Grants

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Affecting over 12 million people worldwide, Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common inherited kidney diseases and is caused primarily by mutations in two genes—PKD1 and PKD2. In the 30 years since the identification of these genes, extensive effort has been made to understand how their protein products function under normal conditions and further, how naturally occurring mutations alter this function in ADPKD. While significant progress has been made to characterize PKD2 as a Ca2+-permeable nonselective ion channel, the function of PKD1 has proven more elusive. PKD1 has been proposed to act as an atypical G protein-coupled receptor (GPCR), but studies to define this function are limited by the absence of a known activating stimulus and lack of a typical 7-transmembrane (7-TM) domain signature present in all known GPCRs. We have discovered secreted WNT proteins as the activating ligands of the PKD1/PKD2 complex, defining this complex as a distinct class of WNT receptors. We asked the question of whether PKD1 functions as a WNT-activated GPCR. Using super-resolution live cell imaging and bioluminescence resonance energy transfer-based G protein biosensors, we find that PKD1 is directly coupled to specific subsets of heterotrimeric G proteins, such as Gαi1-3 and Gαq, but not Gαs or Gα12/13 in a WNT ligand-dependent manner. Specifically, we show that WNT ligands induce: 1) PKD1-mediated dissociation of Gα from Gβγ subunits, 2) time-dependent recruitment of Gα subunits to PKD1, and 3) Gα-GDP to -GTP transition via PKD1. The WNT-induced coupling of PKD1 to Gαi1-3 inhibits basal and forskolin-induced cAMP accumulation. PKD2 has an essential role in enabling PKD1 to function as a GPCR by chaperoning it to the plasma membrane. Naturally occurring mutations in PKD1 or PKD2 that compromise cell surface expression, assembly of the PKD1/PKD2 complex, or disrupt G protein recruitment diminish WNT-induced PKD1-mediated GPCR signaling. These data, which form the premise of this application, define the molecular function of the Polycystin complex and provide a direct mechanistic link of the complex to cAMP metabolism. We propose complementary in vitro and in vivo genetic approaches to take these findings to the next level to determine whether the direct coupling of PKD1 to Gαi and cAMP signaling is critical for the initiation of cystogenesis. Successful completion of the proposed studies will remove previously impossible technical and conceptual roadblocks to determine how naturally occurring mutations alter the function of these complex molecules and lead towards the development of new and more effective treatments for ADPKD.

