Dartmouth College

Fonds de recherche du Québec – Nature et technologies · FY 2023-2024 · 2023-04

Volet: Bourses de recherche postdoctorale; Domaine: Environnement; Objet: Changements climatiques, impacts; Objet: Production végétale; Application: Sciences et technologies; Application: Environnement; Mots-clés: CLIMATE CHANGE IMPACTS, AGRICULTURE, LAND-CLIMATE DYNAMICS, COMPOUND EXTREMES, PLANT PHYSIOLOGY, CLIMATE ADAPTATION

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY The 24-hour cycles of endogenous regulation of physiology, known as circadian rhythms, align organisms to the inherent rotation of the earth. Circadian rhythms influence the regulation of many essential processes, including sleep regulation. Sleep is regulated by circadian rhythms in combination with sleep homeostatic mechanisms in the two-component sleep model. This proposal aims to further our understanding of sleep regulation by investigating key gaps that remain in the molecular mechanism of circadian rhythms, so that we may understand the underlying mechanisms of circadian-based sleep disorders. Filling these gaps will therefore enable us to develop therapies for sleep disorders. Circadian rhythms are defined in part by a ~24-hour period length, and their ability to maintain a consistent period across changes in ambient temperature (temperature compensation – TC). Despite the fundamentality of these properties, questions remain regarding their underlying mechanisms. We will investigate how the feedback loop powering the molecular clock completes a cycle (determining period length) in Aim 1, and the role for kinases and specific phosphorylation events in the TC mechanism in Aim 2. Current evidence suggests that both of these properties are ultimately regulated by phosphorylation. We hypothesize that the mechanism underlying period determination is conserved from Neurospora to mammals, and hence the feedback loop of the mammalian clock is closed by hyperphosphorylation of negative elements, rather than degradation, matching what our lab found in Neurospora. Aim 1 tests our hypothesis that phosphorylation status and not stability of the clock protein PER2 determines period length in mammalian cells. We hypothesize that TC involves precise and dynamic phosphorylation of key clock components by Casein Kinase I and II. Aim 2 uses a combination of phosphoproteomics and epistasis experiments with novel Neurospora strains to establish an integrative TC model that we will then test in mammalian cells in culture, again expecting mechanistic conservation. Overall, this project will shed light on fundamental aspects of circadian rhythms yet to be elucidated, ultimately establishing new models for both period determination and TC. Therefore, successful completion of this work will inform the understanding of all circadian-based disorders, including disorders such as Familial Advanced Sleep Phase Syndrome (FASPS). This fellowship will enable me to fill gaps in my training to become an independent academic scientist. Training will include participation in international conferences, mass spectrometry and modeling courses, meetings with my advisors, thesis committee, and collaborators, and training in teaching techniques, among others. I will take advantage of the rigorous environment at Dartmouth through departmental seminars and be supported in my technical training by our excellent core facilities. My immediate research environment will be the Dunlap/Loros lab that has for decades been a center for studying the molecular basis of circadian rhythms.

NIH Research Projects · FY 2026 · 2023-03

A Cereblon Signaling Network in Wnt-driven Cancers Abstract The long-term objective of this study is to investigate how Cereblon (CRBN) regulates the Wnt signal transduction pathway and to demonstrate how this can be exploited to target Wnt-driven colorectal cancers (CRCs). Wnt signaling is essential for intestinal stem cell maintenance and aberrant activation of this pathway drives the initiation and progression of nearly all CRCs. To date, no drugs that inhibit the Wnt pathway have been FDA- approved, partly due to the lack of druggable targets that can bypass common Wnt pathway mutations in CRC. In a conceptual breakthrough, our recently published findings reveal that Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase that is a drug target in hematological malignancies, plays a critical role in Wnt signal transduction. We found that CRBN promotes the degradation of a subset of substrates, including Casein kinase 1α (CK1α), a negative regulator of Wnt signaling and a key component of the β-Catenin destruction complex. Moreover, Wnt stimulation induces the interaction of CRBN with CK1α to promote its ubiquitination and degradation. Furthermore, we showed that the role of CRBN in Wnt signaling is conserved in human cells, mouse intestinal organoids, zebrafish, and Drosophila. These studies demonstrate the first endogenous mechanism of CRBN regulation and provide a novel means of controlling Wnt pathway activity, with relevance for animal development and disease. The goal of this project is to use in vitro, ex vivo, and in vivo approaches to gain a better understanding of how the clinically relevant anti-cancer target, CRBN, promotes Wnt signal transduction. The three specific aims are to: 1) Identify the mechanisms by which Wnt stimulation activates CRBN; 2) Identify Wnt-stimulated CRBN interactors that regulate tumorigenesis; and 3) elucidate the role of CRBN in Wnt-dependent cancer progression. Because CRBN is a well-studied target for the development of drugs to treat hematological malignancies, the knowledge gained from this study will aid in the development of more selective small molecules as well as suggest innovative treatment strategies for CRC and other Wnt-driven cancers.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY This academic-industrial partnership (AIP) proposal seeks to develop a robust and easy-to-use clinical scanner for tumor oxygen measurement (oximetry) in cancer patients. By knowing quantitative tumor oxygen levels in real time, radiotherapy could be better planned and delivered at times when there will be an optimal therapeutic ratio, thus significantly improving cancer care. The proposed approach seeks to leverage the unique capabilities of the in vivo electron paramagnetic resonance (EPR) oximetry technology that we have developed for measurement in human subjects into a medical device that is ready for routine clinical use. The scanner can make direct and repeated measurements of tumor oxygen by typical end-users in a clinical setting. The EPR research team at the Geisel School of Medicine (Dartmouth College) has successfully demonstrated the clinical feasibility and safety of EPR oximetry for measuring tumor oxygen in the clinic, and now seeks to expand upon that success through a collaborative partnership with ViewRay®, a medical-device company that is engaged in MRI image-guided radiation therapy. The clinical scanner will be designed to make it easier to operate by clinical staff and sufficiently robust and reliable for its intended use in a variety of clinical settings. The following specific aims are proposed to achieve the overall objective of developing an advanced EPR scanner for oximetry that is ready to be manufactured and used in routine clinical care to enhance cancer therapy: (Aim 1) Design and construct a 600-MHz pulse EPR system for clinical oximetry; (Aim 2) Fabricate a new class of compact, lightweight, and advanced resonator designs specifically optimized for pO2 measurements in human tumors; (Aim 3) Develop hardware and software interface with advanced measurement capabilities and user-friendly operation suitable for use in the clinic; (Aim 4) Assemble, test, and evaluate the scanner for repeated measurements of oxygen concentration using tissue phantoms and animal models of tumor; and (Aim 5) Evaluate the efficacy, usability, and safety of the oxygen scanner as a medical device and validate its use to make oxygen measurements in cancer patients and human factors engineering. The EPR scanner, fully integrated with the hardware and software modules, will be evaluated in relation to its ability to meet or exceed regulatory standards and for its practicality as a clinical device. The new first-in-clinical scanner will be a very valuable tool in the clinic for accurate prognosis and development of effective treatment strategies for cancer therapy. It can also be a valuable clinical tool for other clinical conditions where tissue oxygen is a critical variable for decision making, e.g., patients with diabetic peripheral vascular disease.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT Recent studies have revealed an abundance of resident memory T cells (TRM) specific for viral infections in a wide range of tumors, often outnumbering cancer-specific T cells. As these cells lack specificity to tumor antigens, they are spared from the hallmark exhaustion/dysfunction of tumor-specific T cells. To this end, we developed a novel immunotherapy which taps into immunostimulatory virus-specific TRM functions through treatment with viral peptides to break the immunosuppressive tumor environment. Reactivating virus-specific TRM with peptide was sufficient to restrict tumor growth in mice and when combined with checkpoint blockade, promoted durable tumor clearance. Mice that cleared tumors were protected from tumor re-challenge, suggesting anti-tumor immunity was established. Virus-specific TRM represent a major part of the tumor immune environment and can be leveraged therapeutically, yet there is a clinically significant gap in knowledge regarding the mechanisms of tumor clearance and how to optimally harness these cells. The objectives in this proposal are to determine (i) mechanism of tumor cell killing and durable tumor immunity, (ii) the impact of viral specificity on TRM function and therapeutic efficacy, and iii) define the optimal modality to reactivate virus-specific TRM. Using both mouse and human systems, this study addresses an innovative perspective connecting antiviral memory cells to tumor immunotherapy using cutting edge methods. We will complement mouse studies with combinatorial tetramer staining to enable simultaneous profiling of T cells specific for an expanded panel of viruses and vaccines in patient tumors. Completion of these aims will advance our understanding of tumor immunosurveillance and TRM function in mice and humans, and identify new target T cell populations for tumor immunotherapy. This contribution is expected to be significant because it will provide a strong scientific framework to expand the efficacy and utility of virus-specific TRM therapy, and enable the development of novel strategies leveraging these potent immune activating cells.

