Dartmouth College

NIH Research Projects · FY 2025 · 2020-07

Prion diseases are invariably fatal neurodegenerative disorders that occur in sporadic, infectious, and inherited forms, and are caused by the conversion of either wild-type or mutant versions of the cellular prion protein (PrPC) into self-propagating, misfolded conformers (collectively termed PrPSc). There is currently no clinically effective treatment for any form of prion disease. Recently, chemical screens have identified different classes of oral drugs that can significantly decrease the rate of wild-type PrPSc formation, and thereby increase disease-free survival in prion-infected animals. However, in each case, drug treatment did not cure prion infection, which eventually overwhelmed the treated animals. In almost all cases, an alternative PrPSc conformation emerged during therapy, causing prion strain adaptation and, in some cases, drug resistance. Interestingly, prions from drug-treated animals can recover their original strain characteristics and drug susceptibility during serial passage in untreated hosts. In addition, our preliminary work shows that simultaneous co-administration of two different drugs to prion-infected mice failed to create a synergistic effect due to the emergence of an unorthodox new strain that is resistant to the two-drug combination yet susceptible to both drugs alone. Taken together, these observations show that wild-type prions are highly malleable, i.e. able to switch back and forth between different PrPSc conformations in response to changes in selective pressure caused by anti-prion drug therapy. The molecular mechanism responsible for the malleability of wild-type prions is currently unknown. It is also unknown whether mutant prions, which specifically cause the inherited forms of prion disease, are as malleable as wild-type prions. The overall objectives of this proposal are to evaluate novel therapeutic strategies that rationally target prion malleability, and to study the role of cofactor molecules in drug-induced prion strain adaptation. Specifically, we will: (1) evaluate the efficacy of alternating oral drug regimens in a wild-type prion infection model; (2) determine whether cofactor selection plays a role in the mechanism by which wild-type prions acquire drug resistance; and (3) test the efficacy of oral drug regimens in new knock-in mouse models of inherited prion diseases.

NIH Research Projects · FY 2025 · 2020-06

PROJECT SUMMARY Tissue specification, growth, maintenance, and regeneration rely on spatiotemporal cues provided by the evolutionarily conserved Wnt morphogen pathway. Many developmental disorders and cancers, including nearly all colorectal cancers, arise when the normal regulation of Wnt signal transduction is lost; yet key mechanisms that mediate essential steps in this pathway remain poorly understood. An improved mechanistic understanding of Wnt pathway activation is therefore crucial, as no drugs that target Wnt-driven disease have yet been approved by the FDA. The long-term goals of the PI’s research program are to uncover the foundational mechanisms that control Wnt signaling during animal development and to use this knowledge to identify vulnerabilities in the pathway that are susceptible to therapeutic targeting in Wnt-driven diseases. To support these goals, the PI and her laboratory group have developed innovative, cost-effective approaches in the fruit fly Drosophila that provide three major strengths to surmount existing obstacles in the field: cutting- edge genetic tools, limited functional redundancy, and robust in vivo assays for Wnt signaling gradients. Research by the PI’s group identified two novel roles for the tumor suppressor Adenomatous polyposis coli (APC) in Wnt signaling, two novel transcription cofactors required for nearly all consequences of APC loss, the dual mechanisms by which the therapeutic target Tankyrase activates Wnt signaling, and a Wnt receptor regulatory mechanism required to tune signaling strength throughout the morphogen gradient. This project will address major questions centered on the three multi-protein complexes that control Wnt signaling: 1) How is the activity of the membrane-associated signalosome complex controlled by regulation of the Wnt receptor LRP6/Arrow?; 2) How are the components of the nuclear beta-catenin-TCF transcription complex activated by phosphorylation and ubiquitylation?; and 3) How is the activity of the two key kinases in the cytosolic beta- catenin destruction complex, GSK3 and CK1, modulated under basal conditions and following Wnt stimulation? To address these questions, the research capitalizes on state-of-the-art genetic and proteomic screens that have identified six new enzymatic regulators that control the activity of these essential complexes. The function of each will be defined using a combination of genetic, cell biological, and biochemical assays. The impact of this research is enhanced by long-term collaborations that incorporate complementary experimental approaches, including biochemical reconstitution, quantitative phosphoproteomics, and vertebrate models that test evolutionarily conserved functions. The successful completion of this work will provide a strong mechanistic understanding of this fundamental signaling pathway and highlight new therapeutic strategies to target Wnt-driven diseases.

NIH Research Projects · FY 2025 · 2019-08

Center for Quantitative Biology: A focus on "omics", from organisms to single cells PROJECT SUMMARY High-throughput single-cell, spatial, and immune-cell -omic profiling technologies provide a wealth of data to interrogate basic biological processes, changes in cellular processes, and the molecular basis of disease. When combined with genomic data science, these fields are opening new frontiers in biology. Phase 1 established the Center for Quantitative Biology (CQB) with the goal to support and enhance NIH-funded quantitative biological research at Dartmouth and to facilitate integration of single-cell -omic technologies with cutting-edge computational methods. The success of the CQB in Phase 1 is highlighted by the graduation of four Research Project Leaders (RPLs), the onboarding of four new RPLs, and the hiring of seven new tenure-track faculty in CQB focus areas. The IDeA program investment in CQB RPLs and research cores returned >$60 million in total external funding, leveraging NIH funds 5:1. Phase 2 will build on the successes of Phase 1 by mentoring the next generation of CQB COBRE RPLs, continuing growth of research cores, and enabling scientists to engage in cutting-edge scientific inquiry. The CQB will continue to draw upon faculty from Dartmouth’s Geisel School of Medicine, the Thayer School of Engineering, Arts & Sciences, and Dartmouth Health. Through its emphasis on next-generation data, the CQB will synergize genomic data science with ongoing experimental genomic initiatives across campus, a goal accomplished via four specific areas: 1) Enhance the research competitiveness of junior faculty in genomic data science and experimental genomics by providing a research environment that supports and enhances their projects. 2) Expand the breadth and impact of research from NIH-funded quantitative biologists at Dartmouth by: (a) continuing to recruit talented quantitative biology faculty; (b) mentoring junior quantitative biologists; and (c) providing systems for translating single-cell dynamics to applications in human disease. 3) Develop, maintain, and leverage key shared resources to support faculty in the CQB, at Dartmouth, and in the IDeA community by growing the Single-Cell Genomics and Genomic Data Science Cores. 4) Create synergistic interactions between the dry-lab computational scientist and traditional experimental scientist to promote cross-talk, collaboration, and integrative analyses between the disciplines. Dartmouth continues to make a substantial institutional commitment to the success and long-term sustainability of the CQB, including the commitment to hiring five new tenure-track faculty. Institutional program enrichment funds will support research infrastructure, scientific exchange, and a pilot project program to foster a vibrant intellectual community, recruit new project leaders, and enhance the impact and funding competitiveness of all CQB members. With experienced leadership, efficient administrative structures, and a compelling vision, CQB has demonstrated a paradigm that interweaves computational and experimental early-stage translational research, allowing the CQB to grow its portfolio of extramurally-funded investigators and thrive as a nationally- recognized Center of Biomedical Research Excellence.