NIGMS - National Institute of General Medical Sciences

Project Summary Each day, cells experience DNA damage from both external factors and normal cellular processes. To overcome this and maintain genomic stability cells rely on multiple cellular programs dedicated to repairing DNA damage. Failure to repair damaged DNA is detrimental to cells and can contribute to many diseases, including developmental disorders and cancer progression. The Cohesin complex plays a key role in the repair of DNA double strand breaks, though the exact mechanism of how Cohesin contributes to this repair is not well understood. Understanding this role has been hindered by the many cellular functions performed by Cohesin, with roles in cell division, transcription, and DNA replication, where disruption to Cohesin leads to broad and indistinguishable phenotypes. Together, this has left an essential gap in our knowledge of how Cohesin is regulated at DNA damage sites and its contribution to disease. Recently, we identified the Cohesin regulator PRR12 that specifically regulates the population of Cohesin at DNA damage sites, however, the mechanisms of this regulation remain to be defined. Understanding the role of PRR12 provides a unique opportunity to specifically target the population of Cohesin at DNA breaks while leaving its other functions intact, an approach that will allow us to specifically address the role of Cohesin in DNA repair for the first time. Additionally, defining the function of PRR12 is critical as patients with mutations in PRR12 phenocopy cohesinopathies, severe developmental and neurological disorders that result from altered Cohesin function. In this proposal, I will define how PRR12 regulates the localization of Cohesin to DNA damage sites and determine the role of the Cohesin complex in efficient DNA repair. To identify how PRR12 interacts with the Cohesin complex and localizes it to DNA lesions I will first perform a structure function analysis of PRR12. Using biochemistry and genetic tools I will determine if this interaction is direct and test its role in Cohesin stabilization. Next, I will determine if Cohesin regulates DNA architecture surrounding DNA breaks using a chromosome conformation capture technique. Last, I will test if PRR12 regulates Cohesin function in two independent DNA repair pathways using a quantitative fluorescent reporter. The work proposed here will be the first to separate the DNA damage role of Cohesin from its various functions throughout the cell cycle and begin to shed light on how misregulation of this process may contribute to genome instability in disease. This training will take place on the CU Medical School campus, where I have access to a wide variety of core resources and opportunities to interact with cell biologist and biochemists in the genome integrity field that will set me up for the successful completion of this proposal. My training plan will expand my experimental toolkit with training in biochemistry and genome organization techniques that will compliment my current expertise in cell biology and microscopy. In addition, I will prioritize gaining the professional development skills, mentorship experience, and communication skills that I will need to transition into an independent research position.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Coronary artery disease (CAD) remains a rising cause of morbidity and mortality in the United States. Human genetics suggest that a significant portion of the inheritable risk of CAD is related to fibroblast-specific regions of the genome. Using single cell transcriptomics, our group has identified a transcriptionally distinct adventitial fibroblast (AdvFib) population, which undergoes early phenotypic modulation during atherosclerosis development. Specific ablation of AdvFib significantly reduces plaque burden, suggesting their ability to influence atherosclerosis. To identify regulators of AdvFib, we characterized regions of altered chromatin accessibility upon AdvFib activation, revealing the transcription factor Tcf21. Tcf21 is predominantly expressed in AdvFib, and its expression declines upon AdvFib phenotypic activation. Tcf21 knockout in murine AdvFib increases vascular calcifications and upregulation of pro-atherogenic inflammatory cytokines. Given the restricted expression of Tcf21 to AdvFib and phenotypic activation of AdvFib upon Tcf21 knockdown, the central hypothesis underlying this proposal is that overexpression of Tcf21 locks AdvFib in a quiescent state, which then prevents the downstream activation of non-cell-autonomous signals that trigger plaque formation. Using an AdvFib-specific Tcf21 overexpression mouse model, I will characterize histological changes in plaque and alterations in non-AdvFib plaque cells, such as smooth muscle cells and inflammatory cells. I will then use single-cell RNA sequencing to characterize transcriptional changes of AdvFib upon Tcf21 overexpression and downstream transcriptional changes in non-AdvFib plaque cells. Given the putative effect of Tcf21 on the activation/recruitment of inflammatory cells, my goal is to characterize the precise epigenetic regions of Tcf21 binding to understand how Tcf21 regulates inflammatory cytokine production. Overall, this study will elucidate the role of AdvFib in atherosclerosis and characterize how Tcf21 regulates AdvFib.

