University Of Notre Dame

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT Mechanical forces are often modulated in diseased or wounded tissues as a result of inflammatory responses driven by immune cell activity in the affected site(s). In fact, aberrant mechanical force generation in pathological settings may mediate disease progression and treatment resistance. However, little is known about the response of immune cells to these mechanical forces, particularly at the tissue-length scale, and even in normal physiological settings. Thus there is a critically unmet need to fill overlooked gaps in our basic understanding of the interplay between tissue-level mechanical forces and immune cell behavior, both collectively and at the single-cell level. With the support of the NIGMS R35 MIRA for Early Stage Investigators over the next five years, my laboratory will establish the first immune mechanome. We will investigate the impact of tissue mechanical forces on the phenotype and function of innate and adaptive immune cells in a variety of organs. Leveraging engineering-based tools and approaches, we will couple unbiased omics platforms to mechanical testing at multiple scales (e.g., on cells in vivo, tissues ex vivo, and organs in vivo) in order to relate immune response to mechanical forces. During multiscale compression, the trafficking, distribution, motility, cell-cell interactions, and functional behavior of immune cells will be examined and perturbed via: i) intravital and dynamic imaging (e.g., with multiphoton microscopy of fluorescent cells or genetically engineered mouse models); ii) immunocompetent, transgenic, and immunogenic animal models (e.g., OT-I/OT-II antigen systems); and iii) artificial intelligence- based cell state analysis (e.g., from single cell RNA sequencing). We will also explore our hypothesis that beneficial immune activity in the face of pathological conditions is suppressed by heightened tissue mechanical forces. Importantly, the proposed Projects are to be performed in non-specific contexts that are independent of tissue type, organ, or disease in order to maximize the potential for broad impact in the biomedical sciences. The knowledge generated will lay the groundwork for future mechanistic and translational research in both healthy and diseased settings. By operating at the interface of mechanical engineering and immunology in the burgeoning field of “mechano-immunology,” my research program is uniquely suited to reveal new biophysical insights and pathophysiological targets for human disease.

NIH Research Projects · FY 2025 · 2023-09

A Convergent Bioengineered Platform for Multifunctional Therapeutic Exosomes Abstract: The overall goal of this MIRA application is to develop a convergent bioengineered platform for manufacturing and engineering therapeutic exosomes. The platform will allow the loading of drugs into exosomes with high efficiency, biomanufacturing of exosomes in high throughput, and further engineering exosome-based drug delivery systems for various diseases with desired functions including targeted delivery, tracking, and combinational therapies. Exosomes are a subset of extracellular vesicles, with diameters between 50 nm and 150 nm, secreted by most eukaryotic cells. They are very promising drug delivery vehicles due to their small size, biocompatibility, low immunogenicity, and reduced toxicity in comparison with synthetic nanoscale formulations such as liposomes, dendrimers, and polymers. Delivery of anticancer drugs contained in exosomes demonstrated improved pharmacokinetic and pharmacodynamic properties and enhanced anticancer activity in vivo compared to free drug molecules. Loading of therapeutic nucleic acids into exosomes protects the nucleic acids from nucleases and increases cellular uptake and the therapeutic effect due to specific molecular mechanisms of exosome internalization. Exosomes can cross the blood brain barrier and penetrate deep tissues with improved efficacy compared to that of synthetic nanocarriers. Moreover, they play a key role in cancer metastasis and regeneration by inducing transcriptomic and phenotypic changes with their RNA and protein cargoes. Therefore, they can potentially be reengineered for delivery of gene and protein therapeutics. However, there remain fundamental challenges to the utilization of exosomes in the clinic: i) drug loading efficiency into exosomes is very limited; ii) the production of exosomes has yet to reach sufficiently high throughput for clinical tests or even further development; and iii) endowing exosomes with multiple abilities for satisfactory disease targeting, tracking and combinational therapies is highly demanding. To address these challenges, the PI proposes the following three projects: 1) Developing a high-efficiency exosome drug loading technology with chiral graphene nanoparticles; 2) Developing an exosome production bioreactor with stimulating piezoelectric nanofibrous scaffolds; and 3) Engineering hybrid exosomes as a multifunctional targeted delivery system with targeting ligands and functional chiral graphene quantum dots for near-infrared imaging-guided photothermal cancer therapies. The proposed research contains several innovative approaches of exosome production, loading and engineering that, if successful and integrated, will provide a high-throughput and high-efficiency exosome manufacturing platform for drug delivery, and expand exosome-based drug delivery to diverse biomedical and clinic applications by combining the merits of both the native exosomes and synthetic nanoparticles.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Diseases are frequently caused by dysfunction of proteins in the body, perhaps due to maladapted genetics or from a wide variety of other causes. Researchers can gain a glimpse into this function through the study of a protein’s mechanism and dynamics. Ideally, a complete understanding of the role of a protein in biophysical interactions would describe the entire mechanistic pathway on an atomistic and dynamic level. However, this cannot be attained with experimental studies alone with today’s capabilities. Computational studies can provide experimentally inaccessible quantitative and atomistic information so they serve as powerful tools for better understanding diseases and identifying targets for experimental follow-up and potential treatment, but they carry little weight without rigorous experimental validation. We seek to reconcile experimental and computational data, equipping researchers with a method to produce the aforementioned continuous and atomistic information on protein dynamics so that they can elucidate the long timescale dynamics of proteins on an atomic level. When deconvolving time-resolved crystallographic data, I will substitute the typical static crystallographic initial inputs with structures from molecular dynamics simulations and predictive models to improve the continuity and accuracy of deconvoluted data. The objective of this work is to produce the aforementioned ideal dynamics information for a significant portion of the mechanism of PmHMGR as a demonstration and refinement of the proposed Markov State informed Multilinear Singular Value Decomposition (MSiMSVD) method which reconciles experimental and computational data. Application of the MSiMSVD method to slow dynamical events, such as the PmHMGR 2nd hydride transfer, is limited by the ability of molecular dynamics to perform accurate long-timescale simulations. This often requires Transition State Force Fields (TSFFs), but their parameterization for biomolecules often falls into local optimization minima due to high dimensionality. To reduce local minima trapping and make TSFF generation more accessible for biophysical research, I will apply constraints and swarm intelligence techniques to improve current TSFF parameterization. Collectively, these aims will provide a means by which experimental and computational techniques can work synergistically to produce the continuous atomistic protein dynamics information ideal for the investigation of proteins and their related functions and diseases.