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT The gut microbiome is directly impacted by metals exposure and changes in the gut microbiome affect downstream health. An individual’s microbiome also modifies the health effects of toxicants such as arsenic by transforming them into potentially more or less harmful substances. Thus, understanding the impacts of arsenic on the microbiome, and vice versa, is key to achievable prevention and interventions to mitigate the risk of arsenic to human health – a key goal of the NIEHS. Our team’s prior work using the New Hampshire Birth Cohort Study, with ongoing enrollment and a projected size of 3,000 mother-infant dyads, identified an impact of toxic metals on the developing microbiome during the critical window of 0–3 years of age, when microbes are required for immune development. Specifically, our team showed both a sex-specific dysbiosis and a depletion of the immune-training microbe Bacteroides in arsenic-exposed infants/children. Importantly, this work revealed associations between arsenic exposure, changes in the microbiome, and early immune-mediated health outcomes, including respiratory disease. Our team’s recent work indicates a gut-lung link for arsenic-exposed infants depleted for Bacteroides, thus strongly supporting the hypothesis that gut microbiome composition is a key driver of airway health. This project will test the hypothesis that in the sensitive early-life window (0–3 years), when the developing immune system requires interaction with microbes, arsenic affects the developing microbiome, resulting in a paucity of Bacteroides and its secreted metabolites and ultimately associating with increased inflammation and risk of respiratory diseases such as wheeze, upper respiratory tract infection, and pneumonia later in life (out to 12 years of age). AIM 1. Test the hypotheses, using the longitudinal NHBCS and novel bioinformatic tools, that (i) early-life As exposures via food/water are related to sex-specific perturbations in the intestinal microbiome and (ii) the early- life intestinal microbiome modifies or mediates the effects of As exposure on respiratory health outcomes. AIM 2. Test the hypothesis that As enhances secretion of IL-8 from intestinal epithelia due to lack of a key Bacteroides-secreted short chain fatty acid, propionate, ultimately resulting from changes in the epigenome. AIM 3. Test the hypothesis that add-back of Bacteroides can reverse the effect of Bacteroides-depleted stool in a mouse model, thus serving as a proof-of-concept for probiotic interventions.

NIH Research Projects · FY 2026 · 2022-12

ABSTRACT/PROJECT SUMMARY The habenulo-interpeduncular pathway, a forebrain to midbrain conduction system in the vertebrate brain, has been implicated in many essential processes, from sleep to fear/anxiety, pain, learning, motivation, feeding, reproduction, and reward, and in pathological states such as mood disorders and addiction. Despite its importance and diverse roles, little is known about the complete repertoire of neuronal subpopulations and precise connectivity between the bilaterally paired dorsal habenular nuclei (dHb) of the dorsal diencephalon and their major target, the unpaired interpeduncular nucleus (IPN) of the ventral midbrain. A renewed interest in this pathway came from its association with nicotine dependence and withdrawal and from the discovery that, in zebrafish and other vertebrates, the dHb develop with prominent left- right differences in their size, organization, molecular properties and connections to the IPN. In previous work, we described an asymmetric olfactory projection to a subset of neurons in the right dHb that helps mediate the response to aversive cues and determined that altering directional asymmetry of the dHb induces behavioral and physiological changes indicative of enhanced fear/anxiety. Using state-of-the-art genetic, transgenic, imaging, and behavioral approaches, we now aim to identify all of the neuronal populations of the Hb-lPN pathway and further define which groups of neurons are involved in the response to negative cues. We have also extended our studies to characterize the pre-synaptic input and post-synaptic targets of this conduction system to gain a more complete understanding of the underlying neural circuits and their connectivity. The proposed research will shed light on a poorly understood yet highly conserved neural pathway and also address how differential processing of information by neurons on the left and right sides of the brain leads to appropriate behavioral responses. 7