NIH Research Projects · FY 2026 · 2019-08

This R01 renewal application seeks to extend our initial observation that the cytosolic RNA-sensing pattern- recognition receptor MDA5 plays a critical role in regulating the alveolar macrophage-mediated host resistance against the human fungal pathogen Aspergillus fumigatus. Mechanistic understanding of how alveolar macrophages keep fungal infections at bay in immune competent individuals remains ill-defined. Currently, there is a critical gap in understanding how the early host-pathogen interactions between fungi and alveolar macrophages work to drive fungal clearance and host resistance. Our data from the last funding period demonstrate a novel role of live fungal conidia in triggering the cytosolic RNA-sensing MDA5 receptor specifically in alveolar macrophages to initiate the host protective type I and type III interferon response. Thus, our central hypothesis is the lung specific programing of alveolar macrophages enables them to sense multiple vitality features of Aspergillus fumigatus isolates to serve as a central hub of inflammation and host resistance against this menacing fungal pathogen. In SA1 we identify features of alveolar macrophages that enable the effective type I and type III interferon response which are necessary for host resistance against A. fumigatus. This will be done using the adoptive transfer of a novel transgenic fetal-liver derived alveolar macrophage technology developed in our Co-I’s laboratory to our MavsCd11c conditional knock-out mouse line, which has been shown have a critical defect in host resistance against Aspergillus fumigatus. In SA2 we will mechanistically follow-up on our observation that there are fungal dsRNA-dependent and fungal dsRNA- independent mechanisms for triggering MDA5/MAVS activation. This will be done using novel isogenic pairs of Aspergillus fumigatus that are infected or not with a dsRNA mycovirus, as well as Aspergillus fumigatus mutants defective in mycotoxin production to identify the fungal effectors triggering MDA5/MAVS-dependent inflammatory responses. Overall, this research fills a critical knowledge gap regarding the molecular mechanisms of the host- pathogen interaction occurring in the lungs to drive the protective antifungal MDA5-dependent type I and type III interferon response induced by Aspergillus fumigatus in alveolar macrophages in both mice and humans.

NIH Research Projects · FY 2026 · 2019-07

We request support to continue developing and evaluating a combined near-infrared spectroscopic tomography (NIRST) and magnetic resonance imaging (MRI) platform. Our aim is to explore whether this concurrent modal- ity—which does not require contrast injection or involve ionizing radiation—can achieve acceptable diagnostic performance. Research during the current funding period yielded: (i) An MRI-guided (MRg)-NIRST system that can acquire MRI and NIRST data from up to 4,096 source-detector positions over the entire breast at six wave- lengths1; (ii) An MRI-compatible, wearable breast optical interface featuring eight flexible optical strips, each equipped with six photodetectors (PDs) and six side-firing fiber probes, designed to accommodate breasts of various sizes and shapes2; (iii) A new type of tissue phantom that is permanent, easily molded into various shapes, has similar elastic and spectral properties (in the wavelength range of 600–850 nm) to breast tissue, and provides MRI or CT contrast for validating the NIRST system;3 (iv) Reconstructed images of phantoms with inclusions ranging from 10–25 mm in diameter with small errors in estimated inclusion diameter and con- trast in total hemoglobin (HbT); (v) HbT estimates from reconstructed images of healthy subjects which ranged between 8.0–25.2 μM and aligned with previous imaging studies;1,4 and (vi) A deep learning approach (DL) that reconstructs 3D MRg-NIRST images in 1.4 seconds after the network is trained on synthetic data. These achievements have provided valuable technical insights, demonstrating the potential to overcome barriers to clinical acceptance of MRg-NIRST and motivating us to continue the project. The hardware platform proposed in Aim 1 resolves issues caused by MRI surface artifacts (due to the strips) and estimates additional NIRST parameters—such as oxy-hemoglobin, water, lipid and collagen content—by increasing the number of laser sources and extending the wavelength range. Advanced 3D image reconstruction methods, using either deep learning or traditional techniques or their combination, will be developed in Aim 2, and validated on phantoms, healthy volunteers, and patients with known breast abnormalities. Finally, we will evaluate the diagnostic perfor- mance of MRg-NIRST, with and without contrast injection, in a clinical study involving 112 patients with breast abnormalities, as part of Aim 3. This project builds upon an extensive translational breast imaging infrastructure we have developed through which collaborations with experts in diagnostic radiology and biomedical engineering occur that will advance the proposed imaging technology.

NIH Research Projects · FY 2025 · 2018-09

Autophagy is a cellular process in which cytosolic material is captured in double-membrane vesicles, termed autophagosomes. Subsequently, autophagosomes fuse with the vacuole, in yeast, or lysosomes, in mammalian cells, leading to the degradation of the captured contents. Autophagy can capture a diversity of cytosolic cargos, including organelles, large protein aggregates and intracellular pathogens. As such, autophagy plays critical roles in maintaining organelle quality, preventing the accumulation of protein aggregates and clearing intracellular pathogens. Therefore, defects in autophagy have been associated with various human diseases, including cancer, neurodegenerative disease and inflammatory disease. During autophagy, cargo can be captured via a non-selective or selective mechanism. In selective autophagy, cargos are identified by selective autophagy receptors (SARs). SARs are either soluble cytosolic proteins recruited to the cargo upon autophagy initiation or integral membrane proteins embedded in the organelle membrane that will become a cargo for selective autophagy. While the mechanisms by which cytosolic SARs initiate selective autophagy have become increasingly clear, the mechanisms by which integral membrane SARs coordinate with the autophagy machinery to initiate selective autophagy are still not well understood. The proposed work will use mitochondrial autophagy (mitophagy) in yeast as a model system to investigate the mechanisms by which integral membrane SARs coordinate with the autophagy machinery to initiate selective autophagy. Mitophagy in yeast is an ideal system to study selective autophagy initiation as yeast have only a single SAR for mitochondria, termed Atg32. In contrast, mammalian cells have five integral membrane SARs for mitochondria making it more challenging to interpret knockdown or mutagenesis studies of any individual SARs as other related SARs may compensate. This proposal will utilize a cross-disciplinary approach combining cell biology, lipid biochemistry, biochemical reconstitution and structural biology to study mitophagy initiation. The proposed work will help develop a molecular understanding of the mechanisms of selective autophagy initiation by integral membrane SARs, provide a starting point for studying other integral membrane SARs using biochemical approaches and provide novel methods for studying these systems.