NIH Research Projects · FY 2026 · 2023-06

Project Summary Aging is a risk factor for many diseases such as cancer, cardiovascular diseases and neurodegenerative diseases, with the incidence of such diseases peaking between ages 60 and 80. Although both the cellular and the extracellular components of a tissue change with age, current preclinical models have focused on the aging-related changes in cells and overlooked the alterations in the microenvironment, specifically the extracellular matrix (ECM), which is one of the main reasons for the low success rate of pre-clinical to clinical translation. Thus, it is imperative to create disease models that mimic the aging microenvironment to better study disease initiation and progression, as well as reliably test for drug efficacy. Here, as a proof-of-concept aging-associated disease, for the first time in literature, we propose to engineer decellularized aged human ECM (dECM)-based 3D tumor models and implant them into immunodeficient mice to create hybrid mouse models to study the effect of matrix age on tumor progression and drug response. We will follow a bottom-up approach to establish the hybrid mouse model; first we will engineer the aging stroma using aged human breast dECM and aged human stromal cells both derived from healthy donors, then grow aged patient-derived tumor organoids on the stroma to engineer the 3D in vitro tumor models, and finally implant the 3D tumors into immunodeficient mice to create the hybrid mouse models. Hence, we aim to establish reliable and human representative preclinical models, 3D tumor models and hybrid mouse models, which allow us to distinguish the individual and combined effect of aging components (i.e. ECM, stromal cells, and tumor cells) on tumor initiation and progression. We will then mechanistically test the individual effects of aged ECM characteristics, such as the altered stiffness, fiber structure and biochemical composition on tumor progression. The proposed work aims at solving many problems of the current preclinical models. First, we will produce in vitro and in vivo preclinical models that consider the effect of aging human ECM on cancer progression. Second, by creating hybrid models, we will address the lack of systemic response in the 3D models, and the lack of control and inability to discern the effects of individual components in in vivo models. Finally, we will create tumors in mice that better represent the human response and benchmark our hybrid model with actual patient samples. To achieve these goals, we will combine our expertise in tissue engineering, mouse model systems, transcriptomics, primary cell culture model systems, and breast cancer research. Once fully implemented and functionally validated, we expect our state-of-the-art tissue engineered 3D disease models as well as the hybrid mouse models to serve as the next-generation research platform for both basic and translational cancer research and high-throughput drug discovery.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY Depression is prevalent, debilitating, and costly, and derives from complex combinations of genetic and environmental factors. Maternal depression is one of the most established risk factors of depression in offspring, yet the genetic and environmental risk factors tied to intergenerational risk are complex and largely unspecified. Dyadic research examining mother–child neural and behavioral processes is critically needed to identify early pathways and mechanisms of increased depression risk. To this end, the current research strategy suggests a neurobehavioral pathway of early childhood risk development such that depression in mothers is characterized by dampened positive valence systems (PVS) neural activation, leading to decreased engagement in co-experienced positive affect with young offspring, and resulting in dampened PVS neural activation in the young children of mothers with depression. I will assess 125 mother−child (age=18months) dyads. In the first laboratory visit, diagnostic interviews will assess mothers’ lifetime depression history. In the second laboratory visit, dyads will complete a series of tasks to examine neural indicators of PVS activation and mother−child co-experienced positive affect. Electroencephalogram (EEG) and event-related potential (ERP) neural indicators, particularly frontal alpha asymmetry (FAA) and the late positive potential (LPP), reliable measures of approach motivation and motivated attention, respectively, will be used to measure PVS activation at the neural level for both mothers and young children. Co-experienced positive affect will be coded via the Dyadic Interaction Coding System. This project will allow me to examine a potential neurobehavioral pathway from depression in mothers to the development of increased depression risk in offspring. First, I will examine the association between mothers’ depressive symptoms and neural PVS activation in the context of interactions with offspring (Specific Aim 1). Then, I will examine the association between mothers’ depressive symptoms, prenatally and postpartum, and observed co-experienced positive affect in mother–child interactions and test the moderating role of individual differences in mothers’ neural PVS activation on this association (Specific Aim 2). Lastly, I will examine the cumulative and unique effects of co-experienced positive affect and mothers’ neural PVS function on children’s PVS function. These aims leverage a rigorous multimethod research design to provide foundational insight on dyadic neurobehavioral mechanisms of depression vulnerability, crucial for developing more targeted preventions for those at highest risk for depression. In addition, my proposed training goals are to develop advanced skills through rigorous training in: infant and early childhood mental health, dyadic behavioral paradigms and assessment of caregiver–child interactions, dyadic and early childhood neurophysiological assessments, and advanced quantitative methods.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY / ABSTRACT Understanding how intracellular pathogens survive in their host cells has led to better management/pre- vention of their diseases, as well as discovery of fundamental biology. In this application, we aim to start elucidating the strategies and mechanisms used by one of the commonest fungal pathogens, Cryptococcus neoformans, to survive inside macrophages. The treatment of cryptococcal disease is subpar, resulting in mor- talities that range from 20% to almost 90%, depending on the geographical region. The ability of this fungus to survive inside host cells is one of the main drivers of disease progression, and clinical studies show that intra- cellular survival in macrophages correlates with patient’s mortality. However, the molecular mechanisms that govern internalization and the ability to survive intracellularly are not known, hampering the development of more effective therapeutics. Our long-term goal is to understand the cellular and molecular mechanisms behind intra- cellular survival, a significant gap in knowledge in the field. Moving towards that goal, the objectives of this application are to define and characterize the cryptococcal-containing phagosome (CCP) in macrophages, and understand the role of host and fungal factors in the generation of this intracellular niche. We hypothesize that Cryptococcus actively targets Rab GTPases and phosphoinositides to delay the normal maturation of its phag- osome. This allows fungal factors, such as capsular hyaluronic acid and glucuronic acid, to modulate acidification of the CCP, resulting in a fungal permissive niche. Pro-inflammatory signals, mediated in part by Rab20 effects on phagosomal maturation, counter this fungal manipulation and prevent niche establishment. We plan to test this hypothesis by (1) defining and characterizing the intracellular niche of C. neoformans in naïve and activated macrophages; and (2) identify and explore the role of fungal factors in niche establishment. If completed, we will have identified the genetic (fungal genes), phenotypic (CCP properties) and immunological (host’s immune sta- tus) characteristics that enable Cryptococcus to live intracellularly, allowing us to pinpoint potential areas of intervention to block intracellular replication. Completion of these aims will have a positive impact in the field by generating a detailed molecular description of the strategies Cryptococcus uses to survive in macrophages and how host cells respond. Moreover, given the scarcity of well characterized intracellular survival strategies in fungi, the knowledge created here will impact the understanding and studies of other pathogenic fungi as well.