NIH Research Projects · FY 2026 · 2022-11

Dormant Toxoplasma gondii [Toxoplasma] infection is characterized by dormant bradyzoite stage parasites that reside within thick-walled cysts that develop inside neurons in the central nervous system. Cysts provide a structural and physiological habitat that sustains the viability of dormant bradyzoite stage parasites. While many targets and therapeutics have been identified to effectively treat the active Toxoplasma infection that is defined by rapidly replicating tachyzoite stage parasites, therapeutic strategies or drugs that eliminate dormant bradyzoites and their cysts have not been identified. The identification of potential targets to perturb or eliminate dormancy has proven challenging for many microbes, including Toxoplasma, because microbial dormancy is characterized by a reduced metabolic state that sustains viability but not replication. Several lines of evidence support the hypothesis that dormant bradyzoites have markedly reduced mitochondrial functions and rely more heavily on acquiring host glucose not just for energy production but also to meet an increased demand for glucose to build bradyzoite-stage amylopectin and cyst wall glycan biomass. Consistent with this hypothesis, our data has shown that blocking the utilization of host glucose markedly reduced the development as well as the persistence of dormant stage bradyzoites. Here, we propose to define the metabolic basis that underpins the ability of glucose starvation to prevent the development and persistence of dormant bradyzoites. Targeting mitochondrial functions such as the electron transport chain has been shown to have a partial ability to perturb but not to eliminate dormancy. We hypothesize that targeting glucose or glucose + lactate utilization in combination with inhibition of mitochondrial function will accelerate the demise of dormant bradyzoites and their cysts. The work in this proposal charts a way forward to identify a metabolic basis to eliminate Toxoplasma dormancy.

NIH Research Projects · FY 2026 · 2022-11

Virulence gene regulators of enteric bacterial pathogens: Determining the structural and functional mechanisms of small molecule and polypeptide inhibitors Summary: The AraC/XylS family is one of the largest families of bacterial transcription factors with ~16K members distributed amongst 81% of sequenced bacterial species. Family members are present in pathogenic genera including Acinetobacter, Escherichia, Klebsiella, Legionella, Pseudomonas, Salmonella, Shigella, Vibrio, and Yersinia. The regulon of any given family member typically encompasses one of three categories: metabolism, stress response, or pathogenesis. Those involved in metabolism or stress response often have well characterized ligands. For example, AraC regulates the expression of genes involved in arabinose metabolism and its activity is modulated by arabinose. In contrast, small molecule ligands have not been identified for the vast majority of virulence regulators within the AraC family (hereafter referred to as AraC-VRs), which has led to the commonly held belief that the AraC-VR branch of the family has lost the ability to respond to ligands. Published work by us and others –and our preliminary studies– suggest that this assumption is incorrect. Additionally, a large family of endogenously encoded polypeptides, ANRs for AraC negative regulators, have been discovered that inhibit AraC-VRs though an unknown mechanism. The long-term goal of this project is to define the structural and molecular mechanisms underlying virulence gene regulation. The specific objectives of this proposal are to determine how AraC family members including Rns, a primary virulence regulator in enterotoxigenic E. coli (ETEC), are inhibited by 1) small molecule fatty acids and 2) AraC negative regulators (ANRs). Our central hypothesis is that Rns must dimerize in order to bind to DNA and regulate transcription and that these inhibitors block this by distinct mechanisms. The motivating rationale for these studies is that they will identify the molecular and structural requirements for inhibiting virulence gene expression, and will be tested by three specific aims: 1) Determine the structural mechanism by which ligand binding and dimerization regulates Rns activity; 2) Test our hypothesis that the Rns homolog RegA is regulated in the same manner; 3) Determine the mechanism by which ANRs inhibit Rns activity, and clarify if this is distinct from the inhibitory mechanism of small molecule fatty acids. This project is innovative in that the basic molecular mechanisms by which these proteins are regulated are not understood. Our multidisciplinary team, with expertise in microbiology, biochemistry, and structural biology, is uniquely positioned to undertake the proposed studies to determine these mechanisms. This research is significant, not only because it will answer outstanding questions of how AraC proteins function in enteric pathogens, but because we expect to demonstrate that AraC family proteins from a wide variety of enteric pathogens share a common mechanism of being inhibited by fatty acids. This will open up new possibilities for therapeutic strategies to combat global mortality and morbidity.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY Advances in single cell assays have enabled the genome-wide measurement of DNA sequence, RNA expression, chromatin accessibility and protein abundance for tens-of-thousands of cells isolated from a single tissue sample. Methods that capture or infer spatial or temporal information provide additional contextual information to create a detailed, cell-level picture of gene activity and function. These meth- ods give researchers a powerful tool for identifying the cell types in the analyzed tissue, the phenotype of those cells and the network of cell-cell interactions that control tissue structure and function. Although these techniques have revolutionized the study of complex tissues, the significant sparsity and noise of single cell measurements means that statistical analysis is typically performed at the level of large groups or clusters of cells. Although a cluster-based analysis can mitigate sparsity and noise, the re- sults reflect the state of the average cell in the cluster, which may be quite dissimilar from many cells in heterogeneous populations. To fully realize the potential of single cell profiling, bioinformatics meth- ods are needed that can accurately characterize individual cells rather than cell groups. One promising approach for generating cell-level estimates is gene set testing or pathway analysis, which can more effectively capture the state of individual cells by combining the measurements for all genes in a biolog- ical pathway. Unfortunately, statistical and biological differences between single cell and bulk genomic data make it challenging to use existing gene set testing methods, that were developed for bulk tissue, on single cell data. To address this limitation, we will create innovative algorithms for cell-level gene set testing and will use these techniques to support the estimation of cell type, phenotype and interaction potential. The translational application of these methods to study immune cell signaling within the tu- mor microenvironment will help validate our approach and provide important insights into the immune response to cancer.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY Up to eighty percent of clinic visit information is forgotten by patients immediately post visit. This is a significant barrier to self-management, especially in older adults with multimorbidity leading to poor health outcomes. After visit summaries (AVS) can improve recall, yet concerns exist about their layout, accuracy and low patient uptake. A new strategy to augment the AVS is to share visit recordings with patients. When patients receive an audio recording of the visit, 71% listen and 68% share it with a caregiver, resulting in greater recall. Despite growing interest, there is limited research on the impact of recording and sharing clinic visits of patient self- management ability, health outcomes or healthcare utilization of older adults. The objective of this proposal is to conduct a multisite trial evaluating the impact of adding an audio recording of clinic visits (AUDIO) to usual care in older adults with multimorbidity, including diabetes, compared to AVS alone (Usual Care; UC). The specific aims are: Aim 1 Conduct a three-site trial in primary care where older patients with multimorbidity including diabetes (n=336) will be randomized to receive an audio recording as well as AVS (AUDIO) versus AVS alone (UC) for all scheduled clinic visits over 12 months; patients will be assessed at baseline, 1 week, 6 months and 12 months; Aim 2 Identify factors that impact the implementation and sustainable use of visit audio recordings. Applicants Hypothesize (Main Effect) that: compared to those receiving UC, patients randomized to also receive audio recordings (AUDIO) of clinic visits will report a greater self-management ability (Primary Outcome), with improved quality of life, medication adherence, and satisfaction (Secondary Outcomes) at 12 months. Applicants will explore the impact of AUDIO on general medical regimen adherence, diabetes quality of care indicators, healthcare utilization and clinician practice behavior. They will also explore potential moderators of AUDIO, asking whether its impact on self-management is greater for individuals at highest risk of poor self-management including those with less caregiver support, moderate to severe depression, lower health literacy, and high disease burden. In Aim 2, applicants will interview patients, caregivers, clinicians, and clinic staff to identify barriers and facilitators to the implementation and sustainable use of recordings using the Consolidated Framework for Implementation Research (CFIR). The research is innovative: i) it seeks to shift current clinical practice where visit information is provided via AVS, by adding recordings; ii) the routine provision of visit recordings over time moves beyond prior studies focused on one-off recordings of specialty care visits; and iii) a trial in real-world settings of patients with multimorbidity, who are often excluded from trials, is novel and has greater external validity. Results are expected to have a major positive impact as they will increase clinical understanding of the impact and implementation of audio recording on the significant challenge of improving patient self-management in the face of the public health burden of multimorbidity.