NIH Research Projects · FY 2026 · 2018-08

Even with active antiretroviral therapy, some children living with HIV develop neurocognitive deficits and show neurodevelopmental delays. This can affect their ability to read, form relationships, and find productive employment. Detecting HIV-associated central nervous system (CNS) effects early, and following their progress reliably, is critical for studying, assessing, and treating this disabling HIV co-morbidity—particularly in the lower- and middle- income countries where most cases exist and resources are limited. We have been tracking literacy development and cognitive performance longitudinally in a cohort of children living with and without HIV (CLWH/CLWOH) in Dar es Salaam Tanzania. The focus is determining if tests of central auditory function (i.e., tests of the brain’s ability to process complex sounds) are associated with, or predict, the development of literacy, behavioral problems, and neurocognitive abilities. Results show that CLWH have worse neurocognitive performance, poorer literacy skills, and more behavioral problems on average than their non-infected age and socioeconomic status matched peers. Interestingly, central auditory test performance correlates with literacy and neurocognitive outcomes. Children who consistently perform better than their peers in detecting speech in background noise are more likely to read earlier than those who do not. Similarly, children with poor central auditory performance also have worse performance on cognitive tests of memory, attention, and processing speed. These results support our previous work showing that performance on tasks assessing the central auditory system correlate with tests of neurocognitive abilities. These longitudinal results suggest the tests might be predictive, where a central auditory test at a young age might help predict the subsequent development of literacy and at a later age. Children entered the current study at ages 3-8, now they will be entering adolescence. A key question is whether these tests might predict critical life outcomes such as school performance, mental health, and quality of life. Very few studies exist that follow CLWH into adolescence when CLWH take more responsibility for their care. This renewal will show how auditory tests relate to educational outcomes, mental health, and quality of life in a well-studied cohort of CLWH/CLWOH as they progress into adolescence. We have assembled an international team with experience in central auditory, neurocognitive, and neuropsychological testing. Dr. Nina Kraus and her team at Northwestern are internationally recognized experts in auditory electrophysiology. The Dar es Salaam team has extensive experience in performing both central auditory and neurocognitive tests and managing large-scale studies. Dr. Jonathan Lichtenstein is expert in assessing neurocognitive function and developmental delays in children. Together this team will track educational, mental health, and quality of life outcomes in CLWH and determine the role central auditory testing could play in predicting or assessing the neurocognitive effects of HIV infection. These tests may offer a new and improved way to assess critical life outcomes in adolescents living with HIV.

NIH Research Projects · FY 2025 · 2018-07

In cystic fibrosis (CF), mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) unleash a cascade of clinical disorders, including chronic airway infections, systemic inflammation, microbial virulence, diabetes, malnutrition, and kidney and liver disease. Great progress has been made in some areas of disease, and CFTR modulators provide dramatic benefits to many people with CF (pwCF). Still, there is growing evidence of the interconnections among lung and gut dysbioses and CF pathogenesis. As pwCF live longer, formerly rare symptoms are becoming more common. Thus, there is a pressing need to both understand and systematically treat the functional relationships between CFTR function, commensal and pathogenic microbes, metabolic states, epithelial function and immune responses. Dartmouth has an interactive CF research team of 49 faculty members with extramural funding of $14.4M/year, studying epithelial biology, CFTR correction, host-microbe interactions, gut dysbiosis and systemic immunity, as well as airway infections and antimicrobial strategies. The Dartmouth CF Research Center (DartCF) will build on progress made in the past six years. We will deploy P30 and institutional funds to recruit new CF faculty, strengthen our research base, and foster interdisciplinary discovery. Our aims are: 1) to catalyze new research in CF basic and translational research in areas of interest to NIDDK; 2) to develop integrative strategies to understand and address CF pathobiology; 3) to create new research tools and support CF research through outstanding shared services; and 4) to build research capacity in CF locally, regionally, and nationally. We will focus P30 resources on 1) forging collaborations between CF and data-science researchers to mine CF datasets for systems-level perspectives, 2) expanding access to sophisticated GI model systems, and 3) building on unique Dartmouth longitudinal patient cohorts to explore microbial community structure in the gut, host-microbe signaling, the effects of existing therapies, and implications for whole-body disease. A key theme is that dysbioses are interconnected, and that parallel investigations, coupled by powerful new data-science strategies can understand this complex underlying biology and reveal new therapeutic approaches. In parallel, we will leverage our research base to investigate extra- pulmonary CF disease and treatments. DartCF supports a variety of mechanisms. First, we fund a Pilot Project Program (P3) to develop new scientific opportunities in NIDDK-relevant areas and to recruit new faculty members to the Center. Second, we fund three scientific cores to support studies in CF: a Gastrointestinal Model Systems Core (GI-MSC), a Clinical and Translational Research Core (CTRC), and a CF Bioinformatics & Biostatistics Core (CF-BBC). Finally, we support an Enrichment and Research Administration Core (ERAC) to foster an interactive scientific community, sponsor retreats and courses, and track program progress. These efforts will identify new CF therapeutic opportunities, develop novel interventions, monitor the resulting changes body-wide, and track outcomes. DartCF will intensify the translation of research into improved CF outcomes at Dartmouth.

NIH Research Projects · FY 2025 · 2018-03

Project Summary Emotions play a critical role in organizing human experience and behavior, and emotion dysregulation lies at the heart of psychopathology and functional impairment across disorders. To measure and understand emotion dysregulation, advances in understanding the fundamentals of how the brain generates and represents emotional states are vitally needed. This proposal develops and validates brain models underlying emotional states in naturalistic, narrative contexts. We combine Functional Magnetic Resonance Imaging (fMRI), multimodal latent factor models, natural language processing, and pattern recognition techniques to develop models of brain activity that characterize how individuals generate emotional experiences. Over the previous project period, we have developed several technical innovations to identify dynamic emotional states from multimodal data and how they vary moment-by-moment during fMRI scanning. These models allow different individuals to experience and express latent emotional states at different times, accounting for the idiosyncratic interpretations of events that are a hallmark of human emotional responses. We elicit emotional experiences using dynamic, naturalistic movies and autobiographical stories. In Experiment 1, we infer latent emotional states using a novel application of the Shared Response Model (SRM), a latent factor model that integrates multiple simultaneously acquired measurement modalities including: moment-by-moment subjective ratings inferred using an innovative collaborative filtering approach, automatically decoded facial expressions using computer vision techniques, and psychophysiological signals. We then use these emotion signals to identify distributed patterns of brain activity that track distinct emotional states. In Experiment 2, we characterize how cortical-subcortical circuits involved in appraisal–and particularly the ventromedial prefrontal cortex–generate interpretations of unfolding events that give rise to emotional experiences. We leverage high-dimensional semantic embeddings of participants’ appraisals as revealed by a think-aloud protocol. The resulting brain models of specific emotion categories afford several potentially transformative advantages. Such models can (a) provide insight into which systems are necessary and sufficient for emotion generation; (b) be shared and tested across studies, allowing us to evaluate their generalizability across contexts; and (c) provide targets for psychological and neurological interventions. Together, these studies will yield generalizable models of the dynamic brain patterns underlying specific emotional experiences. Such models could transform the study of emotion by providing ways of capturing the moment-by-moment dynamics of emotional states, and clinical research by allowing investigators to test effects of psychological interventions on brain targets related to specific emotions.