NIH Research Projects · FY 2025 · 2023-05

Project Summary Organs of the human body rely on complex integration of multiple neuronal circuits in order to maintain homeostasis. For example, the heart must respond to internal and external stimuli and produce sufficient blood flow to meet environmental demands. Glia play critical roles to support nervous system function but are also indispensable for establishment of appropriate circuit structure and physiology. Our lab recently identified a cell type in the heart with many similarities to astroglia populations found elsewhere in the body. This cell type, which we termed cardiac nexus glia, is present as the heart is being formed and is critical for establishment of heart rhythm. Ablation of these glia causes cardiac arrhythmia and ventricular fibrillation, both serious physiological conditions. This project uses the zebrafish animal model to investigate the developmental ontogeny and morphological characteristics of cardiac nexus glia in the developing hearts of living animals (Aim 1). Additionally, it probes the functional relationship between these glia and cardiomyocytes and their roles in modulating heart rate and rhythm (Aim 2). The results of these experiments will contribute crucial detail to our understanding of the multi-faceted roles of glia in the peripheral nervous system and provide important and specific insight about cardiac nexus glia function in heart development and disease. This research will be undertaken in a supportive and collaborative laboratory environment within the Biological Sciences Department at Notre Dame University. It will rely on expertise from staff of the Center for Zebrafish Research and the Integrated Imaging Facility. In addition to the development of bench skills and technical expertise inherent to this project, the applicant will also hone teaching, mentoring, and management skills that will be critical for her future success as a PI running her own research group at a primarily undergraduate institution.

NIH Research Projects · FY 2024 · 2023-05

ABSTRACT Microglia are the resident immune cells of the Central Nervous System (CNS). They are positioned at the center of brain development and function by playing crucial roles in neuronal network architecture and homeostatic surveillance. Unlike other CNS-resident cells, microglia originate outside the brain, specifically in the embryonic yolk sac (YS). Before microglia can serve their crucial functions, they must first migrate to and infiltrate the developing brain. The cellular and molecular dynamics governing this process are not fully understood, and what we do know is based on YS- derived microglia. However, cre/lox fate mapping studies only map ~30% of all mouse microglia to the YS. This suggests additional sources and populations of microglia could exist. This has been confirmed in zebrafish, where microglia have additional, non-YS origins. The discovery of additional microglia populations leaves us with even less understanding of how microglial precursors seed the brain, especially given that what we do know is from YS-derived microglia only. To begin to fill this critical gap, we investigated the microglia that seed the embryonic brain. Because microglia seeding the brain is a highly dynamic process, we utilized timelapse imaging in zebrafish to watch the cells live. We identified an undescribed cell in the brain that expresses canonical microglia markers, clears debris, and expands in injury. These microglia-like cells are labeled with Mannose Receptor C, type 1a (mrc1a) and colonize the brain earlier than known microglia precursors. mrc1a+ microglia are dependent on the mrc1a+ lymphatic vessels sitting just outside the CNS boundary. These mrc1a+ cells are located within the brain parenchyma and do not associate with vessels or the brain-border, suggesting they’re not macrophages, perivascular cells, or brain lymphatic endothelial cells. Despite our discovery of this early-infiltrating population, the dynamics of how this mrc1a+ population colonizes the brain and expands as microglia are unknown. The implication of mrc1a+ brain-border lymphatics in these processes is also undetermined. The goal of the proposed study is to investigate the cellular and molecular characteristics of a novel, mrc1a+ microglia subpopulation as it colonizes, expands, and differentiates in the developing brain. I will accomplish this by using a combination of in vivo imagine, optogenetic tools, and photoactivatable drugs to carry out the following aims: 1. Determine if brain-border lymphatic vessels contribute microglial progenitors to the developing brain. 2. Characterize the molecular profile of mrc1a+ cells as they differentiate into microglia. This work has the potential to impact a broad spectrum of fields including basic neurodevelopmental biology, neurodevelopmental disorder and disease, glial biology, and CNS homeostasis by investigating the fundamental dynamics behind early microglial precursors as they infiltrate the developing brain, expand, and turn on canonical microglia markers.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY/ABSTRACT Metastatic breast cancer (BC) remains largely resistant to immune checkpoint blockade (ICB) therapy. Tumor-infiltrating neutrophils (TINs) with immunosuppressive activity represent a major component in the tumor microenvironment (TME) to drive immunotherapy resistance. Therapeutic debilitation of immunosuppressive TINs is a promising approach to elicit synergistic efficacy when combined with immunotherapy. However, therapeutic targeting of TINs faces challenges in selectivity and safety, highlighted by the lack of TIN-specific targets. Recently, we used single-cell RNA sequencing to compare TINs and circulating neutrophils in BC models and identified aconitate decarboxylase 1 (Acod1) to be uniquely upregulated in TINs. Acod1 catalyzes the production of itaconate, a metabolite with anti-inflammatory activity in macrophages. But Acod1 function in neutrophils is poorly defined. Our preliminary results in BC mouse models suggest that TINs rely on Acod1 to sustain survival in the TME, and Acod1 loss leads to reduced TIN infiltration and metastasis. There are still significant knowledge gaps about the function, mechanism and therapeutic potential of Acod1 in TINs. Our central hypothesis is that Acod1 upregulated in TINs that infiltrate metastatic BC is essential for the TINs to persist in the TME and exert the immunosuppressive function, thus Acod1 ablation debilitates TINs, favors anti-tumor immunity, and sensitizes metastatic BC to immunotherapy. We propose to accomplish three Specific Aims: (Aim 1) Validate the pro-metastasis function of Acod1 in TINs in syngeneic and spontaneous murine mammary tumor models. (Aim 2) Identify the upstream and downstream molecular mechanisms underlying the upregulation and function of Acod1 in TINs. (Aim 3) Improve metastatic BC response to immunotherapy by Acod1 ablation and validate ACOD1 expression in clinical samples. To achieve our research goals, we have developed both whole-body and neutrophil-specific Acod1 knockout mice as hosts for BC syngeneic models, both mouse and human in vitro cell models of TINs, single- cell technologies for gene profiling and immune cell phenotyping, injection techniques that generate mammary tumors and metastases to lung and bone in the mice, and multiplex immunofluorescence staining techniques suitable for validation studies using clinical samples. We have assembled a strong research team with complementary expertise, which further ensures that the studies proposed are highly feasible to accomplish. Upon completion of the project, we expect to uncover the previous unknown function and mechanism of Acod1 in immunosuppressive TINs that are enriched in BC metastases, provide novel links between metabolic rewiring and immunoregulatory function of TINs, and generate the key preclinical evidence for targeting Acod1 to improve immunotherapy. In the long term, we envision that the bench-to-bedside translation of our findings through development of Acod1 inhibitors may accelerate the therapeutic application of combining agents that reprogram immunometabolism and immunotherapeutics to the curative treatment of BC and other cancers.