NIH Research Projects · FY 2025 · 2022-09

Understanding the forces that direct cell fate is a central goal of biology. Many fundamental questions remain unanswered and vast tracts of vertebrate development are almost completely unexplored. What are the developmental origins and lineage relationships of cell types? What, where, and when are the key molecular events that underlie cell fate transitions? These questions remain unanswered as we lack the technology to map cell fate in both high-throughput and high-resolution. The overall objective of the proposed work is to develop and apply novel technologies that track and translate the cell fate forces into human-decipherable recordings. Our proposal combines innovative use of new CRISPR systems, molecular recording technologies, and improvements to our established lineage tracing technology to reconstruct high accuracy, annotated lineage trees. We will apply these cell fate technologies in mouse embryonic stem cells (mESCs), where we will track differentiation into various progeny cells, generating both transcriptional and lineage maps describing the probabilistic relationship between intestinal cell types at high-resolution. We will then expand these systems into recording mouse lines, where both molecular and lineage recordings can be captured from terminally differentiated cells. In aggregate, the resulting high-resolution lineage maps and tools will provide new insight into the plasticity of cell fate, and these recording mouse lines will be a valuable resource to the biomedical community. We believe these trailblazing technologies will ultimately spur the development of novel fate modulators with application to human disease.

NIH Research Projects · FY 2025 · 2022-09

The overall goal of this proposal is to develop new chemical technologies based on ring-forming reactions of oxyallyl cations followed by fragmentation of the resultant products to deliver novel molecular entities that are of biomedical interest. This contribution is significant because it will fuel cross-disciplinary discoveries by providing synthetic approaches to structurally unique indolines alkaloids with important biological properties. This project is innovative because we will develop a unified strategic vision for making both our target molecules and also compounds belonging to several important classes of monoterpene indoline alkaloids. Thus, we will pursue the following three specific aims. Aim #1: Complete the total synthesis of strychnochromine and cabucraline, Aim #2: Develop an efficient route to structurally-informed vinblastine variants with a novel C7 carboxylate, and Aim #3: Invent new (3+2) annulation and Beckmann fragmentation reactions. Strychnochromine is the only molecule found in nature or of anthropogenic origin that possesses the densely-functionalized pentacyclic ring system comprising its core. It was found to have anti-protozoal activity and no total synthesis of strychnochromine has been reported to date. Cabucraline is an akuammiline alkaloid that is distinct from all the other members previously synthesized at the configuration of the C2 stereocenter, and it has never been made before. Its synthesis will require the development of a novel regioselective (3+2) annulation reaction with a heterocyclic oxyallyl cation. The proposed vinblastine analogue is unique by virtue of the unprecedented C7 carboxylate and structural modifications at C3−C5. Natural vinblastine is an anti-cancer drug that is on the WHO list of essential medicines, but its clinical use is accompanied by undesirable side-effects. In addition, vinblastine-resistant cancer cell lines are becoming increasingly problematic. Due to its predicted greater efficacy, we hypothesize that these variants may require smaller therapeutic dosages vis-à-vis vinblastine, thereby resulting in lower occurrences of adverse side- effects. Structural differences of the analogues may also address issues related to vinblastine resistance. All of the proposed syntheses in the first two specific aims will feature the novel dearomative (3+2) indole annulation followed by fragmentation discovered by our research group. In the third aim, we seek to develop strategies that significantly broaden the scope of our dearomative (3+2) annulation technology by applying innovative methods for generating key reactive oxyallyl cation intermediates. In short, we will invent new tandem processes that result in the formation of densely-functionalized tetrahydrocarbazoles products with unique and stereodefined substitution patterns.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY/ABSTRACT T cells were first observed in postmortem brains of Alzheimer’s disease (AD) patients decades ago, yet very little is known about their role in disease progression. AD is marked by oligomerization of specific proteins that can accumulate into extracellular plaques, driving research to understand innate immune cells in AD due to their potential for reacting to and clearing these plaques. More recent evidence suggests that adaptive immune cells play a significant role in AD; numbers of CD8+ T cells are significantly increased in AD brains, and expansion of a subset of memory CD8+ T cells in the cerebrospinal fluid of AD patients is negatively associated with cognition. To date, T cell depletion and knock-out studies in mice have yielded conflicting data on the influence of T cells in AD. However, a population of T cells that has long been overlooked due to their only recent discovery and the historic view of the brain as immune privilege are tissue resident memory T cells (TRM), which reside in the brain and are optimally positioned to exert local immune activation. As there is currently no defined T cell antigen in Alzheimer’s disease, we will take an innovative approach to model TRM activation in the brain using a model antigen. This will be the first study to examine TRM in context of Alzheimer’s disease and to examine the impact of T cell activation in a defined manner on AD progression. The objectives of this proposal are to i) test the ability of brain CD8+ TRM to trigger innate immune cell activation, including NK cells, microglia and macrophages, in a mouse model of AD, ii) determine the impact of brain CD8+ TRM activation on Alzheimer’s disease progression in mice, including cognition and pathology, and iii) determine the migration dynamics, abundance and clonality of CD8+ TRM in mouse and human AD brain. These goals will be attained by complementing classical immunologic techniques for studying TRM in mouse models with a continuing collaboration with onsite neuropathologists to study T cells from human postmortem tissue from AD and non-neurologically involved brain tissue at a single cell level. Collectively, these experiments will enhance our understanding of the role of CD8+ TRM in Alzheimer’s disease and has potential to provide insight into T cell functions in other neurological diseases, reveal novel therapeutic targets, and overall build a foundational understanding of TRM functions in the brain.