NIH Research Projects · FY 2025 · 2018-02

ABSTRACT Resident memory (Trm) cells are a long-lived tissue localized CD8 T cell compartment that serves as a critical immune barrier against cancer progression and metastasis. Our work on the prior cycle of this R01 has shown that melanoma-specific Trm populations become distributed across skin and tumor-draining lymph nodes (TDLNs) where they provide protection against primary and metastatic melanoma in mice. In humans, we found that Trm populations with similar characteristics are sustained for years in the skin of melanoma survivors. However, the field lacks a clear understanding of mechanisms governing the establishment, function, and interconversion of Trm populations across different tissue locations. The overarching goal of this renewal application is to define the programming requirements, trafficking requirements, and potential clinical benefit of tumor-specific Trm cells that localize to tumor-draining lymph nodes. Based on our preliminary data that Trm populations in lymph nodes (LNs) uniquely require type-1 interferon (IFN) signals for their generation, Specific Aim 1 will define the mechanisms and timing of type-1 IFN production that are crucial for LN Trm formation. We will define the critical window of type-1 IFN sensing, and the cellular interactions that govern this sensing during Trm cell programming and maintenance. We will also determine the importance of the cGAS/STING pathway in providing IFN signals to Trm cells in TDLNs. Thus, we expect to reveal type-1 IFN as a key cytokine for promoting LN-restricted Trm generation. Second, based on our finding that Trm cells traffic from skin to draining LNs during primary and recall responses to melanoma, Specific Aim 2 will define the dynamics between these tissue- restricted compartments. By limiting T cell homing to peripheral tissue we will define a role for tissue access in supporting Trm seeding in LNs. We will also determine the relative contribution of skin vs. tumor egress to Trm generation in LNs. Then we will define Trm cell fate and differentiation trajectory between skin and LNs to test the provocative hypothesis that skin serves as a reservoir for functional T cell responses against melanoma. Finally, Specific Aim 3 will define the function and prognostic significance of LN Trm populations in patients with melanoma and non-small cell lung cancer (NSCLC). These studies will test the hypothesis that regional LN Trm populations correlate with resistance to metastasis to LNs. Our studies will define the function, stemness, and plasticity of human LN Trm populations that associate with protection from metastasis. By defining mechanisms underlying tissue-specific Trm generation, this work will support our overarching goal of generating Trm responses throughout tissues where cancers grow and metastasize.

NIH Research Projects · FY 2025 · 2017-09

Project Summary. Fungal mediated disease progression is highlighted by populations of fungal cells that form a community referred to as a biofilm. For therapeutic success, contemporary antifungal therapies must be effective at the site of infection in the context of an established fungal biofilm. Critically, it is now clear that emergent properties arise from fungal biofilms that directly alter virulence, disease progression, and antifungal drug susceptibility. However, the mechanisms through which filamentous fungal biofilm emergent properties impact virulence, disease progression, and antifungal susceptibility remain a significant knowledge gap. The long-term goal of this project is focused on defining the molecular mechanisms of Aspergillus fumigatus biofilm mediated disease progression mechanisms to inform contemporary and novel therapeutic approaches. In the prior funding period, we made important progress that surprisingly revealed heterogeneity in A. fumigatus biofilm morphology across clinical isolates. Differences in biofilm morphology altered virulence and disease progression in vivo in murine models of aspergillosis. We discovered that long term growth in a low oxygen environment gives rise to a biofilm morphology we termed H-MORPH and that a novel fungal specific gene cluster that contains a protein with unknown function was sufficient for H-MOPRH formation. Significantly, we identified H-MORPH clinical isolates from both acute invasive aspergillosis patients and patients with chronic aspergillosis, suggesting H-MORPH can arise in human disease. We observed that H-MOPRH occurs in vivo in a murine model of invasive pulmonary aspergillosis and contributes to worse disease outcomes compared to the contrasting N-MORPH isolates. In aim 1, we will define the genetic pathway(s) that regulate A. fumigatus the development of this unique population level morphotype utilizing the newly discovered biofilm architecture factor (baf) gene family as a tool to dissect the underlying mechanisms. In aim 2, we will define the differences in fungal metabolism that underly N-MORPH and H-MORPH morphotypes and test the hypothesis that H- MORPH biofilms are carbon catabolite de-repressed which leads to increased fitness in vivo. In aim 3, we test the hypothesis that H-MORPH strain metabolism is immune modulatory through alterations in fungal pathogen associated molecular pattern exposure. Taken together, our proposed studies will fill significant knowledge gaps related to the discovery of distinct A. fumigatus morphotypes that directly impact virulence. Advancing our understanding of this knowledge gap is expected to lay the foundation for new diagnostic and therapeutic strategies to combat highly virulent and drug resistant strains of this important human fungal pathogen.

NIH Research Projects · FY 2025 · 2017-09

The ‘actin cytoskeleton’ is not one structure but a number of distinct structures assembled and disassembled for different purposes. In mammalian cells, a few abundant and easily recognizable structures dominate our view of the actin cytoskeleton, including: stress fibers, lamellipodia and filopodia. However, a growing number of less abundant and/or highly transient actin-based structures have been revealed, controlling important cellular processes. Two such actin structures are the subject of this application: 1) CIA, calcium-induced actin; and 2) ADA, acute depolarization-induced actin. Though highly transient, both structures are extensive in the cytosol and affect important processes. In addition, both CIA and ADA impact the structure and function of mitochondria. CIA depends on calcium activation of the formin protein INF2, which stimulates actin polymerization on the endoplasmic reticulum and throughout the cytosol. Downstream effects of CIA include increased mitochondrial calcium and increased mitochondrial fission. The importance of CIA is illustrated by the fact that INF2 mutations link to two diseases, focal segmental glomerulosclerosis (FSGS) and Charcot-Marie-Tooth disease (CMTD). ADA is triggered by mitochondrial depolarization (either pharmacologically-induced or hypoxia-induced), which activates two parallel pathways: 1) mitochondrial calcium release activates protein kinase C-, activating in turn Rac, WAVE complex, and Arp2/3 complex; and 2) decreased ATP activates AMP-dependent protein kinase (AMPK) through LKB1, activating in turn Cdc42 and FMNL formins. The ADA actin network is tightly associated with mitochondria. An exciting new result is that one immediate consequence of ADA is rapid stimulation of glycolysis. Additionally, ADA temporarily inhibits longer-term consequences of mitochondrial depolarization such as mitochondrial reorganization and recruitment of the mitophagy protein Parkin. The goals in this grant period are to elucidate both the mechanisms triggering CIA and ADA, as well as their downstream effects. These goals will be accomplished using a combination of cellular approaches (live-cell microscopy, proteomics, metabolic analysis) and biochemical approaches (cell-free reconstitution, analysis of purified proteins on model lipid membranes). The questions to be asked include the following. 1) How is INF2 activated by increased calcium? 2) How are INF2-polymerized filaments organized into a network by myosin II and fascin? 3) How does CIA interface with known mitochondrial fission proteins such as Mff and Drp1 to stimulate fission? 4) How do PKC and AMPK activate Rac and Cdc42, respectively, during ADA? 5) How do Arp2/3 complex and FMNL formins work together during ADA? 6) How does ADA stimulate glycolysis? These questions address fundamental mechanistic questions important to a wide range of mammalian cells, and occupy an exciting frontier between cytoskeletal biology, mitochondrial biology, and metabolism. 1