NIH Research Projects · FY 2026 · 2023-03

Summary TCR recognition of peptides bound and presented by MHC proteins underlies cellular immunity. TCR recognition of pMHC is most often viewed through the lens of traditional receptor-ligand theory, where cellular responses are presumed to be governed by solution binding affinities or kinetics. While this is often the case, work over the past several years has shown that complexities from mechanical forces exerted on membrane bound TCR and pMHC can profoundly influence T cell signaling. Of notable interest are catch bonds: force dependent enhancements of the lifetimes of TCR-pMHC complexes formed between interacting cells. Catch bonds can lead to large changes in signaling output and can greatly enhance T cell sensitivity. Demonstrating the importance of mechanical forces in tuning T cell responses, ligands that are recognized with strong affinity but fail to result in catch bonds yield altered or even no T cell signaling. Force-dependent behavior has been implicated in a wide range of T cell biological processes, including thymic education, responses to viral or tumor antigens, and viral escape. Although the importance of mechanical force in TCR recognition has been demonstrated, we have only a rudimentary understanding of how TCRs form catch or revert to slip bonds. We (PI Evavold) have had recent success in manipulating TCR catch bonds (published in Science this year) but this was achieved through screening libraries and without an understanding of mechanism. We thus lack predictive models for force dependent behavior in TCRs and in turn how this affects biology, which in turn impacts our ability to predict immunogenicity, assess the consequences of mutations, and hinders our ability to understand T cell specificity. Recently, however, we developed a comprehensive framework to identify, manipulate, and predict force dependent behavior in TCR-pMHC interactions. Unlike prior efforts, our framework directly addresses mechanism. Here, we will further develop, refine, and apply our framework. Our driving hypothesis is that viewing force dependent behavior through the lens of energy will provide the missing mechanistic detail of how and why catch bonds emerge in TCRs, allow their rational prediction and manipulation, and permit force considerations to be included in assessments of T cell recognition of antigen. Our three Aims are to 1) further develop our mechanistic framework for force dependent TCR behavior; 2) explain how changes to catch bonds emerge from natural variations in TCR interfaces and how catch bonds regulate T cell biology; and 3) Use rational catch bond engineering to better control viral infection in mice. Overall, the work in this proposal will illuminate the opaque mechanisms that underlie T cell mechanobiology, place catch bonds on a formal mechanistic footing, and provide the means to predict and productively manipulate TCR catch bonds and ultimately T cell biology.

NIH Research Projects · FY 2026 · 2023-03

Summary: Hsp90 is a molecular chaperone that is responsible for the conformational maturation of signaling proteins associated with all ten hallmarks of cancer, making it a promising target for the treatment of cancer, as multiple signaling nodes can be simultaneously derailed as a consequence of Hsp90 inhibition. Moreover, researchers have shown that Hsp90 inhibitors accumulate in tumors with high differential selectivity, making Hsp90 a highly sought after target for cancer. Unfortunately, clinical trials with 17 small molecule inhibitors have led to multiple detriments that have significantly dampened enthusiasm for Hsp90 inhibitors, as increased levels of Hsp90 were observed in the clinic, which led to dose-escalating toxicities among other concerns. Consequently, Hsp90 remains a desirable target for the development of cancer chemotherapeutics, but new approaches to inhibit the protein machinery are needed that do not induce Hsp90 levels. Through a number of seminal studies, it has been shown that inhibitors of the Hsp90 C-terminal domain can segregate Hsp90 inhibition from induction of Hsp90 levels, and therefore, we propose in this application to optimize these compounds and to perform a number of pre-IND studies on the best molecules in an effort to move them toward clinical evaluation.

NIH Research Projects · FY 2026 · 2023-02

Project Summary: Retinal degenerative diseases are a major medical issue for society. One potentially exciting approach to restore vision is the regeneration of lost retinal neurons from an endogenous population of retinal cells, the Müller glia. We are studying this process in zebrafish, which unlike mammals, exhibits a natural Müller glia- dependent retinal regeneration response. However, there are two major gaps in our understanding of this retinal regeneration response. The first is why rapid acute damage exhibits a regeneration response and a slow chronic damage, which is what is often observed in human retinal degenerative diseases, does not induce a regeneration response in zebrafish. The second gap in our understanding is the role of the microglia, the immune cells of the central nervous system, which are the major source of inflammation resulting from damage and a known regulator of the Müller glia-dependent retinal regeneration. We will address these two gaps in three Specific Aims. Aim 1 will determine the potential of two different chronic zebrafish retinal degeneration mutants (gosh, an early onset rapid cone photoreceptor degeneration mutant and cep290, a late onset slow cone degeneration mutant) to induce Müller glia proliferation and regenerate lost cones using different stimuli. We will determine to what extent either a secondary acute damage or the introduction of molecules that stimulate Müller glia proliferation can induce cone regeneration in chronically damaged fish and how complete the regeneration process is. In Aim 2, we will conduct a comprehensive and unbiased, comparative analysis of gene expression and chromatin accessibility in Müller glia and microglia using a multiomic single-nuclear RNA- Seq and ATAC-Seq analysis in these two chronic degeneration mutants, along with two mouse chronic retinal degeneration mutants. We will determine the similarities and differences in gene expression and chromatin accessibility in the Müller glia and microglia between the acutely and chronically damaged retinas. These bioinformatic analyses will reveal transcription factors and signaling (cytokine, growth factors, ligand/ receptor pairs) molecules that are essential for regeneration following acute damage and blocking regeneration in the chronically damaged zebrafish retina. We will also determine the differences and similarities between the chronically damaged zebrafish and mouse retinas to determine how similar these regulatory components are between the zebrafish and mouse. Aim 3 will then functionally test the roles of the candidate regulators previously identified in our scRNA-Seq datasets or in Aim 2 by either modifying their expression or their activity in the chronically and acutely damaged zebrafish retina. This work will be the first molecular analysis of how retinal regeneration is regulated in the chronically damaged zebrafish retina and will be critical in the translation of Müller glial-dependent retinal regenerative therapies into human retinal degenerative diseases.