NIH Research Projects · FY 2026 · 2022-09

PROJECT SUMMARY/ABSTRACT Pathology is focused on providing medical diagnoses and prognoses based on laboratory methods to guide patient treatment and management. Microscopy is fundamental for pathologists to examine tissues and cells. Despite numerous advancements, there have not been many changes in the last century in terms of how microscopy images are used in pathology. The current approach in anatomic pathology lacks standardization and relies on the cognitive burden imposed on pathologists to manually evaluate millions of cells across hundreds of slides in a typical workday. Deep learning-based methods have recently shown encouraging results for analyzing microscopy images. However, they rely on standard computer vision architectures and pipelines, which are limited due to the required time and cost of slide digitization and the computational constraints of analyzing huge high-resolution images. Furthermore, developing accurate deep learning models requires having access to large databases of labeled microscopy images, which is challenging. In this application, new methodologies are proposed to take advantage of the unique characteristics of histopathology datasets and the range of features in histology microscopy images to address these limitations. This project presents a novel approach based on generative adversarial networks for difficulty translation to generate augmented data with realistic, rare, and hard-to-classify histopathological patterns. This approach will mitigate data imbalances in annotated histology datasets and improve the performance of deep learning models for histological classification, particularly for uncommon and difficult-to-classify cases. Furthermore, a novel curriculum learning approach for histology image classification will be developed based on the range of classification difficulty among histopathological patterns and multi-annotator labeled datasets. This approach trains on progressively harder- to-classify images, as determined by annotator agreement, and significantly improves the performance of the resulting deep learning models without requiring additional data or computational resources. In addition, a self- supervised knowledge distillation method will be developed to enhance the efficiency of histology image classification. As large, labeled datasets are scarce, this method uses a self-supervised approach to distill feature extraction capabilities at a high resolution into a student model operating at a lower resolution by leveraging unlabeled datasets. The resulting distilled student models can achieve high classification accuracy on low- resolution histology images while saving a significant amount of time and resources on digitization efforts and required computational resources. The proposed methods in this application remove current bottlenecks in deep learning applications for digital pathology. Therefore, the results from this project could have a major impact on new opportunities that use deep learning technology in clinical workflows and integrate histopathological information with other clinical and molecular data to improve patients' diagnoses, prognoses, and treatments.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Shared information in cortical functional architecture is embedded in topographies that are idiosyncratic, posing a major impediment for functional brain imaging research. Hyperalignment resolves this problem by projecting information from individual brains into a common model information space. The proposed research project will create HyperBase – research infrastructure that will enable the brain imaging research community to leverage hyperalignment to greatly enrich their data, enable analyses of shared information and individual differences embedded in idiosyncratic fine-scale cortical topographies, and create a data sharing platform for data in the hyperaligned common model information space. The infrastructure will be an optimized, standardized template common model space based on a normative database, turnkey software tools for hyperaligning new brains and estimating individual functional topographies, and a framework for sharing hyperaligned data. These data and tools will provide community infrastructural support for research on a broad range of topics in clinical neuroscience, brain aging, and basic cognitive neuroscience. The proposed database will consist of fMRI data in 60 participants collected during movie viewing, story listening, at rest, and during a large set of functional localizers, augmented with demographic information and cognitive and personality test scores. Specific aims 1. Produce an optimized, standardized template for hyperalignment based on a normative database with open-source software that will allow mapping numerous functional topographies, based on standard localizer data in the normative sample, into new participant brains using only fMRI data collected while the new participants watch a movie, listen to a story, or are at rest. 2. Adapt hyperalignment algorithms to work with a standard template and estimate functional topographies via the template and normative localizer data. Develop new hyperalignment algorithms that increase power, precision, and flexibility. 3. Create a system for sharing functional brain imaging data that are projected into the common information space model, allowing accumulation of data in a framework that affords at a fine-grained level of detail. Hyperalign existing public datasets.