NEI - National Eye Institute

Project Summary: Neurodevelopmental disorders, including syndromic disorders of retinal and brain development, are a major cause of morbidity in children. A subset of these disorders results from abnormal vascular development, and microcephaly and chorioretinopathy (MCCRP) is a recently identified disease that may belong in this category. It is characterized by small head circumference, brain anomalies, developmental delay, and vision loss due to chorioretinopathy and abnormal retinal vasculature. Autosomal recessive MCCRP results from defects in TUBGCP4 or TUBGCP6, which encode components of the gamma-tubulin ring complex (γ-TuRC), a ubiquitous structure necessary for microtubule nucleation and spindle formation in cells. However, γ-TuRC has not previously been implicated in vascular development and it is unknown why defects in γ-TuRC lead to blindness and microcephaly. We demonstrated that Tubgcp4 and Tubgcp6 expression is highly upregulated in murine vascular endothelial cells (EC) from the retina and brain (relative to EC from other tissues), and that murine EC deficiency of TUBGCP4 results in embryonic lethality, indicating a critical role for TUBGCP4 in EC. Our long-term goal is to identify the role of the γ-TuRC in retinal and brain development. The objective of this application is to define the pathophysiology of TUBGCP4 and TUBGCP6-associated MCCRP. We will test the hypothesis that TUBGCP4 and TUBGCP6 serve critical, EC-specific roles in the retina and brain, and that deficiency of these proteins results in MCCRP due to a primary vascular developmental defect. Since retinal and cerebral vascular development is not complete until several weeks after birth, we will use novel conditional knockout mouse models of Tubgcp4 and Tubgcp6 and a tamoxifen-inducible EC-specific Cre recombinase to eliminate expression of these genes in EC postnatally. Ophthalmic studies will demonstrate the necessity of EC-specific TUBGCP4 and TUBGCP6 in retina and retinal vascular development, including optical coherence tomography (OCT), OCT-angiography, electroretinogram, optokinetic response, and retinal histology (Aim 1). Neurologic studies in mice and/or embryos lacking TUBGCP4 or TUBGCP6 in EC will demonstrate the necessity of these proteins in cerebral and neurovascular development, including MRI brain imaging, neurobehavioral studies, and brain immunohistochemistry (Aim 2). Elucidating the pathophysiology of MCCRP will improve our understanding of the genetic mechanisms controlling retinal and brain vascular development, and may reveal new therapeutic targets for more common blinding retinal vascular diseases. The career development objective of this proposal is to develop the mentorship and expertise needed to become a productive independent clinician-scientist and international leader working at the intersection of inherited retinal diseases (IRD) and disorders of vascular development. OHSU is a center of renowned expertise in IRDs, in vivo retinal vascular imaging, and the neurosciences; it provides state of the art resources and world-class faculty to support Dr. Everett’s scientific and career development goals for this proposal.