NIH Research Projects · FY 2025 · 2017-07

Project Summary During chronic fungal infections, heterogeneous subpopulations can arise. While these diversified populations can pose significant challenges for treatment, they also provide an opportunity to identify pathways under selection in vivo. This proposal focuses on the study of unique longitudinally-collected sets of Candida (Clavispora) lusitaniae isolates from three individuals with cystic fibrosis (CF). In each case, the C. lusitaniae infections replaced prior chronic bacterial infections, were associated with high levels of airway inflammation, and were resistant to treatment. C. lusitaniae is an emerging agent of candidiasis known to develop resistance to antifungal drugs and is a close relative of Candida auris, a multidrug resistant pathogen that has repeatedly caused hospital associated outbreaks with high mortality. Through the genomic and phenotypic analysis of variable traits in these chronic infection populations, via a productive collaboration between the Hogan and Stajich Labs, we found striking heterogeneity in two genes: MRR1, which encodes a transcription factor known for its ability to confer resistance to azoles, bacterial toxins, and host antimicrobial peptides, and MRS4, a mitochondrial iron transporter that affects metabolism and metal uptake. We propose that these genes strongly impact host interactions and fungal physiology in vivo. Our studies revealed that Mrr1 controls a large regulon of resistance, metabolic and metal acquisition genes, and we discovered the first endogenous inducer of Mrr1, methylglyoxal (MG), which spontaneously forms from intermediates in glycolysis. Further, we found that repeated loss-of-function mutations in a second gene, MRS4, biases cells towards a glycolytic metabolism, increased MG production, and increased Mrr1 signaling. We propose that these changes promote survival in an inflammatory environment. Specifically, we propose to test the hypotheses that (Aim 1) endogenous MG directly stimulates inducible Mrr1 variants, (Aim 2) that MRS4 loss-of-function mutations induce MG Mrr1 signaling through increased glycolysis, and (Aim 3) that increased glycolysis decreases ROS accumulation in co-culture with activated neutrophils. As we show in published and preliminary data, the pathways and mechanisms studied here are conserved broadly across diverse Candida pathogens including C. auris. Through this work, we aim to further develop our understanding of C. auris, and ways in which C. lusitaniae can be used as a highly tractable, parallel system with an expanded tool kit including genetic, genomic, transcriptomic, and metabolomic resources. We propose that the studies will reveal new broadly relevant mechanisms by which fungi adapt to the host environment which can inform new treatment strategies.

NIH Research Projects · FY 2025 · 2017-06

PROJECT SUMMARY/ABSTRACT We propose to continue our Training Program in Surgical Innovation (TPSI) to provide pre-doctoral engineering students the knowledge, skills and experience necessary for becoming leaders in surgical innovation. During the current funding period, we established a pipeline of quality applicants, implemented an effective review and selection process, developed new courses, and trained 10 predoctoral students (2 of whom are new trainees). Program evaluations (performed independently by Dartmouth’s Center for Program Design and Evaluation, CPDE) found our innovation curriculum was praised, mentor-mentee relationships were strong, and merging of training in biomedical engineering, surgical translation and innovation & entrepreneurship was valued highly by trainees and mentors, alike. Outcomes to date are also strong: graduates hold industry positions in innovative biomedical technology companies (two in medical start-ups), patents were filed and papers published in peer- reviewed journals. Current trainees are also exceling: 2 were selected among 6 finalists (from more 50+ appli- cants as far away as Canada) in a regional pitch competition; two are interning at biomedical start-ups. TPSI links the Thayer School of Engineering (TSE) PhD Innovation Program (PhD-I) with a state-of-the-art NIH- sponsored surgical research facility, the Center for Surgical Innovation (CSI). PhD-I provides trainees with na- tional recognition, a well-developed curriculum, experienced faculty, approved advanced degree requirements and a proven track record of success. CSI provides trainees access to experienced clinician-scientists and state-of-the-art operating rooms equipped with advanced intraoperative imaging, designed for human and ani- mal use. The funding request will cover tuition and stipends for 6 pre-doctoral positions per year. We will retain TPSI’s administrative structure of three Program Directors (now Multi-PIs) with expertise in Biomedical Engi- neering, Surgical Translation, and Innovation & Entrepreneurship, respectively. Participating mentors will con- tinue to be organized similarly into Biomedical Engineering (BE), Surgical Translation (ST) and Innovation & Entrepreneurship (IE) groups, and each trainee is assigned to a trio for guidance through the program. Train- ees satisfy all of the elements of PhD-I – technical proficiency, technical breadth, specialization, professional competence, original research and innovation skills development – tailored to the surgical setting, including specialized courses in surgical innovation and commercialization. Methods for trainee recruitment and reten- tion, diversity enhancement, program evaluation, instruction in responsible conduct of research and methods for enhancing reproducibility are in place, and will continue to be monitored by CPDE.

NIH Research Projects · FY 2026 · 2017-03

In this application, we propose continued funding to the Dartmouth Cystic Fibrosis Training Program (DCFTP). Continuing support is sought for a comprehensive, highly interactive, interdepartmental training program with an emphasis on CF basic and translational science. The overall philosophy of the program is to use a disease-centered approach to teach fundamental concepts of basic and translational science in the context of a dynamic, multidisciplinary research and team-based mentoring environment. In this submission, we propose to directly fund 4 Trainees annually. Our program has pooled the talents of 22 dedicated Faculty Trainers from a wide range of departments—our current program includes faculty in the departments of Biochemistry and Cell Biology, Chemistry, Molecular Systems Biology, Medicine, Microbiology/ Immunology, Epidemiology, Pediatrics, and Engineering—to provide instruction in CF-related research to PhD and MD/PhD students. The DCFTP will function in the context of a Research Base already rich in basic, translational, and clinical CF-related studies. The CF research program at Dartmouth has grown from three investigators and one NIH grant in 1997 to a community of world-class scientists that currently secure $21.1M per year in direct costs of research funding ($0.96M per investigator average). Dartmouth will also provide ~$39.6M in institutional support, including direct support to DartCF, our training program and the affiliated graduate programs, over the next 5 years. Our Training Faculty use approaches including the study of clinical cohorts, quantitative methods, and laboratory-based and animal studies; thus, our trainees are exposed to a broad range of scientific strategies to answer impactful scientific questions. The DCFTP participating Training Faculty, which include PhD, MD, and MD/PhD researchers, work in a range of disciplines, with CF research as the central focus; all Training Faculty conduct research, are well funded, and teach and mentor our Trainees. This group of basic and physician-scientist researchers is actively engaged in CF-related research, with expertise in cell biology, structural biology, microbiology, immunology, microbiome, genetics, bioinformatics, statistics, and proteomics. A hallmark of our program is the close and consistent interaction of our Training Faculty with our DCFTP Trainees via DCFTP-sponsored general and Trainee-specific activities, including weekly research meetings, courses, journal clubs, and our annual retreat. Training-grant funds and DCFTP- sponsored, Trainee-specific enhanced activities provide a unifying base that greatly facilitates the strikingly interactive nature of this group (evidenced by 38 collaborative publications over the past ~5 years among our 22 Training and 9 Affiliated Faculty) and of the collaborative grants held by these faculty.