NIH Research Projects · FY 2024 · 2022-08

Human B cells play a fundamental role in the adaptive immune response to infection, development of protective immunity from vaccination, and pathology of many autoimmune diseases. Central to all of these processes are migration of B cells among different tissues and differentiation of B cells into functional subtypes. Currently, understanding B cell migration and differentiation is limited because these processes are dynamic and difficult to directly observe, particularly in humans. Our previous work has demonstrated that it is possible to detect migration and differentiation events along evolutionary trees inferred from B cell receptor (BCR) sequences, which are subject to rapid somatic hypermutation and antigen-driven selection during adaptive immune responses. This is analogous to viral phylogeography, the use of evolutionary trees to track the spread of viruses during epidemics. However, there are key differences in the biology of B cells and viruses that require modifying and extending existing approaches to make them appropriate for B cells. The goal of this proposal is to enable B cell phylogeography. We will develop novel computational methods that leverage recent advances in single B cell sequencing technology to infer how B cells migrate between tissues and differentiate into cellular subtypes based on their activation states during immune responses. The aims of this proposal focus on solving the roadblocks to the development of phylogeographic methods for B cells. These methods will be validated by simulations and experimental analysis. We will work with established experimental collaborators to translate these novel methods into meaningful outcomes in the research of influenza vaccine response and the treatment of autoimmune diseases, including lupus and myasthenia gravis. We will implement these methods in widely available free software, which will greatly increase their potential to inform vaccination strategies against other pathogens like HIV and SARS-CoV-2, as well as treatment of other B cell-mediated conditions such as multiple sclerosis and asthma. The K99 phase of this proposal will be guided by Prof. Steven Kleinstein at Yale School of Medicine, a world leader in computational methods development for BCR sequence analysis, and Prof. Kevin O’Connor, a leading experimental biologist in the B cell pathology of neurologic autoimmune diseases. The candidate, Dr. Kenneth Hoehn, has a strong background in genetics and evolutionary biology, and has an established record of developing evolutionary models to study B cell populations from BCR sequence data. The work detailed in this proposal will fill gaps in the candidate’s training in B cell biology, single cell analysis, and software development. The R00 phase will build off of this work to develop a highly generalizable framework for characterizing the complex migration and differentiation patterns that underlie B cells’ role in vaccination and autoimmunity. This will support new, medically-relevant discoveries about B cell biology, and serve as a foundation for the candidate’s future as an independent computational immunologist.

NIH Research Projects · FY 2025 · 2022-08

ABSTRACT Protein phosphorylation is an essential post-translational modification that plays pivotal roles in the regulation of protein function, localization, and stability and governs most cellular processes, including cell division. Phosphorylation by protein kinases, including the master regulators Cdk1, Plk1, Aurora kinase A and Aurora kinase B, are required to coordinate the functions of a wide array of substrates to promote faithful transit of cells through mitosis. Indeed, dysregulation of mitotic kinase functions underlies many human diseases such as birth defects and cancer. In particular, the kinase Plk1 promotes mitotic entry by regulating Cdk1/cyclin B activity, centrosome separation and maturation, as well as spindle assembly. Plk1 is required for the removal of sister chromatid cohesion and spindle assembly checkpoint (SAC) signaling, and fine-tunes microtubule-kinetochore attachment dynamics. Plk1 also contributes to mitotic exit and cytokinesis by regulating the anaphase promoting complex/cyclosome (APC/C) and recruiting proteins to the central spindle and the midbody. Example Plk1 substrates that facilitate these functions include docking/scaffolding proteins, motor proteins including kinesins, structural proteins, proteases, E3 ubiquitin ligases, and other kinases. While entry into mitosis is driven by the activation of kinases and a net increase in phosphorylation, dynamic regulation of phosphorylation levels is needed for mitotic progression and exit. We found that Plk1 inhibits PP6 to promote its own activation. However, PP1 and PP2A-B56 restrain Plk1 activity to silence the SAC. Thus, complex regulatory interactions between kinases and phosphatases control mitosis. Our overarching goals are to identify and uncover the principles that govern phosphorylation signaling in mitosis as a means to better understand cell division. Specifically, we will focus on the role of Plk1 as an apical mediator in mitosis and it is regulatory interactions with other kinases and phosphatases. To accomplish this, we will develop and apply a combination of proteomic, bioinformatic, cell biological and biochemical strategies to connect kinases to their substrates, investigate substrate function, and uncover new regulatory interactions of kinases and phosphatases Our past studies have enabled us and other researchers to functionally interrogate specific mechanisms that underlie many biological processes of interest. We are dedicated to sharing our findings with the research community and will continue to do so as publically available resources.

NIH Research Projects · FY 2025 · 2022-08

Immigrants are arriving in the United States at an unprecedented rate, with variation in experiences of adversity during and following immigration to this country. Such adverse social experiences have been linked to the development of poor health. My post-doctoral project will investigate how the process of immigration and assimilation are associated with mental health and biological aging within immigrants that arrived to the United States within the last six years (since January 1, 2019). I will do this through a longitudinal study following 100 immigrants living in the United States across one year. I will investigate two questions: 1) How does baseline mental health and biomarkers of aging vary relative to immigration and adverse social experiences for recently arrived immigrants? And 2) How do experiences of structural and informal support influence mental health and biological aging within the first year of living in a new country? This research aims to elucidate mechanistic links between immigration experiences, mental health, and biological aging using validated mental health assessments, ethnographic interviews, and molecular biomarkers of aging. Additionally, the inclusion of social support as a metric in evaluating health outcomes moves this project toward solution-oriented research, in which identifying specific forms of support could help in bolstering these networks and resources across all populations in the United States.