NCI - National Cancer Institute

ABSTRACT Neuroendocrine prostate cancer (NEPC) is an aggressive and lethal subtype of prostate cancer (PC). While NEPC can develop as de novo disease, more commonly it develops as a resistance mechanism to Androgen Receptor (AR) targeted therapies in castrate resistance prostate cancer (CRPC) patients. NEPC patients often present with high metastatic burden. The prognosis of NEPC is poor with a 5-year survival rate of less than 20% with limited treatment options. Thus, new tractable molecular targets to combat NEPC are urgently needed. In our preliminary studies we have identified Tyrosine Threonine Kinase (TTK) as a new high value NEPC molecular target. TTK, also referred to as Monopolar Spindle 1 (Mps1), is a dual specificity protein kinase that plays a significant role in mitosis and the spindle assembly checkpoint (SAC). TTK expression is undetectable in normal cells and upregulated in transformed cells, making it an ideal therapeutic target. Moreover, TTK is a highly translational target, which as five small molecule inhibitors in clinical development. TTK expression progressively increases throughout PC disease progression and is highest in NEPC. The TTK expression trend holds in PC cell lines and patient-derived xenograft (PDX) models, wherein TTK expression is highest in cell lines and PDXs of NEPC origin. Genetically and pharmacologically targeting TTK slows NEPC cell growth in vitro and in vivo. These data support that TTK is an actionable target that may impede NEPC tumor growth. The objective of our proposal is to test the potential of TTK as a target for NEPC and define TTK mechanisms of action. For the latter, we conducted high resolution differential phospho-proteomic analysis in TTK targeted NEPC cells. We have prioritized Rab-Like Protein 6 (RABL6) as a candidate TTK NEPC phospho-target. In specific Aim 1, we will evaluate TTK phospho-target RABL6 and test the role of RABL6 in NEPC. In Aim 2, we will utilize a combination of NEPC cell line, patient derived xenografts and syngeneic NEPC models to rigorously test the effects of targeting TTK in vivo. Along with genetic approaches we will utilize clinical grade TTK inhibitors and preclinical TTK proteolysis targeting chimeras (PROTAC). In Aim 3 we will evaluate the therapeutic efficacy of combining leading TTK clinical inhibitor with first line NEPC chemotherapies. Transcriptomic analysis and immune cell profiling of tumors will be conducted to assess therapeutic response and identify biomarkers. Successful completion of these studies will confidently determine if TTK is a high value target which may ultimately help to lessen the morbidity of NEPC.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Non-typhoidal Salmonella enterica (NTS) is a leading cause of bacterial foodborne gastroenteritis and leading cause of death amongst foodborne pathogens. NTS elicit neutrophilic inflammation both during gut colonization and in systemic infection. Neutrophils produce toxic products to kill invading pathogens. One toxic product produced by activated neutrophils during severe bacterial infection is sulfite. Sulfites are reactive sulfur species that are toxic to all life forms. In addition to their presence during severe bacterial infection, sulfites are endogenously produced during metabolism of sulfated amino acids and are ingested in food products. Sulfites are also added to food products as antimicrobial agents. Therefore, enteric pathogens such as NTS must resist sulfite toxicity in several stages of infection. Our preliminary data link yeiH with sulfite stress resistance in Salmonella Typhimurium. We show a ∆yeiH mutant is defective for growth in sulfite stress conditions and yeiH expression is specifically activated by sulfite. yeiH encodes a highly conserved putative inner membrane protein with no documented function. The purpose of the proposed work is to establish the role of YeiH in sulfite stress resistance. We hypothesize that YeiH exports sulfites to allow Salmonella to resist neutrophil-derived sulfite stress during infection. We will test our hypothesis in two aims. In Aim 1, we will establish the role of yeiH in infection. We will use animal models of colitis and sepsis to establish whether yeiH is needed for Salmonella fitness during infection. Using a yeiH transcriptional reporter as a novel sulfite biosensor, we will determine the tissues in which yeiH is expressed to establish the spatial organization of sulfite stress during enteric and systemic salmonellosis. In Aim 2, we will determine how YeiH contributes to sulfite stress resistance. We will determine whether the proton motive force drives YeiH function and establish the amino acid residues important for YeiH role in sulfite stress resistance. We will also establish whether YeiH function is to export sulfite from the cell. YeiH orthologs are present in many bacteria, including in different pathogens with importance to human health. Successful completion of the proposed experiments will allow us to establish how YeiH contributes to Salmonella sulfite stress resistance in vitro and in vivo. This work will lead to future studies to define the mechanism of YeiH function and establish how sulfites contribute to human health and resistance to bacterial infections.

NIAID - National Institute of Allergy and Infectious Diseases

Project summary Enterovirus D70 (EVD70) is an understudied human enterovirus that causes acute hemorrhagic conjunctivitis, a highly contagious and painful ocular disease and that can, in rare instances, progress to polio-like paralysis. The molecular mechanisms underlying EVD70 infection, including 1) the host factors required for the viral life cycle and the 2) cellular tropism within the eye, remain poorly understood, due in part to a lack of eye-specific models. EVD70 attaches to ocular cells, at least in 2D cell culture, via sialic acids. There is, however, limited evidence suggesting the existence of an as-yet-unidentified proteinaceous receptor for EVD70 that triggers entry and uncoating in ocular cells. Through a genome-wide genetic knockout screen, I have identified host factors necessary for EVD70 infection in vitro, including the orphan receptor Jumping translocation breakpoint (JTB), which has limited demonstrated cellular functions. Notably, genetic knockout of JTB confers resistance to EVD70. I hypothesize that JTB serves as a receptor critical for EVD70 entry and uncoating in ocular cells and that JTB expression contributes to cellular tropism for corneal and conjunctival epithelial cells in the eye. The objectives of this proposal are to 1) determine the mechanism by which JTB is required for EVD70 infection and 2) define the cell-specific tropism and host immune responses to EVD70 in corneal organoids, which recapitulate the cellular diversity and organization of the ocular surface. Aim 1 will systematically evaluate if JTB is an entry and uncoating receptor for EVD70. Aim 2 will define the tropism of EVD70 and investigate host responses, such as interferon signaling, in corneal organoids using unbiased single cell RNA- sequencing and multiplexed cytokine analysis. Together, these aims will provide critical new insights into EVD70 biology and its interaction with ocular cells. Completing the proposed aims will advance our understanding of EVD70 molecular biology and pathogenesis and provide training to better equip me to progress towards independence as a scientist.