NIH Research Projects · FY 2024 · 2022-09

Sleep is important for cognitive well-being, yet many adults do not get enough sleep. Sleep is especially important for selectively strengthening memories that are emotionally salient, associated with rewards, and relevant for future use; and sleep’s effect on memory selectivity may exacerbate some mental disorders. However, the neural mechanisms of sleep-facilitated selectivity are still being identified, and whether the same mechanisms generalize across different cognitive domains such as emotional memory and problem solving is unknown. The current research will examine 1) the interaction and unique contribution of sleep to emotional memory selectivity and problem solving; 2) the neural signatures of sleep’s effect on selectivity; and 3) whether targeted memory reactivation during rapid eye movement sleep similarly facilitates emotional memory selectivity and problem solving. We will record participants’ brain activity using EEG while they sleep in one of four state-of-the-art laboratory bedrooms, allowing us to precisely identify participants’ sleep stages and associated neural signatures. This research will lead to a better understanding of the neural mechanisms of sleep’s effect on cognition, allowing future research to examine the effect of sleep disruption on sleep-facilitated selectivity. The fellowship training plan will enable the applicant to conduct the proposed research and prepare her for a future career as an independent researcher. Through coursework, workshops, and mentorship, the applicant will extend her prior training in problem solving to include expertise in the cognitive neuroscience of sleep and memory, including EEG analysis techniques such as spectral power analysis, sleep spindle detection, and slow- oscillation-spindle coupling. In addition, the training plan provides extensive professional development in areas of academic communication, research management, and research ethics. Results from the proposed research will be widely disseminated in scientific meetings and publications, and will inform novel research questions at the intersection of sleep, memory, and problem solving, providing a solid foundation from which the applicant can launch her independent research career.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract The physical laws that govern the universe also govern the healthy functioning of living tissues, as well as the genesis and development of diseases. Historically, the biomedical research community has overlooked the role of mechanics in many developmental, pathological, and adaptive processes. In my research group, the Compu- tational Mechanics of Morphology at Notre Dame (CoMMaND Lab), we work at the intersection of mechanics, computation, and biology, to investigate the coupled bio-mechanical behaviors of tissues and organs, particularly during growth and remodeling. In this proposal, we aim to extend our work to the study of inflammatory swelling. Similarly to growth, inflammation and swelling involve local changes in mass (for instance, due to an influx of cells) which can manifest as changes in volume. While external swelling is often used as an indicator of under- lying inflammation, constraints from surrounding tissue can also restrict swelling and instead result in increases in pressure. In addition, inflammation can drastically change the cellular composition of a tissue. Inflammation is widespread among different tissues, and these sequelae can have important implications for the diagnosis, de- velopment, and treatment of different diseases. We will developing novel computational models of inflammation and swelling that 1) allow for cell behavior to vary spatially, temporally, and by cell type; 2) account for mechanical interactions between the swelling tissue and surrounding tissues; and 3) report results in a way that facilitates calibration, validation, and comparisons with experimental. This work will provide tools for probing small-scale phenomena beyond large-scale swelling, exploring the effects of individual parameters, and testing hypotheses regarding the biomechanics of inflammation in silico. 1

NIH Research Projects · FY 2025 · 2022-09

Multi-drug resistant (MDR) Acinetobacter baumannii infections present an enormous ongoing challenge to public health. Due to the frequent occurrence of multidrug resistance, current treatment options for A. baumannii infections are limited. ß-Lactam antibiotics, especially carbapenems, represent the treatment of choice for susceptible infections. However, carbapenem resistance is increasingly common, and for such infections there is no consensus on the optimal alternative treatment. Because resistance has hitherto been relatively uncommon, colistin has become a favored treatment in spite of the fact that deleterious side effects are common. However, resistance to colistin in A. baumannii is becoming more frequent with the recent dissemination of plasmid-borne colistin resistance genes (mcr-1-10) into healthcare facilities. Unfortunately, the recent track record of discovery of new antibiotics that are active against Gram-negative bacteria is exceedingly poor, which, coupled with the exit of Big Pharma from antibiotic discovery, has made the development of new therapies and non-traditional therapeutic approaches vital. To combat this growing threat, we initiated a research program to identify small molecules, termed antibiotic adjuvants, that potentiate the activity of macrolides against MDR A. baumannii. To this end, we have successfully identified molecules that lower the minimum inhibitory concentration (MIC) of clarithromycin up to 512-fold against all members of a panel of primary clinical A. baumannii isolates from the Walter Reed Army Institute of Research (WRAIR) that encompasses nearly all clinically relevant A. baumannii clades. Adjuvants also potentiate the activity of vancomycin up to 256-fold. Both macrolides and vancomycin are typically viewed as “Gram-positive” selective antibiotics due to their inability to cross the outer membrane of Gram-negative bacteria, Mechanistic studies have led to a working hypothesis that these compounds overcome this barrier by increasing permeability of the outer membrane through inhibiting lipooligosaccharide (LOS) production. Combinations of adjuvant with clarithromycin are effective in a Galleria mellonella model of infection, which has been shown to predict outcome in murine models of infection in the context of MDR A. baumannii. Therefore, combinations of such adjuvants with either clarithromycin or vancomycin may form the basis for an efficacious approach to treating MDR A. baumannii infections for which there are no effective antibiotics.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY / ABSTRACT Over the past two decades, suicide rates have increased nearly 35% in the U.S., with up- ward trends in nearly all demographic groups. Further increases have occurred since the COVID-19 pandemic began. Despite ambitious goals for reducing suicides and significant fed- eral and private investment, suicide rates continue to rise unabated. To date, the predominant approach to mitigating suicide risk in the U.S. is secondary prevention. Typically, these pro- grams identify risk of recurrence among those who have already attempted suicide at least once. Although secondary prevention is crucial, the majority of deaths by suicide occur on first attempt. Thus, targeted primary prevention earlier in development is essential. Most current pri- mary prevention programs are intensive, expensive, and delivered by highly trained mental health providers, who are in short supply. Traditional face-to-face therapy is also unavailable to many who live in underserved communities, and disliked by adolescents, who much prefer digi- tal delivery on their devices. This high-risk, high-reward proposal addresses these limitations and needs. We use an experimental therapeutics approach to evaluate the independent and combined efficacies of two unconventional but scalable interventions: transcutaneous vagus nerve stimulation (tVNS) to target emotion dysregulation, and a peer-support smartphone app to combat social isolation. These low-cost interventions, which hold strong promise but have not been used before, can reach large numbers of adolescents, with much potential to reduce pro- spective suicide risk. We will enroll 212 adolescents, ages 13-17 years, who show elevations on at least two prominent risk factors for suicide (e.g., self-injury, maltreatment). Using a 2 × 2 de- sign, adolescents will be assigned randomly to receive 30 days of treatment with (1) tVNS to tar- get emotion dysregulation, (2) a peer-support phone app to target social isolation, (3) tVNS + a peer-support phone app, or (4) enhanced treatment as usual with monitoring and access to re- sources. Intervention effects on mechanisms (emotion dysregulation, social isolation) proximal efficacy signals (e.g., physiological reactivity, self-harm) and target outcomes (suicidal ideation, suicidal behaviors) will be evaluated immediately post-intervention and at one-year follow-up. Treatment data will be monitored daily to fine-tune dosing of both interventions. This transforma- tive and innovative proposal tests two novel, scalable preventive interventions designed to “meet adolescents where they are" by using digital technologies to address core mechanisms of suicide risk.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY / ABSTRACT Over the past two decades, suicide rates have increased nearly 35% in the U.S., with up- ward trends in nearly all demographic groups. Further increases have occurred since the COVID-19 pandemic began. Despite ambitious goals for reducing suicides and significant fed- eral and private investment, suicide rates continue to rise unabated. To date, the predominant approach to mitigating suicide risk in the U.S. is secondary prevention. Typically, these pro- grams identify risk of recurrence among those who have already attempted suicide at least once. Although secondary prevention is crucial, the majority of deaths by suicide occur on first attempt. Thus, targeted primary prevention earlier in development is essential. Most current pri- mary prevention programs are intensive, expensive, and delivered by highly trained mental health providers, who are in short supply. Traditional face-to-face therapy is also unavailable to many who live in underserved communities, and disliked by adolescents, who much prefer digi- tal delivery on their devices. This high-risk, high-reward proposal addresses these limitations and needs. We use an experimental therapeutics approach to evaluate the independent and combined efficacies of two unconventional but scalable interventions: transcutaneous vagus nerve stimulation (tVNS) to target emotion dysregulation, and a peer-support smartphone app to combat social isolation. These low-cost interventions, which hold strong promise but have not been used before, can reach large numbers of adolescents, with much potential to reduce pro- spective suicide risk. We will enroll 212 adolescents, ages 13-17 years, who show elevations on at least two prominent risk factors for suicide (e.g., self-injury, maltreatment). Using a 2 × 2 de- sign, adolescents will be assigned randomly to receive 30 days of treatment with (1) tVNS to tar- get emotion dysregulation, (2) a peer-support phone app to target social isolation, (3) tVNS + a peer-support phone app, or (4) enhanced treatment as usual with monitoring and access to re- sources. Intervention effects on mechanisms (emotion dysregulation, social isolation) proximal efficacy signals (e.g., physiological reactivity, self-harm) and target outcomes (suicidal ideation, suicidal behaviors) will be evaluated immediately post-intervention and at one-year follow-up. Treatment data will be monitored daily to fine-tune dosing of both interventions. This transforma- tive and innovative proposal tests two novel, scalable preventive interventions designed to “meet adolescents where they are" by using digital technologies to address core mechanisms of suicide risk.