NIH Research Projects · FY 2025 · 2022-08

A hallmark of biological intelligence is the ability to learn from a remarkably small set of experiences to select appropriate behaviors in novel contexts. This ability arises from inductive bias, referring to the assumptions used in generalizing prior observations to novel data. In machine learning, inductive bias is essential for efficient learning from a small number of examples as animals frequently do. In neuroscience, inductive bias has been mostly considered as shaped by evolution to produce an innate inductive bias. Although an agent’s inductive bias should be adaptive to changing task demands, how brains develop inductive bias, use it to guide learning, and adapt it to task statistics, remain poorly understood. The overall goal of this project is to study the neural and computational bases of inductive bias adaptation by training non-human primates on a novel learning task, characterizing choice behavior and neuronal activity, and developing computational models of learning in neural networks. Our theoretical framework of neural kernel learning makes precise predictions for behavior and neural activity that will be experimentally tested in the proposed studies. We will characterize behavior in a newly designed “crosstalk” task, which is designed to characterize inductive bias and to drive a subject’s adaptation through well-defined and variable task statistics (Aim 1). In this task, the subject learns through experience to categorize stimuli composed of multiple features. Within a block of trials, certain features are differentially informative of the category. Next, we will record simultaneous spiking activity from neurons in dorsolateral prefrontal cortex and the dorsal striatum during task performance (Aim 2). In parallel, we will develop and refine algorithmic and artificial neural network models of adaptive learning (Aim 3) to generate testable predictions for behavior and neural activity. Results from these studies will impact paradigms used to study the computational and neural bases of learning and generalization in humans and animals.

NIH Research Projects · FY 2025 · 2022-08

Project summary Mammalian prion diseases, such as Creutzfeldt-Jakob disease (CJD), Chronic Wasting Disease (CWD), bovine spongiform encephalopathy (BSE), and scrapie, are a group of infectious neurodegenerative disorders caused by the autocatalytic conversion of the host-encoded prion protein, PrPC, into a group of misfolded, infectious conformers collectively termed PrPSc. Multiple lines of evidence from biochemical, genetic, and cell biological studies suggest that PrPC and PrPSc employ specific pathways for biosynthesis, trafficking, and degradation in living cells. However, full elucidation of these pathways has been hindered the lack of tractable model organisms for genetic screening. To overcome this obstacle, we recently developed methods to detect both PrPC and PrPSc in brain-derived CAD5 cells using fluorescence-activated cell sorting (FACS). We will use these sensitive sorting methods to perform CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 whole genome library screens in CAD5 cells infected with different prion strains. These studies will be the first to fully map the molecular pathways that control the biosynthesis, trafficking, and degradation of both PrPC and PrPSc molecules, and thereby greatly advance our fundamental knowledge of prion cell biology. Our unbiased screens will also reveal the rate limiting steps of prion formation and clearance, thereby identifying the most susceptible targets for drug therapy.

NIH Research Projects · FY 2025 · 2022-07

With NIH support, the Ubongo Sikivu cohort of both people living with HIV (PLWH) and uninfected controls was established in Tanzania and has had regular assessments of their peripheral hearing ability over 10+ years with detailed central auditory and neurocognitive assessments added over the last 5 years. This cohort is uniquely positioned to address the issues posed by PAR-20-127 “Advancing HIV/AIDS Research within the Mission of the NIDCD.” Data from the cohort has already answered important questions about the ototoxicity of anti-retrovirals, the effects of HIV infection and treatment on both peripheral and central hearing parameters, and the relationship of central auditory test (CAT) results to neurocognitive performance. By following this cohort longitudinally questions about how CATs could be used to predict or track neurocognitive performance and how age and long-term anti-retroviral treatment affect the auditory system can be answered. To date, the most significant result has been demonstrating that CAT results correlate with cognitive performance. This suggests CAT results might be useful for forecasting or tracking cognitive decline over time. The next important steps are determining whether worsening CAT performance precedes the later development of cognitive deficits in PLWH and which central auditory tests and other variables can predict neurocognitive deficits accurately. Neurocognitive screening tests are often sensitive to education, literacy, and culture. Full neurocognitive test batteries can be difficult to employ, particularly in the developing world where clinician time is limited, few trained personnel are available, and normative data often do not exist. Using CATs would be a major advance for following HIV+ patients because the CATs can be short (a gap detection test takes 5 minutes), easy to explain (the hearing-in-noise test and triple digit task involve identifying words or numbers in background noise), or effortless for the subject (the FFR test requires no subject input at all). This project’s goal is tracking the trajectory of peripheral auditory, central auditory, and neurocognitive performance over time by continuing to follow this cohort. With these longitudinal data machine learning and other statistical techniques will be applied to assess which factors forecast the subsequent development of cognitive deficits and which factors or combination of factors identify those with existing neurocognitive deficits. An international team with experience in central auditory testing and neurocognitive testing in PLWH has been assembled. Dr. Nina Kraus and her Northwestern team are internationally recognized experts in the auditory FFR. The Dar es Salaam team has extensive experience in otolaryngology and performing peripheral auditory, central auditory, and neurocognitive tests. Drs. Roth and Boivin are experts in assessing neurocognitive function. Dr. Gui has diverse biostatistical experience. Dr. Niemczak is an expert in peripheral and central auditory processing. This team and longitudinal cohort offer the unique ability to assess the use of CATs in evaluating cognition as well as the effects of aging and medications on both central and peripheral auditory function in PLWH.