NIGMS - National Institute of General Medical Sciences

DNA binding to chromosomal DNA is essential for many fundamental biological processes including: transcriptional regulation, DNA replication and repair, recombination, and chromosome segregation. There is a fundamental gap in understanding how transcription factors (TF) bind regulatory regions located in compacted, high-order chromatin. Therefore, there is a fundamental need to determine the mechanistic rules defining TF binding to chromatin. Our long-term goal is to define the biological rules dictating TF binding to chromosomal DNA in a cell. All TFs, even pioneer factors, are not able to bind all their targets throughout the genome, indicating that there are binding constraints imposed on all binding events. Multiple factors appear to impact how TFs identify TF binding sites (TFBSs) including: TFBS positioning within a nucleosome, TFBS variants, sequence of the nucleosome, cooperativity, histone modifications, DNA methylation, and chromatin remodeling. To accomplish our goal, we will combine in vitro with in-cell assays to allow the systematic dissection of factors required for initial and sustained/functional TF binding to chromatin. At the nucleosome level, we will use our in vitro Pioneer-seq assay, which allows the direct comparison of TFBSs located in all possible nucleosome positions, with differing nucleosome sequences, across many TFBS variants, with modified histones. At the regulatory region level, we will ectopically express pioneer TFs in comprehensively characterized cell lines, with cell models of disease, to uncover the chromatin characteristics, chromatin remodelers, or cooperativity required for TF binding. With this MIRA our lab will close these knowledge gaps by answering three fundamental questions: 1) How do TFs recognize their binding sites within a nucleosome? 2) How do histone modifications and nucleosome positioning regulate TF binding? 3) How does chromatin organization regulate TF binding? Our results are expected to have an important positive impact on our mechanistic understanding of how all TFs regulate transcription and direct gene expression during normal development and disease. Our findings will likely be transformative by providing quantitative models for how proteins bind to their targets within or near nucleosomes.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract While the advent of antiretroviral therapy (ART) has improved the lives of many people with HIV (PWH), HIV infection remains persistent for the lifetime of most individuals. ART interruption leads to viral rebound and renewed disease progression, thus highlighting the existence of the latent HIV which exists even when viral RNA is undetectable in plasma during ART. To date, there is no broadly accessible cure for HIV. This requires most PWH to stay virally suppressed with a constant ART regimen. As such, there remains an urgent need to find functional cures of HIV and alleviate the need for a lifetime of costly ART and its associated long-term side effects. However, the complex nature of the HIV reservoir has impeded our collective efforts to understand the reservoir for elimination strategies. Viral transcription decreases over time on ART and can be nonexistent in many HIV+ cells, thus leading to evasion of immunosurveillance. The leading concept in the field for eliminating the reservoir is to reverse latency using pharmacological agents so that viral antigen is produced to render HIV+ cells permissive for elimination by an interventional therapy. However, current attempts at latency reversal are neither efficient nor complete as only a fraction of HIV+ cells are reactivated. This suggests that transcriptional regulation of the various proviruses (i.e. the integrated viral DNA) is highly heterogeneous. Evidence further suggests that proviral epigenetics, defined as the regulation of transcription at the chromatin level independent of sequence, is critical in understanding how the latent HIV reservoir is established, regulated, and disrupted. Some proviruses are situated in more condensed (heterochromatic) DNA without transcription, while others are in more accessible (euchromatin) DNA that is more conducive to transcription. This spectrum of reactivation potential means that reservoir elimination will first require a single-cell investigation to precisely define these proviral epigenetic states and assess their contribution to latency reversal. Therefore, the primary objective for this project is to define the ex vivo proviral epigenetic landscape at single-cell resolution and to develop a pioneering strategy to selectively reverse latency. The underlying central hypothesis is that HIV+ cells with heterochromatin-associated proviruses are more resistant to reservoir disruption and latency reversal. The central hypothesis will be tested through two specific aims: 1) define the proviral epigenetic landscape and its susceptibility to reservoir disruption from ART-treated PWH; and 2) determine the efficacy of mRNA-LNP delivery of HIV-specific transcription activator-like effectors (TALEs) for latency reversal. This proposal is highly innovative because it represents a major shift in how the field studies the HIV reservoir by using new single-cell methodology to circumvent the challenges inherent to studying the HIV reservoir. The outcomes will uniquely define the identities of the latent HIV reservoir and will offer additional opportunities to target and eliminate the HIV reservoir.