Oklahoma Medical Research Foundation

MOLECULAR AND IMMUNOLOGIC ANALYSIS OF THE PATHOBIOLOGY OF ANTHRAX

ACCELERATING MEDICINES PARTNERSHIP-AUTOIMMUNE AND IMMUNOLOGIC DISEASE TISSUE RESEARCH CORE - PROJECT SUMMARY THE SUCCESSFUL ACCELERATING MEDICINES PARTNERSHIP IN RA/LUPUS (AMP1) PROGRAM FOCUSED ON DECONSTRUCTING DISEASE TISSUES THROUGH SINGLE-CELL TRANSCRIPTOMIC TECHNOLOGIES. AMP IN AUTOIMMUNE AND IMMUNE-MEDIATED DISEASES (AMP AIM) IS POISED TO EXPAND THE UNDERSTANDING OF THE CELLULAR COMPONENTS AND INTERACTIONS AT PLAY IN FOUR AUTOIMMUNE DISEASE TARGET TISSUES WITH NEW SPATIALLY-ORIENTED MODALITIES OF SINGLE-CELL ANALYSES. AS IN THE AMP1, THE AMP AIM WILL REQUIRE HIGH-QUALITY SAMPLES FOR INTERROGATION, STANDARDIZED METHODS TO ASSESS SAMPLE QUALITY AND COMMON MOLECULAR/PHENOTYPIC BIOMARKER TESTING ACROSS ALL SUBJECTS FOR A CLINICALLY AND MOLECULARLY WELL-PHENOTYPED COHORT TO APPLY NEW CUTTING-EDGE TECHNOLOGIES TO RE-CONSTRUCT DISEASE. THE OMRF TISSUE RESEARCH CORE (TRC) IS UNIQUELY POSITIONED TO SUCCESSFULLY DEPLOY A CENTRALIZED TRC ACROSS ALL DISEASE TEAMS FOR LUPUS (SLE), RA, SJOGREN’S DISEASE (SJD), AND PSORIATIC SPECTRUM DISEASES (PSD). WITH A ROBUST, EXISTING INFRASTRUCTURE, CAPABILITIES, AND LEADERSHIP, THE OMRF TRC WILL PROVIDE THE NETWORK WITH: 1) STANDARDIZED PROTOCOLS AND MANUALS TO BE USED ACROSS ALL PARTS OF THE NETWORK, 2) CENTRALIZED LOGISTICS FOR COLLECTION, TRANSPORT, STORAGE AND DISSEMINATION, 3) CENTRALIZED, TRANS-DISEASE QC AND INITIAL TESTING OF ALL SAMPLES IN A CONTINUOUS MANNER AS SAMPLES ARE COLLECTED FOR EARLY QUALITY MANAGEMENT AND IDENTIFICATION OF SAMPLES OF HIGH IMPORTANCE, 4) TISSUE IMAGING (BASIC HISTOLOGY & INITIAL HIGH-DIMENSIONAL) UTILIZING SAMPLE SPARING WORKFLOWS THAT PROVIDE SCORING AND QC OF TISSUE, PLUS INITIAL MULTI-OMIC, SPATIALLY-INFORMED DATA SETS, AND 5) INDUSTRY STANDARD REPOSITORY AND IMAGE DATABASE SYSTEMS. THE OMRF TRC IS CENTRALLY LOCATED, MAKING IT AN IDEAL LOCATION FOR LOGISTICS MANAGEMENT AND, SINCE IT WAS THE REPOSITORY FOR AMP1 CARRIES NETWORK KNOWLEDGE ALONG WITH THE EXTENSIVE EXPERIENCE AS REPOSITORY FOR MULTIPLE OTHER NATIONAL CONSORTIUMS, PROVIDES FOR CONTINUITY DURING TRANSITION TO AMP AIM. THE OMRF TRC ALREADY HAS INFRASTRUCTURE IN PLACE AND THE REPUTATION THAT FOLLOWS FROM STRONG LEADERSHIP TO CONTINUE TO ACT AS AN IDEAL HONEST BROKER FOR THE NETWORK. THE OMRF TRC HAS STATE-OF-THE-ART FACILITIES THAT ARE INTEGRATED WITH ADVANCED TECHNOLOGIES WITHIN ITS ASSOCIATED CORES TO RECEIVE, STORE, COLLECT MEANINGFUL QC AND INITIAL DATA SETS, AND DISSEMINATE SELECTED SAMPLES TO TECHNOLOGY AND ANALYTIC CORES (TAC) POISED TO PROVIDE DATA FROM EMERGING TECHNOLOGIES TO BUILD UPON THE DEEP CLINICAL AND INITIAL CHARACTERIZATION DATA, TO MAKE EXTREMELY HIGH- DIMENSIONAL, ROBUST DATASETS. CONSIDERING THE LONGSTANDING HISTORY OF THE OMRF TRC IN BIOBANKING SAMPLES FOR AUTOIMMUNE DISEASE STUDIES, THE OMRF TRC IS POISED TO A PRODUCE A SOLID FOUNDATION OF BANKED SAMPLES, ORGANIZED LOGISTICS, AND INITIAL MULTI-OMIC DATASETS THAT WILL SETUP THE NETWORK FOR SUCCESS IN RECONSTRUCTING OUR MECHANISTIC UNDERSTANDING AND CLINICAL APPLICATION OF THESE NEW DATA TO BETTER TREAT THESE AUTOIMMUNE DISEASES.