NIH Research Projects · FY 2025 · 2022-09

PROJECT SUMMARY/ABSTRACT Few evidence-based programs exist to support children and families affected by conflict, despite documented evidence of their heightened risk for emotional and behavioral adjustment problems associated with exposure to conflict and violence at multiple levels of the social ecology (e.g., political, community, and family). Thus, a critical need exists for an evidence-based program to ameliorate the impact of political violence on the overall well-being of children and families. The current study will conduct a rigorous evaluation of a theoretically driven, family-based intervention program in Palestine, including both the West Bank and Gaza. The long-term goal of this project is to provide a family-focused intervention program (Promoting Positive Family Futures; PPFF) that may facilitate individuals’ sense of safety and support in the context of chronic adversity. The objective is to evaluate this intervention program in the context of a randomized clinical trial (RCT) in the West Bank and Gaza (N=300). The central hypothesis is that the program will have direct positive effects on family conflict, parent psychopathology and parental security in the family as well as on adolescent emotional security in the family, with cascading effects on adolescent adjustment. Consistent with family systems theory, we further hypothesize that treatment effects on parents will mediate on the effects of the treatment on adolescent adjustment. The rationale is that bolstering resilience in family systems is a key approach to promoting positive functioning in families exposed to violence. The hypothesis will be evaluated with three specific aims: 1) evaluate the efficacy of an evidence-based family support program; 2) examine process models of treatment change, and 3) examine interrelations between parent and child functioning. To achieve these aims, the study will be an RCT employing a longitudinal design (N=300) with multi-method assessments at baseline (T1), post-test (T2), 6-month follow-up (T3) and 12-month follow-up (T4). Families will be randomized into the intervention condition (PPFF) or treatment as usual (TAU). Implementing partners and investigators will work together to ensure the study procedures are implemented in parallel across sites. Data collection will be conducted by trained research staff from a third-party survey and policy research organization. The proposal seeks to shift current research and clinical paradigms in these contexts by employing novel theoretical concepts, approaches, and methodologies. The contribution will be significant by 1) further developing new directions for empirically based interventions in these high-risk contexts, and 2) advancing a relatively brief, cost-effective program that can be readily implemented to help children and families, with the potential to be brought to scale in other contexts, including in the United States.

NIH Research Projects · FY 2025 · 2022-08

Adapting a point of use test card, the chemoPAD, for protecting chemotherapy drug quality in sub-Saharan Africa Project Summary/Abstract Goal: validate a new technology for detecting bad quality chemotherapy products at the point of use. Motivation: Chemotherapy medicines form the backbone of affordable cancer treatment in low- and middle- income countries (LMICs), yet LMICs often lack technical and regulatory capacity to evaluate the quality of chemotherapy products. There is currently no commercial technology to screen for bad quality chemotherapy products at the point of use in LMIC settings, and the drug regulators in Ethiopia, Malawi, Kenya, and Cameroon do not conduct post-market surveillance testing on chemotherapy products. Activities: The technology that will be validated, called SpotCheck, consists of an inexpensive paper test card (the chemoPAD) and a cell phone app. We will first adapt the chemoPAD to screen eight types of injectable chemotherapy drugs. The phone app’s neural network will be trained to identify products that are falsified or contain less than 65% of the stated API content. Clinical, academic, and supply chain partners in Ethiopia, Malawi, Cameroon, and Kenya will conduct annual situation awareness and quality surveys of 320 chemotherapy products per year; the results will enable a team of researchers at U. North Carolina to model the markets for chemotherapy products and evaluate the cost-effectiveness of the SpotCheck system. After a technical performance milestone is passed, we will tailor the clinical validation of SpotCheck to suit the local needs, clinical workflows, and regulatory capacity in each site. The validation of the SpotCheck system will proceed through a planning and ethical approval milestone (Y3 in Ethiopia and Malawi and Y4 in Kenya and Cameroon) and three clinical phases: proficiency study, clinical validation, and implementation pilot. Proficiency testing will demonstrate that oncology pharmacists and nurses can use SpotCheck with accuracy >85% to detect SF products. Clinical validation will establish whether SpotCheck works correctly in a clinical setting on authentic products, rather than proficiency samples. The implementation pilot study will probe SpotCheck’s ability to test the drops left over in product vials after patient treatments are prepared in the hospital; this method of use would allow sustainable implementation of SpotCheck in many hospitals and clinics in low-resource settings. Technology transfer efforts will empower LMIC partners to produce the chemoPAD locally and integrate the cell phone app into regulatory reporting systems. Impact: This project will help to fill the huge evidence gap about the quality of chemo drugs in LMICs, make it harder for manufacturers and distributors to sell bad quality products, and improve the quality of products that are used to treat patients in LMICs. 1