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Allergic diseases persist throughout a person’s lifetime, and the mechanisms underlying maintenance of the Th2 cells that drive these diseases are poorly understood. Chronic inflammation canonically drives T cell exhaustion in chronic infection and cancer, while Th2 cells conversely persist despite chronic inflammation We recently created a single-cell transcriptomic atlas from human tissues with allergic type 2 inflammation. A conserved set of Th2 cells was identified across tissues, including an aberrant memory-like population co-expressing TCF1 and LEF1, with evidence of chronic activation. This transcriptional profile mirrors the progenitor/stem-like CD8+ T cells that have been described in cancer and chronic infection, and we hypothesized that this Th2 cell population may serve as the reservoir that perpetuates Th2 responses. We named this cell the Th2 cell multipotent progenitor (Th2-MPP). Ex vivo functional studies established that these cells couple self-renewal with terminal differentiation in Th2, TFH- like, and Treg cells. We propose that aberrant cell-cell interactions and alarmin signaling networks in allergic tissues orchestrate an epigenetic state in Th2-MPP that enables self- renewal in the face of chronic antigen burden. The specific aims of this research strategy will investigate the epigenetic mechanisms and cell- cell interactions driving human Th2-MPP maintenance (Aim 1). Moreover, we will functionally interrogate candidate pathways through the development and characterization of a mouse model of chronic type 2 pulmonary inflammation, with temporal conditional genetic ablation of putative regulators of Th2 maintenance (Aim 2). These complimentary human and mouse immunology approaches will yield fundamental insights into the chromatin states of type 2 lymphocytes in barrier tissues and signaling mechanisms that drive this profile. Concurrently, the mechanistic studies of candidate pathways in mice will establish new avenues for the development of therapeutic modalities. This study combines mechanistic mouse models and primary human tissue analyses with cutting-edge single cell bioinformatics to provide the candidate new training in key elements of translational immunology. Through support from the co-mentors and Advisory Committee, the candidate will be well-positioned to transition to independence as an investigator, with the goal of identifying new therapeutic targets for the treatment of chronic inflammatory diseases.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY The temporomandibular joint (TMJ) is a complex joint system critical for dental occlusion, mastication, respiration, and speech. The TMJ is comprised of a network of muscles, ligaments, and a fibrocartilaginous disc and condyle. TMJ osteoarthritis (OA) causes cartilage loss and pain. TMJ OA poses a major clinical problem, but there are no regenerative therapies. Understanding stem/progenitor cells is critical knowledge in organ tissue regeneration. We previously defined TMJ Lgr5-expressing cells as secretory niche cells critical for supporting TMJ cartilage progenitor cells in a canonical Wnt inhibitory niche. We also defined two secreted Wnt inhibitors, SOST and DKK3, as critical parts of the TMJ cartilage progenitor cell niche. We showed that therapeutic intra- articular injections of SOST ameliorate TMJ OA in a preclinical, mini-pig post-traumatic TMJ OA animal model. However, the identity of the TMJ cartilage progenitor cells regulated by Lgr5+ secretory niche cells and therapeutically targeted by cWnt inhibition is unknown. This R01 renewal proposal builds logically on our previously funded R01. We will investigate candidate TMJ cartilage progenitor cells and their regulatory niche critical for TMJ development, homeostasis, and regeneration. This proposal will: 1) establish the TMJ condyle as a sophisticated model for studying cartilage progenitor cells; 2) leverage basic science to design TMJ OA drugs. These foundational studies are critical for the advent of novel minimally invasive TMJ regenerative therapies.