NIH Research Projects · FY 2025 · 2022-07

Adoptive T cell therapy for cancer has proven remarkably successful and is capable of producing high response rates and dramatic remission in some patients. Currently it is more effective against liquid than solid tumors, due to the environment within tumors being hostile for T cell survival and function. As is true of many tumors, melanomas are highly glycolytic, and deplete the local glucose concentration. Melanomas that develop resistance to Vemurafenib undergo metabolic remodeling to become dependent upon glutamine. As both glucose and glutamine are essential for effector CD8 T cell differentiation and anti-tumor effector functions, the T cell response is compromised in the tumor microenvironment. Advances in adoptive T cell therapy have focused mostly on better targeting and activation of tumor-specific T cells, however they may still fail to thrive in this metabolically challenging environment. Engineering cells with the flexibility to use multiple carbon sources, with less reliance on glucose and glutamine, is one very promising approach that could be layered onto any tumor targeting strategy. In this application we show that CD8 T cells lacking in the transcriptional repressor Zbtb20 display enhanced glycolytic and mitochondrial metabolism, and increased fuel flexibility relative to wild- type cells. Single cell transcriptional profiling confirmed these metabolic changes and confirmed phenotypic studies showing a skewing toward the memory fate. Consistent with these attributes being favorable for anti- tumor immunity, we found adoptive transfer of Zbtb20-deficient CD8 T cells conferred superior immunity upon challenge with melanoma or adenocarcinoma cells. Therefore, suppression of Zbtb20 is a very promising approach to improve T cell metabolism for adoptive immunotherapy. In this proposal we determine the precise molecular mechanisms underlying enhanced anti-tumor immunity, mechanisms for elevated glycolytic and mitochondrial metabolism, and the extent to which each change is responsible for anti-tumor immunity. We also determine the extent to which a dominant negative mutant of Zbtb20 can replicate enhanced protection observed in Zbtb20 deficient cells, as this is more readily translatable to human T cells. These studies will reveal the mechanism(s) by which Zbtb20 deficiency enhances anti-tumor immunity and investigates strategies that can be translated into human T cell adoptive therapy.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT Metabolic dysregulation is the major preventable risk factor for leading causes of chronic disease-related deaths. More specifically, chronic obesity is correlated with adipocyte hypertrophy and hyperplasia, both of which may be circadianly regulated. All circadian clocks are cell-intrinsic, and circadian oscillators that are tissue-specific control metabolic homeostasis by fine-tuning nutrient utilization; adipose tissue responds to microenvironmental changes in a clock-dependent manner. Thermogenic adipocytes can redirect energy away from ATP production during nutrient excess by disrupting the electrochemical proton gradient, producing heat in a process called Non- Shivering Thermogenesis (NST). Thermogenic adipocytes are sometimes capable of cell- autonomously sensing ambient temperature and adopting a reversible thermogenic profile. The circadian clock's importance in this thermogenic plasticity is not well understood, nor the cellular decision to adopt this state. The objective of this work is to understand how circadian rhythms affect adipocyte biology, especially thermogenic plasticity. To delineate the relationship between the cellular circadian system and adipocyte biology in the absence of organismal cues, circadian output will be characterized in Specific Aim 1 by transcriptionally profiling in vitro differentiated adipocytes from inguinal adipose tissue over 3 circadian days with a 2-hour resolution via RNAseq. In this way I will determine what aspects of adipocyte biology and environmental stimuli can be influenced by time-of-day. Though multilocularity and mitochondrial abundance are not indicators of thermogenic potential per se, these two organelles are intricately involved in NST. To extend the hypothesis that thermogenic plasticity is clock-controlled, I will use quantitative fluorescence live cell microscopy to characterize lipid droplet and mitochondrial spatial patterning and thereby describe organelle morphology as a function of circadian time. In Specific Aim 2 I will determine the cell-autonomous clock’s role in heat production, the quintessential component of thermogenesis, using infrared thermal imaging to identify rhythms in heat production (1) during a state of decreased bioenergetic efficiency via uncoupling with BAM15 and (2) by suppressing UCP1 with purine nucleotides. The long-term goal of this proposal is to determine the clock’s role in regulating thermogenesis. Findings from this study will increase our understanding of clock- controlled energy metabolism and adipocyte dysfunction, advancing our understanding of the non-linear association between weight, energy expenditure and risk in chronic disease.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY Biases in whether and how social interactions are perceived are a hallmark symptom of most major mental illnesses (e.g., people with autism tend to under-perceive social interactions, people with paranoid schizophrenia tend to over-perceive social interactions, and people with depression tend to perceive social interactions in a negative light). These biases likely reflect the extreme end of a continuum that exists in the normative population, suggesting that characterizing individual differences in social-perceptual styles is critical to furthering our understanding of disease. That humans are primed to perceive social interactions even in stripped-down, unlifelike stimuli (e.g., animations of geometric shapes) is a phenomenon that has long been recognized and exploited to study social cognition in both normative and patient populations. However, when it comes to these basic stimuli, while we may have the intuition that we “know it when we see it”, we do not understand what it is about stimuli deemed social that makes them social—in other words, which specific visual features are required, and in what doses. Furthermore, because task paradigms are often a simple binary choice (i.e., ‘social’ or ‘random’), we do not understand heterogeneity across individuals in terms of their thresholds for deciding if a given stimulus represents a social interaction, and if so, what kind of social interaction (i.e., positive or negative). A critical step toward understanding and correcting biased social cognition in mental illness is to define the fundamental sensory features of basic social interactions, and determine how and why different people compute differently on these features to give rise to different social percepts. This will open the door to interventions that can prevent an individual from going down a biased path. In this project, we will establish a social stimulus class for which we have precise, parametric control over low-level visual features. This will allow us to construct individual “social tuning curves” for various types of social interactions and determine how variability in these tuning curves relates to trait phenotypes. Combining these stimuli with simultaneous neuroimaging (fMRI) and eye-tracking will shed light on where in the processing hierarchy percepts diverge within and across individuals, and allow us to test the hypothesis that social percepts emerge earlier in the cortical hierarchy than previously thought. This would indicate that idiosyncratic social cognition is more closely linked to automatic, sensory-driven processes than controlled reflection, a distinction that is important for informing diagnostic and interventional tools. Finally, within a set of densely sampled individuals, we will directly test causality between stimulus features, brain activity, and percepts using real-time fMRI to implicitly steer individuals toward a given percept based on ongoing patterns of brain activity. The outcome of the proposed research will be a causal model of how stimulus features and brain dynamics interact to give rise to a given social percept within a given individual. This model will provide testable hypotheses regarding targeted therapies to normalize biased cognition in mental illnesses.