NIH Research Projects · FY 2025 · 2022-08

Project Summary The long reach of early life remains one of the most enduring puzzles in human health. From famine to poverty and neglect, adverse early life experiences lead to higher mortality and elevated risk for obesity, diabetes, and chronic heart disease. These effects are likely mediated, in part, by the stress response, a cascade of neuroendocrine, metabolic, and cardiac responses to challenge involving the hypothalamic– pituitary–adrenal (HPA) axis and the autonomic nervous system. In support, several leading hypotheses propose that repeated social and environmental stressors—both in early life and adulthood—cause over- activation of the stress response, chronic stress, and accelerated aging. Long-term studies of natural animal populations offer compelling models for testing these ideas because they often have fine-grained, prospective, longitudinal data on social and environmental stressors from individuals across the life course. However, natural animal models also face considerable challenges in measuring multiple facets of the stress response: most are constrained to measuring glucocorticoids (GCs) as the sole measure of stress responses, reflecting just one aspect of the HPA axis with no information on autonomic responses. This limitation has led many to call for expanded tools to measure stress responses in natural animal models of aging. Our objectives in this proposal are to: (1) expand the tools for measuring the cardiometabolic consequences of stress in natural animal models by validating insertable cardiac monitors (ICMs) with accelerometry to measure heart rate, heart rate variability, and physical activity; and (2) test the social and environmental drivers of the autonomic stress response and its metabolic consequences. We will develop and validate ICMs using captive baboons at the Institute of Primate Research in Kenya, and a well-studied, natural population of baboons in Amboseli, also in Kenya. Prior work in Amboseli has already shown that an accumulation of harsh conditions in early life and social isolation in adulthood exert profound effects on adult mortality, setting the stage to probe the stress responses underlying these links. While early life adversity and social isolation lead to elevated GCs in adulthood, and animals with high lifelong GCs have lower survival, GCs do not mediate the link between early adversity and life span. Gaining a broader perspective on individual stress responses and their consequences is an essential next step. The results will contribute the first prospective, longitudinal data in any species to understand how early life adversity and adult social conditions interact to shape acute autonomic stress responses, chronic stress, and energy expenditure. This study will provide a direct link between socio-environmental circumstances, stress responses, and adult health, helping to identify key targets to mitigate the effects of stress over the life course on aging.

NIH Research Projects · FY 2025 · 2022-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The Chemistry-Biochemistry-Biology Interface (CBBI) Program at the University of Notre Dame is an established NIH-funded program that supports the training of scientists who conduct multidisciplinary research at the interface of chemistry and biology-related disciplines. The goal of the CBBI Program is to produce PhD scientists who work effectively at the interface of chemistry and biology and who are able to speak the language of two or more disciplines. The CBBI Program has established an outstanding training environment for our predoctoral trainees. Since 2007 we have trained 84 students, 57 of whom have completed PhD degrees in an average of 5.14 years (time to PhD degree from CBBI trainees is 4.98 in the past 10 years) with 19 still in training. Our 84 trainees have 362 publications, of which 159 are first-author publications. This represents 4.3 publications per trainee and 1.9 first-author publications per trainee. During the past 14 years, our attrition rate has been low, with only eight of 84 trainees (9.5%) who did not complete a PhD degree. The key highlights of the CBBI Program are: 1) a large pool of highly qualified candidates that are selected to be CBBI fellows through a solely merit-based review; 2) a strong track record of collaborative and multidisciplinary research by both trainees and mentors; 3) a faculty training group that serves as research mentors and are experienced, productive, and federally-funded; 4) an intensive, cross-disciplinary research internship in the other field outside the mentor’s laboratory; 5) multidisciplinary seminars that supplement training; 6) individual and group meetings with trainees; 7) the annual CBBI symposium that features both oral and poster presentations at the chemistry/biology interface; 8) program administration with an established track record of training students at the chemistry/biology interface; 9) a plan and mechanism for continuous evaluation and refinement of the training program; 10) professional development and career placement; 11) outstanding research facilities; and 12) a strong institutional commitment. We propose to continue to train PhD scientists with the skills and expertise to solve challenging biomedical problems, regardless of discipline. The University of Notre Dame enthusiastically supports this training program and will continue to provide a generous fellowship match to the CBBI Program and additional resources, including the cost of the research internship for all trainees, upon renewal of funding by the NIH.