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Allogeneic hematopoietic stem cell transplant (HCT) recipients are at high risk of severe morbidity and mortality due to respiratory viral illnesses, including COVID-19. Despite two mRNA SARS-CoV-2 vaccines that are highly effective for the general population, HCT recipients have reduced immune responses to vaccines and remain at risk. This population’s ongoing risk is amplified by the emergence and circulation of SARS-CoV- 2 variant strains, relaxation of mask mandates and other public health measures, and waning effectiveness of SARS-CoV-2 monoclonal antibodies. New vaccination strategies are urgently needed for patients with hematologic malignancies, both to protect these patients and to reduce viral reservoirs that otherwise would allow for generation of new variants. To inform these strategies, we will evaluate the unique role of adoptive transfer of humoral and cellular immunity in HCT recipients, which will simultaneously use vaccine response as a mechanism to evaluate immune reconstitution. These studies will test our overarching hypothesis that these patients have distinct and altered immune responses to mRNA SARS-CoV-2 vaccination, resulting from their underlying disease, past immunomodulatory therapies, and the unique donor-to-recipient immune dynamics. The Specific Aims of this proposal are: Aim 1: Characterize the relationship between donor and recipient SARS-CoV-2 immunity in matched-related donor allogeneic HCT patients through analysis of humoral responses and transfer of immunity post-transplant from banked samples; Aim 2: Describe the correlation between donors’ pre-harvest cellular responses and recipients’ post-transplant humoral SARS-CoV-2 vaccine responses through creation of a prospective study of HCT recipients and their matched-related donors; and Aim 3: Evaluate donor v. recipient Ag-specific T and B cells and SARS-CoV-2 clonal breadth and depth after re-vaccination post-transplant to assess immune reconstitution, using T and B cell receptor repertoire analyses methods. We expect that completion of these aims and the associated training objectives will generate clinically applicable preliminary data to inform vaccine development and policy for patients with hematologic malignancies. My long-term career goal is to become an independently funded clinician-scientist and leader in translational vaccinology with a focus on developing precision vaccines for immunocompromised adults. I will achieve this through: 1) investigation of immunologic pathways that promote or inhibit vaccine-induced responses for patients with hematological malignancies, and 2) translation of these findings into novel vaccines via early-phase clinical trials. Brigham and Women’s Hospital and the Precision Vaccines Program at Boston Children’s Hospital are highly collaborative research environments within the broader Harvard Medical School community that will provide abundant resources and institutional support to complete the described aims.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Pathogenic bacteria have developed an array of strategies to undermine the host's defense mechanisms. In the context of non-Typhoidal Salmonella Typhimurium, a notable strategy involves the confinement of a single bacterium within a host vacuole called the Salmonella Containing Vacuole (SCV). This approach potentially provides an evolutionary advantage: by ensuring one bacterium per SCV, the host must target each SCV individually to eliminate the bacterial load, as opposed to confronting a vacuole harboring multiple bacteria clustered together. This mechanism could potentially extend the time required to combat the infection. Moreover, a single bacterium residing within an SCV exploits all accessible nutrients for replication and division. Conversely, multiple bacteria clustered within a single vacuole may engage in nutrient competition. Hence, understanding the fundamental mechanism of bacterial division in conjunction with vacuolar scission is imperative for gaining deeper insights into the pathogenic strategies employed by Salmonella enterica. Prior to this proposal we engineered a series of genetically minimal pathogenic strains that eliminates redundancy within the complex SPI-2 effector gene repertoire of Salmonella enterica serovar Typhimurium. Using this unique resource, here we will determine how a small network of SPI-2 T3SS effector proteins coordinate the complex events involved in SCV membrane scission and bacterial division within the host cell. This includes determining the location and host substrates of effector proteins at the SCV membrane using single cell particle tracking and live cell imaging (Aim 1). We will also investigate the molecular mechanisms of individual effector proteins that target a novel Rab-family GTPase defense system of the host (Aim 2). The resulting cellular and biochemical theories will be tested in murine models of systemic disease that are designed to evaluate effector protein functions at single cell resolution (Aim 3). Developing new drugs that target bacterial effector – host enzyme complexes would be an innovative approach to combat emerging infectious disease. While this idea holds great potential, the paucity of mechanistic information gleaned from deep studies into virulence factor functions has so far hampered their development as suitable drug targets. As a means to this end, the work performed here will allow us to predict new mechanisms of action for understudied Salmonella effector proteins and provide a glimpse into the structural-based evolutionary progression of a related pathogen groups.