NIH Research Projects · FY 2025 · 2016-09

We are advancing the success of the Environmental Influences on Child Health Outcomes program that seeks to “enhance the health of children for generations to come” as one of the Pregnancy and Pediatric Cohort Study Sites. Activities include accruing data and biospecimens on participating adults and their children. Data and biospecimens from our cohort contribute to Concept Proposals, including Opportunity and Innovation Fund (OIF) awards to early career investigators. The cohort’s biospecimens are leveraged for ECHO-Wide laboratory analyses led by our Dartmouth team and other investigators. This includes the ECHO Genome Wide Association Study. Non-DNA samples shipped to the ECHO Biorepository include urine, hair, blood, and teeth. We actively take part in ECHO program leadership, committees and working groups, designing and sharing protocols, and creating instructional videos and participant feedback materials. We are recruiting additional pregnancies, enlisting current partners, and following children previously accrued into the cohort and newly born in this study phase. We adhere to the ECHO Cohort Protocol and rely on the program’s REDCap Central database and single IRB. We capitalize on ECHO’s unprecedented data and samples, along with Dartmouth’s robust infrastructure and expertise. Thus, our study site fills a critical niche in the ECHO program and will continue to provide leadership and collaboration in support of its overall mission.

NIH Research Projects · FY 2026 · 2016-08

ABSTRACT Reversible protein phosphorylation is a major regulatory mechanism that controls most cellular processes. Indeed, most human proteins are phosphorylated at one or more residues. A ‘tug of war’ between kinases and phosphatases controls protein activity and function by dynamically changing phosphorylation states of proteins. Despite substantial progress in deciphering kinase-mediated phosphorylation and its functional consequences, much less is known about phosphatases and how dephosphorylation is regulated. The long-term goals of our research program are to uncover the mechanisms that coordinate and integrate PPPs, kinases, and their shared substrates in the signaling networks that control cell division. The majority of cellular dephosphorylation is carried out by the seven catalytic subunits of the phosphoprotein phosphatase (PPP) family. Despite the apparent simplicity suggested by the small number of catalytic PPP enzymes, complexity and specificity arise through the formation of holoenzymes. Each holoenzyme functions as a distinct entity. The non-catalytic subunits modulate the activity of the catalytic subunits, enabling substrate specificity, dictating subcellular localization, and ensuring appropriate regulation. Combinatorially, there are several hundred functional PPP holoenzymes. There are key gaps in knowledge and challenges in studying PPP function and mechanisms of dephosphorylation. Although hundreds of thousands of phosphorylated sites have been identified, only a few hundred have been matched to be dephosphorylated by a specific PPP holoenzyme. This is in part due to the lack of holoenzyme-specific inhibitors. To overcome this obstacle, we will continue to develop new approaches to identify holoenzyme-specific substrates to comprehensively map the PPP substrate space, providing a foundation for studying PPPs for our lab and the signaling community. Furthermore, the mechanisms that PPPs use to recruit substrates and catalyze site-specific dephosphorylation are still emerging. We will dissect these mechanisms to connect PPPs, their substrates, and opposing kinases. Finally, determining regulatory inputs governing PPP holoenzyme formation, activity, and function is needed to understand their role in regulating cellular processes. To fill these gaps in knowledge, we will use cell biological and biochemical strategies to (i) identify and functionally annotate PPP holoenzyme-specific substrates, (ii) dissect substrate recruitment mechanisms, (iii) decipher dephosphorylation preferences, and (iv) identify regulatory inputs governing PPP holoenzymes. We combine quantitative measurements of the proteome and phosphoproteome with the reconstitution of minimal signaling units in vitro and in cells to precisely determine phosphatase function and regulation in mitosis.

NIH Research Projects · FY 2025 · 2016-07

Project Summary/Abstract Membrane fusion is essential for cell growth, hormone secretion, neurotransmission, and cell invasion by pathogens. Membrane fusion mechanisms are conserved from yeast to humans. We have developed yeast vacuole fusion as a model system, identifying genes for membrane fusion, establishing an in vitro fusion assay with purified vacuoles, and purifying each relevant protein and lipid for reconstitution into proteoliposomes which faithfully reconstitute each aspect of fusion. These studies have revealed novel elements, most recently: 1. A dazzling array of proteins and lipids which cooperate for orderly lipid bilayer strain and rearrangement, giving fusion without lysis. 2. The assembly of complexes among membrane-bound proteins termed "SNAREs" isn't spontaneous, as heretofore believed. Their assembly is actually catalyzed by a large hexameric complex termed HOPS, which recognizes each of the individual SNAREs and assembles them in active intermediates, poised for rapid fusion. 3. Chaperones to the SNAREs, termed NSF/Sec18 and aSNAP/Sec17, which had been believed to only function to disassemble SNARE complexes after fusion, also promote fusion. 4. Lipids have a vital and active role in fusion. Each of these mechanistic insights will be pursued; our goals for the next 5 years are to understand the intermediates of HOPS-catalyzed SNARE assembly, their structures, the roles of chaperones Sec17/Sec18, and how these proteins trigger the lipid rearrangements of fusion. The importance of understanding this pathway is underscored by the central role of HOPS in the invasion of human cells by pathogenic viruses and bacteria.

NIH Research Projects · FY 2026 · 2016-06

Virtually all eukaryotic organisms appropriately examined have been shown to possess the capacity for endogenous temporal control and organization known as a circadian rhythm. The cellular machinery responsible for generating rhythms is collectively known as the biological clock. A healthy circadian clock underlies both physical and mental health. Because of the ubiquity of its influence on human mental and physiological processes - from circadian changes in basic human physiology to the clear involvement of rhythms in work/rest cycles and sleep - understanding the clock is basic to prevention and treatment of many physical and mental illnesses, from metabolic disorders to sleep/wake dysfunction and cancer. Our research uses genetic and molecular studies of the model eukaryote Neurospora, as well as mammalian cells in culture, to further our understanding of the organization of the circadian oscillator, a one- step transcription-translation feedback loop whose regulatory architecture is conserved from fungi to mammals. Planned research lies within three foci. Focus #1 builds upon our understanding of the interplay between structure and function in core clock components. We will determine how phosphorylations and interactions among clock components lead to repression within the feedback loop; address a controversy as to whether negative element turnover has a role in the mammalian oscillator; probe how clock-controlled phosphorylation guides essential interactions and activities of clock components leading to the canonical circadian property of temperature compensation, and how modulation of RNA metabolism and gene expression contribute to nutritional compensation. Focus #2 pioneers new territory and exploits recently developed techniques, expanding the use of cell biological tools to complement genetics in defining the spatio-temporal dynamics of clock components within the cell. We will show how, as well as where in the cell the clock operates. Focus #3 will build upon our strong grounding in the genetics and genomics of light-regulation, using computational and informatic tools to define the hierarchical network of transcription factors that govern the response of Neurospora to light and time. The aim is to provide the first concrete model for global circadian control of a eukaryotic genome. Our long term goals are to describe, in the language of genetics and biochemistry, the feedback cycle comprising the circadian clock, how this cycle is synchronized with the environment, and how time information generated by the feedback cycle is used to regulate the behavior of cells and organisms. These projects are complementary and mutually enriching in that they rely on genetic and molecular techniques to dissect, and ultimately to understand, the organization of cells as a function of time.