NIH Research Projects · FY 2025 · 2022-05

Project summary: Acute myeloid leukemia (AML) is a devastating cancer with limited options, despite decades of intensive searches for curative therapeutics. The average 5-year survival of AML is about 30% (for all patients) but for most elderly patients over 60 years, the 2-year survival rate is less than 5%. About 30% of AML patients harbor a mutated FLT3 kinase and these patients have the worst outcome. Midostaurin and gilteritinib, FLT3 inhibitors were approved in 2017 and 2018 respectively. Crenolanib another FLT3 inhibitor is in advanced phase III clinical trials, whereas quizartinib was approved in Japan but failed to gain FDA approval. However patients on all of the four FLT3 inhibitors ultimately relapse due to secondary mutations in the FLT3 kinase (such as FLT3-ITD, D835 and F691 mutants) and other compensatory resistance mechanisms. Breast cancer has an average 5-year survival rate is 93% for stage I and II but for a small but significant percentage (15-20%) of breast cancer patients, who harbor hormone refractory cancer (called triple negative breast cancer, TNBC), there are few therapeutic options. Clearly new therapeutics, which are effective against AML and TNBC cancers, are needed. The PIs have identified novel 3H-pyrazolo[4,3-f]quinoline-based kinase inhibitors, synthesized in only a single flask operation, that potently inhibit FLT3 and/or CDK2 or CDK12/13 or CDK18. The selectivity for these kinases depend on the substitution pattern of the 3H-pyrazolo[4,3-f]quinoline core. CDK12/13 and CDK18 are involved in the cell's response to DNA damage and the inhibition of these kinases lead to BRCAness in various cancers, making such cancers sensitive to agents that damage DNA or inhibit DNA damage repair, such as doxorubicin or PARP inhibitors respectively. The overall goal of this project is to optimize these interesting new class of kinase inhibitors for possible translation into AML and breast therapeutics. In aim 1, second-generation 3H- pyrazolo[4,3-f]quinoline-based compounds, (first-generation compounds have already shown impressive in-vivo efficacy against AML in vivo), will be biochemically characterized and evaluated in vivo (Aim 3). Additionally new chemistries will be used to make third-generation 3H-pyrazolo[4,3-f]quinoline-based kinase inhibitors that have better drug-like properties. Aim 2 characterize how these new potent anti-proliferative compounds affect protein phosphorylation in cancer cells and to determine the potencies of the compounds at killing various AML cell lines, which are resistant to current therapies such as gilteritinib, and triple negative breast cancer cell lines in vitro. In aims 3, the PIs will evaluate the in-vivo efficacies of lead compounds against AML and breast tumors. project, novel agents against AML and breast cancer, which inhibit traditional FLT3 (AML) as well as CDK12/13 and CDK18, which are interesting new cancer targets with no approved drugs that target them.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY - Decoding the regulation of protein folding by synonymous codon usage Synonymous mutations are widespread in complex, polygenic diseases but are typically regarded as phenotypically silent, as they preserve the amino acid sequence of the encoded protein. Yet, synonymous mutations can significantly perturb protein homeostasis through a variety of mechanisms, including perturbing the folding mechanism of the encoded protein. Recently, my lab discovered that synonymous codon-induced changes to protein folding can be large enough to (a) exceed the protein homeostatic buffering provided by molecular chaperones and (b) lead to a dramatic two-fold decrease in cell growth rate. For these proteins, changing the codon usage pattern produces a folded protein with an altered structure, which leads to changes in activity and/or susceptibility to degradation by cellular proteases. The profound implication of these results is that codon usage represents another level of information encoded within genomes, linking together “silent” genetic differences with proper protein function and regulation of a potentially broad range of cellular mechanisms. Historically, however, studying perturbations to protein folding mechanisms in vivo has presented immense technical challenges. For this reason, to date only a few examples have been identified of connections between codon usage and protein folding. As a result, we lack a comprehensive picture of the extent to which synonymous codon usage contributes to the production of a functional proteome and how synonymous mutations perturb protein homeostasis. This Pioneer Award project is designed to break through existing technical challenges, developing a novel approach to (i) broadly measure for the first time the number and types of proteins with folding mechanisms sensitive to synonymous codon usage, across an entire proteome and (ii) deeply interrogate which codon usage patterns and features best support proper protein folding in vivo. The ambitious, overarching goal of this project is to enable a next generation of genomic inference by developing a predictive understanding of the synonymous codon usage patterns that best support production of a functional proteome and the dysregulation that leads to human genetic disease.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Public health faces threats from a multitude of pathogens on an ongoing basis, yet pathogens associated with different diseases are typically compartmentalized with respect to surveillance, management, and research. This compartmentalized approach ignores the many ways that pathogens interact, in some cases leading to the exacerbation of their collective burden on public health. These interactions can be biological (e.g., cross-reactive immunity), behavioral (e.g., prompting adherence to good hygiene), or clinical (e.g., misdiagnosis). Modern, data- driven approaches to mathematical modeling have the potential to resolve the dynamics of co- circulating pathogens by accounting for these interactions. In doing so, modeling also has the potential to improve pathogen-specific disease forecasts by borrowing information across surveillance data for different diseases. To date, this potential remains largely untapped. In this project, I will develop a generalizable framework for modeling the dynamics of co-circulating pathogens. The first component of this framework will use Bayesian hierarchical modeling to fuse mechanistic descriptions of pathogen transmission dynamics with statistical descriptions of surveillance processes, allowing for maximal leveraging of heterogeneous data streams to inform biological inferences. The second component of this framework will involve validating model inferences through forecasts of future disease dynamics. Both components of this framework will involve the use of multiple models that represent competing hypotheses about pathogen interaction, as well as other forms of model uncertainty. This framework will be applied in two settings: mosquito-borne viruses in Brazil and respiratory pathogens in Indiana. In both of these settings, co-circulation of recently emerged and endemic pathogens poses new challenges for surveillance and control activities, making the development of new modeling tools to address these challenges especially timely.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Background: The metastasis of cancerous cells to distant and vital organs is responsible for in excess of 90% of cancer mortalities. Given this extraordinarily high mortality rate, there is a significant need for the development of novel therapeutic approaches that either eliminate metastatic cancer cells or eradicate incipient cancer cells prior to metastatic dissemination. An important barrier to tumor progression and metastasis is anoikis, a caspase- dependent cell death program induced by loss of integrin-mediated attachment to extracellular matrix (ECM). However, it has become clear that ECM-detachment can induce anoikis-independent mechanisms that can compromise cell viability. More specifically, we have discovered that detachment of non-cancerous epithelial cells from ECM triggers a significant elevation in the levels of reactive oxygen species (ROS) which compromises cell survival in an anoikis-independent fashion. The understanding of the cellular changes that contribute to the elevation of ROS during ECM-detachment remains rudimentary and the strategies utilized by cancer cells to combat ROS during ECM-detachment are insufficiently explored. Therefore, these points represent significant knowledge gaps that this grant proposal aims to address. Discernment of mechanistic information regarding the links between ECM-detachment, ROS, and cell survival could provide targets for the design of the therapeutic approaches aimed at specifically eliminating ECM-detached cancer cells; an outcome that may have significant impact for patients with metastatic disease. Objective/hypothesis: In aggregate, our preliminary studies have unveiled a novel cell death mechanism (with a tumor suppressive function) that compromises the viability of ECM-detached cells: the induction of mitophagy as consequence of RIPK1 signaling. As such, these data have motivated our central hypothesis that RIPK1- mediated mitophagy during ECM-detachment functions as a barrier to breast cancer progression. Specific Aim I: To elucidate the molecular mechanism by which ECM-detachment promotes RIPK1-dependent mitophagy and initiates cell death. Specific Aim II: To assess the capacity of RIPK1-mediated mitophagy to antagonize tumor formation in vivo and to evaluate antioxidant inhibition as a novel strategy to limit tumorigenesis in cancers that are deficient in RIPK1-mediated mitophagy Anticipated Outcomes: Following the completion of these studies, we will have accumulated significant mechanistic knowledge regarding the relationship between ECM-detachment, RIPK1 mitophagy, and cell death. Thus, the completion of these studies will unveil fundamental biological insights regarding cancer cell survival during ECM-detachment that may ultimately lead to the development of therapeutic approaches to limit the dissemination of breast cancer cells.