NSF

Complex manifolds are higher-dimensional geometric spaces defined using the complex numbers. This project focuses on two special kinds of such spaces, namely Calabi-Yau manifolds, and Kähler-Ricci solitons. These types of complex manifolds have a wide array of applications throughout physics and mathematics, and for this reason have attracted a great deal of attention in recent years. This project will focus on constructions and classifications of such complex manifolds. The results will deepen our understanding of the geometry of complex manifolds as a whole and more broadly will further advance our knowledge through connections to other subjects ranging from algebraic geometry, topology, and analysis to physics. The project will also be used to help support researchers interested in contributing to these active and competitive fields. More technically, the project aims to further our understanding of the singularity development both of the Kähler-Ricci flow and of collapsing Calabi-Yau manifolds. Characterizing the development of singularities along the Kähler-Ricci flow is one of the fundamental questions in geometric analysis and is expected to reveal deep ties between geometric analysis, algebraic geometry, and topology. On the other hand, the analogous collapsing Calabi-Yau picture has many applications, including to physics. These two programs fit into a common framework, each with a given singularity model (respectively Kähler-Ricci solitons and complete non-compact Calabi-Yau metrics) and a corresponding type of degeneration (respectively the Kähler-Ricci flow and families of collapsing Calabi-Yau manifolds). The project will (1) construct examples of non-compact singularity model geometries, (2) describe the moduli of these spaces and (3) analyze the corresponding degenerations. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Labor Department

In this notice, OSHA announces the application of DEKRA Certification Inc., for expansion of the recognition as a Nationally Recognized Testing Laboratory (NRTL) and presents the agency's preliminary finding to grant the application. Additionally, OSHA proposes to add one test standard to the NRTL Program's List of Appropriate Test Standards.

Labor Department

In this notice, OSHA announces the final decision to expand the scope of recognition for DEKRA Certification Inc., as a Nationally Recognized Testing Laboratory (NRTL). Additionally, OSHA announces the final decision to add one test standard to the NRTL Program's List of Appropriate Test Standards.

City of Kansas City

Housing

Delaware Aviation Museum Foundation

Delaware Aviation Museum Foundation is an active grantmaking foundation based in Mardela Spgs, MD. Focus: arts. Contact the foundation directly for current funding opportunities.

Delaware Community Foundation

Delaware Community Foundation ($350M in assets) supports nonprofits in DE through grants focused on education, community development, health, arts. Awards range from $2,000 to $100,000.

Delaware Community Foundation

Delaware Community Foundation is an active grantmaking foundation based in Wilmington, DE. Focus: philanthropy. Contact the foundation directly for current funding opportunities.

Delaware

Delaware County

Delaware

Community Planning

Delaware

Delaware County

Delaware

Imminent Threat