NIH Research Projects · FY 2025 · 2016-05

The Institute for Biomolecular Targeting Overall Grant Abstract: The Dartmouth Institute for Biomolecular Targeting (bioMT) infuses mechanistic investigations with a sophisticated awareness of disease pathology and therapeutic need, enhancing the quality of even the most fundamental research. At the same time, it helps orient mechanistic investigations towards long-term translational goals for complex diseases. In phase I, our progress was strong. All six of our research project leaders (RPL) with more than two years’ support have received R01-equivalent funding. Our cores provide unique protein biochemistry resources and ‘navigators’ to access microscopes campus-wide. Our cores and seminars have created a vibrant and interdisciplinary scientific community. Here, we propose to deploy phase II COBRE and institutional program enrichment funds to build on this foundation and fill key gaps to prepare bioMT for the transition to support independent of COBRE resources. Aim 1 is to increase our team of funded bioMT investigators to fill strategic roles in our research portfolio. Our two most recent RPLs (3–15 months’ support) are continuing into phase II, joined by two outstanding new hires. Their projects explore basic signaling and immunological mechanisms with potential relevance to therapeutic targets, interconnected by shared scientific and technical interests. All receive professional skills development from dedicated mentoring teams to help them achieve independent extramural funding, and all receive technical support from scientific cores offering state-of-the- art technologies directly relevant to their bioMT research projects. With institutional support, we will also hire five new faculty members working in the areas of discovering and exploiting molecular targets, and selected to create disease-relevant research areas of critical mass. As starting RPLs graduate, we will recruit new hires for EAC consideration as replacements. Aim 2 is to enhance our core facilities and prepare them for a transition to COBRE independence during phase III. The cores are fully staffed and have invested heavily in phase I instrumentation. We will add key new technologies based on user input, including parallel protein expression, mass spectrometry, advanced microscopy, and cryoEM. Cost-recovery models will be developed for incremental deployment in phase III to enable core financial independence. Aim 3 continues enriching our scientific exchange, including mini-symposia and pilot awards to foster new multi-PI and program-project applications, which will contribute to core utilization. Overall, these aims will leverage proven COBRE strategies – junior faculty hiring based on scientific excellence, academic mentoring, excellent administrative and scientific core support, and interdisciplinary research forums – to enhance bioMT’s scientific impact in targeted areas of therapeutic impact. This will prepare us for the transition to phase III funding and ultimate independence as an interdisciplinary and nationally visible research institute, spanning three schools and 10 departments at Dartmouth and deeply connected to local centers and regional IDeA partners.

NIH Research Projects · FY 2026 · 2015-09

This proposal is a competing renewal application of the Northeast Node of the National Drug Abuse Treatment Clinical Trials Network (CTN), which has been part of the CTN since 2015. The Northeast Node has engaged a network of hundreds of community, healthcare, researcher, policy, and payor partners across the states of New Hampshire, Vermont, and Maine. These states have among the very highest rates of substance use and overdose mortality in the U.S. and include some of the most rural communities in the U.S. The Node has brought significant expertise to the CTN in successfully leading multi-site clinical trials as well as feasibility, efficacy, effectiveness, implementation, and hybrid implementation/effectiveness research studies focused on improving the prevention and treatment of substance use disorders (SUDs); community-informed research; digital health, Artificial Intelligence, data science, data analytics, machine/deep learning, digital phenotyping and digital therapeutics for SUDs and related issues; and collaborating with health systems, research networks, community organizations and our SUD policymakers, state authorities and congressional delegates. The Node has also been a leader in many national CTN-wide initiatives, including launching and leading the first-of-its kind national Community Representative Council (CIRCL) to inform the work of the CTN across the nation and leading the strategic planning of the CTN, with PI Marsch serving as Chair of the national CTN Steering Committee. This renewal application builds on the Node’s strong infrastructure, track record of innovation and impact, and expertise of direct relevance to the priorities in RFA-DA-25-027 for the next iteration of the CTN. Since the Northeast Node’s inception 9 years ago, the Node has led or partnered on 17 CTN studies, 6 of which will continue into the renewal phase. And we plan to expand our research contributions to the CTN to promote advances in scientific discovery that offer great promise for increasing the reach and effectiveness of treatments for SUDs that address both key and emerging substance use needs. We seek to generate evidence to inform healthcare policy and payor decisions and scale-up and sustain best practices. We will also enhance our infrastructure and processes to best ensure our broad community informs the priorities for the research and dissemination agenda of our Node as well as the national CTN by formalizing a Northeast Node Partner Exchange and increasing the deliverables of CIRCL to enhance its reach and impact. And we will enhance our infrastructure and processes to expand our training program to provide trainees in our Node the opportunity to gain valuable hands-on experience in designing, participating in, and managing clinical studies in the CTN.

NIH Research Projects · FY 2026 · 2014-08

Project Summary/Abstract: Meiosis is the cell division process in which a diploid cell undergoes one round of DNA replication followed by two rounds of chromosome segregation to ultimately produce haploid gametes. Errors in meiotic chromosome segregation can result in miscarriage and trisomy conditions, such as Down syndrome. Therefore, studying meiotic chromosome segregation is important for understanding how errors in this process occur. The objective of this proposal is to determine the mechanisms that regulate proper chromosome segregation, focusing on unique events in meiosis I and meiosis II. These studies leverage the model organism S. cerevisiae, due to the ease of developing tools to address mechanistic questions. These innovative tools will allow the investigation of how cells establish microtubule-kinetochore attachments, how cells correct improper attachments, and how cells monitor the attachments through spindle checkpoint activity, which delays the cell cycle in the presence of unattached kinetochores. The rationale for the proposed research is that the questions focus on processes that are unique to meiosis but are likely to be highly conserved, allowing the findings in budding yeast to uncover general mechanisms of meiotic regulation. Strong preliminary data guide the following three specific aims: 1) determine how the number of crossovers and crossover position along the chromosome affects the establishment of correct kinetochore-microtubule attachments in meiosis I; 2) investigate how cells prevent persistent spindle checkpoint activity during meiosis; and, 3) determine how the phophoregulation of proteins at the meiotic kinetochore ensure proper kinetochore-microtubule attachments in meiosis II. In the first aim, strains have been developed to target crossovers at particular locations on a chromosome arm. Using time-lapse imaging, the strains will be monitored for the timing of establishing bioriented kinetochore microtubule attachments, and for the number of rounds of error correction of improper attachments. The second aim tests the novel hypothesis that cells have a developmentally programmed mechanism to overcome persistent spindle checkpoint activity in meiosis to ensure the production of gametes. The third aim analyzes how protein phosphatases counteract kinase activity to specifically ensure kinetochore assembly and the establishment of kinetochore-microtubule attachments specifically in meiosis II. The innovative approach of combining the latest imaging technologies to monitor kinetochore-microtubule attachments in engineered strains allows the testing of novel hypotheses about cell-cycle regulation. The proposed research is significant because the results are expected to reveal general principles of meiotic regulation important for proper chromosome segregation. Ultimately, the results will further our understanding of how errors in meiosis facilitate developmental abnormalities.

