if more kinetochores then take on large deformations. Together, this work will help define the mechanisms underlying mammalian kinetochore structural maintenance (Aim 1 and Aim 2) and the impact of incorrect attachments on kinetochore structure (Aim 3). Looking forward, this will help us elucidate how the kinetochore’s functions emerge from its structure, and how its malfunctions can emerge from structural failures. Through this work, I will get training in cellular biophysics, computational image analysis, molecular approaches, and how to rigorously answer questions, mentor and communicate science. These will help me towards my goal of being a group leader in industry or academia.