NIH Research Projects · FY 2025 · 2014-07

This renewal application proposal aims to sustain and enhance a unique predoctoral and postdoctoral training program in an emerging area of transdisciplinary research, co-occurring substance use and other psychiatric and medical disorders (COD). The need for this scientific focus continues to be vital given that COD are more the norm than the exception among those with addictive disorders. To effectively impact the problems of addiction and common psychiatric and health disorders, one must be fully aware of their co-existence, phenomenology, and clinical manifestations. Our evolving program leverages unique opportunities available at Dartmouth. Five research groups (Center for Technology and Behavioral Health - CTBH, The Dartmouth Institute, Quantitative Biomedical Sciences, Psychological and Brain Sciences, and Dartmouth Cancer Center Population Science) provide CODs-focused mentored research and training opportunities in treatment development and evaluation, digital health innovations for assessment and intervention, mechanisms of change, implementation science, and health services research. The program has evolved such that the preponderance of our faculty’s research occurs in the digital health space related to the emergence of our NIDA-funded P30 Center, the CTBH, as an international leader in innovative digital approaches to the study of COD. Our T32 Leadership Team and most of our other training faculty are also leaders and affiliates of the CTBH. Accordingly, our training activities have increasingly focused on digital health research methods and applications. Trainees are exposed to a broad, transdisciplinary agenda that combines a rigorous course of didactic seminars, training in the responsible conduct of research and rigor and reproducibility in research, and career development training and experiences. Each trainee works directly with a primary mentor and at least one co- or secondary mentor to assure exposure to a diverse range of research approaches. During the first 9.5 years of the program, high quality trainees have filled program slots. We have trained or are in the process of training 9 predoctoral and 18 postdoctoral trainees, plus 6 affiliated trainees funded by other sources. The great majority have had outstanding publication and presentation records, and all have continued in research intensive positions. Program evaluations have been excellent. We recently engaged an External Advisory Board, and the first meeting yielded a highly positive report; and more importantly, excellent suggestions for enhancement of the program. This renewal proposes to maintain an active census of 3 predoctoral and 4 postdoctoral trainees, which has worked well to date. The program will provide an enhanced, rigorous training environment that will continue to prepare young scientists to engage in collaborative, cutting edge, transdisciplinary research on COD.

NIH Research Projects · FY 2025 · 2013-04

ABSTRACT This Academic-Industrial Partnership (AIP) will further the clinical development of fluorescence guided surgery (FGS) through the utilization of the FDA exploratory Investigational New Drug (eIND) pathway. While recent advances in both imaging agents and technology for FGS has been impressive, significant limitations remain in detecting low dose administration of imaging agents and obtaining true molecular contrast between the tumor and normal surrounding tissues. The basis of this application and the underlying tenet of the proposed AIP is that a cost-effective, risk-diluted approach to clinical translation of imaging methodologies is needed in order to realize the promise of FGS. Utilization of our previously developed bench-to-clinic pipeline stands to accelerate surgical oncology to revolutionize both the procedures that are possible and the surgical outcomes that will result. In the first funding cycle our AIP implemented a low-cost testing pipeline for ABY-029, a fluorescent epidermal growth factor receptor-targeted synthetic peptide. We established the necessary protocols to bring ABY-029 from pre-clinical animal models into three Phase 0 clinical trials using the FDA eIND pathway (eIND #122681). We will build upon our previous success to apply for Investigational New Drug (IND) status of ABY-029 for economical, investigator-led Phase II testing of detection accuracy in glioma and head and neck cancers. Sim- ultaneously, we will utilize the pipeline to achieve eIND approval of paired-agent imaging. The proposed AIP between Dartmouth (Engineering and Medical Schools), Affibody AB, LI-COR, and DoseOptics brings together 4 partners who have the intellectual property (IP), expertise and infrastructure to develop, test and advance molecular-targeted fluorescent tracers and imaging systems for surgical guidance. Clinical translation and testing of FGS will be achieved through the completion of the following aims: Aim 1 - Advance near-microdose ABY-029 FGS for resection of primary high-grade glioma in a Phase 2 trial; Aim 2 - Adapt FGS system for minimally invasive resection of head and neck cancers for Phase 2 testing; and Aim 3 - Leverage the Dartmouth fluorescent agent development pipeline to bring quantitative molecular paired- agent imaging (PAI) to Phase 0 trial using a cocktail of ABY-029 and IRDye 680LT carboxylate (pABY-IR680). The translational INNOVATION will include advancement of ABY-029 into Phase II clinical trials, first-in-human implementation of paired-agent imaging for true molecular contrast via the established bench-to-clinic eIND pipe- line, and the integration of a highly sensitive, multi-channel near infrared surgical camera imaging system capa- ble of imaging in full surgical lights. The SIGNIFICANCE of this AIP will be to demonstrate improved margin detection for surgical resection of glioma and head and neck cancers using a low-cost, efficient testing pipeline.

NIH Research Projects · FY 2025 · 2012-05

Project Summary/Abstract Bacterial pathogens use type III secretion systems to translocate effectors into host cells to promote virulence. Type III secretion can also activate compensatory innate immune responses that are host protective. For example, type III secretion can trigger inflammasome assembly in host cells, resulting in release of the cytokine IL-1b. Virulent pathogens can inhibit compensatory protective immune responses triggered by type III secretion but how this is achieved at the cellular and molecular levels in vivo remains poorly understood. To address this knowledge gap, this project seeks to determine at the cellular and molecular levels how type III secretion effectors in virulent Yersinia species inhibit a protective inflammasome pathway in vivo. Yersinia uses two effectors, YopM and YopJ, to inhibit the pyrin inflammasome. It is not known if YopM and YopJ promote virulence by inhibiting the pyrin inflammasome in a cell specific manner. During invasive infections of lymphoid tissues Yersinia grow as extracellular microcolonies in direct contact with neutrophils within an organize immune structure known as a pyogranuloma. Pyogranulomas can be considered battlefields where Yersinia virulence factors combat protective immune responses in neutrophils acting as foot soldiers. Yersinia mutants lacking YopM and YopJ have a significant survival defect in lymphoid tissues suggesting that these effectors inhibit the pyrin inflammasome in pyogranuloma neutrophils. Additionally, IL-1b is important for host protection against infection by Yersinia lacking YopM and YopJ. Based on these published data and preliminary results we hypothesize that YopM and YopJ promote Yersinia virulence by inhibiting the pyrin inflammasome in neutrophils to prevent release of IL-1b in pyogranulomas. This hypothesis will be tested in Aim 1. YopM binds to pyrin in infected host cells and in purified form, but the molecular basis of this interaction is undefined. Based on published and preliminary we hypothesize that YopM targets the pyrin domain to inhibit the inflammasome in vivo and promote Yersinia virulence. This hypothesis will be tested in Aim 2. Completion of these aims will fill important knowledge gaps, move Yersinia pathogenesis research forward, have a broad impact on the field of neutrophil inflammasomes and inform new therapeutic strategies aimed at augmenting protective neutrophil inflammasome responses to bacterial pathogens.