EXPANDING EXCELLENCE IN DEVELOPMENTAL BIOLOGY IN OKLAHOMA

CENTER FOR CELLULAR METABOLISM RESEARCH IN OKLAHOMA - ALTERED CELLULAR METABOLISM CONTRIBUTES TO THE PATHOGENESIS OF MANY COMMON DISEASES, SUCH AS CARDIOVASCULAR DISEASE, CANCER, DIABETES, AND AUTOIMMUNE DISORDERS, WHICH ARE PREVALENT IN OKLAHOMA. HOWEVER, METABOLISM- RELATED RESEARCH RESOURCES AND INFRASTRUCTURE ARE CURRENTLY INSUFFICIENT AND SCATTERED IN OKLAHOMA. TO ADDRESS THESE DEFICIENCIES, THIS CENTER FOR CELLULAR METABOLISM RESEARCH IN OKLAHOMA (CMRO) PHASE I COBRE WILL CREATE AND NURTURE EXCELLENCE IN CELLULAR METABOLISM IN OKLAHOMA. THE CMRO IS LOCATED AT THE OKLAHOMA MEDICAL RESEARCH FOUNDATION (OMRF), A LEADING BIOMEDICAL RESEARCH INSTITUTE. THE CENTRAL FOCUS OF THE CMRO IS TO SUPPORT FIVE NEW ASSISTANT MEMBER-LEVEL FACULTY (EQUIVALENT TO TENURE-TRACK ASSISTANT PROFESSOR AT A RESEARCH UNIVERSITY) AS PROJECT LEADERS FROM THREE DIFFERENT OMRF RESEARCH PROGRAMS: CARDIOVASCULAR BIOLOGY (CVB), AGING & METABOLISM (AM), AND ARTHRITIS & CLINICAL IMMUNOLOGY (ACI). THESE NEW JUNIOR INVESTIGATORS ARE THE FOUNDATION FOR EXPANSION AND UNIFICATION OF CELLULAR METABOLISM RESEARCH. THEY WILL RECEIVE AN APPROPRIATE LEVEL AND FORM OF MULTIDISCIPLINARY MENTORING FROM EXPERIENCED AND COMPETITIVE SCIENTISTS. THE CMRO COBRE AIMS TO DEVELOP A CRITICAL MASS OF INVESTIGATORS WHO CAN SECURE EXTERNAL FUNDING TO SUPPORT THEIR RESEARCH WHILE CREATING A NEW, EXCITING, MULTIDISCIPLINARY, AND COLLABORATIVE ENVIRONMENT FOR SUSTAINED COMPETITIVE RESEARCH ON CELLULAR METABOLISM IN OKLAHOMA, WHICH WILL BE ACHIEVED THROUGH FIVE AIMS: 1) SUPPORT FIVE PROMISING YOUNG PROJECT LEADERS TO SCIENTIFIC INDEPENDENCE THROUGH INCORPORATION OF CELLULAR METABOLISM RESEARCH IN THEIR LABS; 2) PROMOTE NOVEL MULTIDISCIPLINARY RESEARCH FOCUSING ON CELLULAR METABOLISM THROUGH MENTORING THE JUNIOR PROJECT LEADERS FROM THREE DIFFERENT OMRF RESEARCH PROGRAMS; 3) INTEGRATE EXISTING AND NEW RESOURCES THROUGH CORE FACILITIES THAT SUPPORT CELLULAR METABOLISM RESEARCH; 4) PROMOTE AN ENVIRONMENT THAT SUPPORTS THE SUCCESS AND DEVELOPMENT OF AN INDEPENDENT SUSTAINABLE CENTER. THE COBRE SUPPORT WILL BE CRITICAL TO CREATE A NEW, MULTIDISCIPLINARY AND COLLABORATIVE ENVIRONMENT FOR SUSTAINED COMPETITIVE RESEARCH ON CELLULAR METABOLISM IN OKLAHOMA.

PROTEIN-GLYCAN INTERACTIONS IN THE VASCULAR SYSTEM

MOLECULAR MECHANISMS AND GENETICS OF AUTOIMMUNITY

GENOMICS OF LUPUS

COMPREHENSIVE GENOME INTERROGATION OF AFRICAN AMERICAN SARCOIDOSIS FAMILIES

OKLAHOMA SJOGREN'S SYNDROME CENTER OF RESEARCH TRANSLATION

INTERDISCIPLINARY RESEARCH IN VASCULAR BIOLOGY

OKLAHOMA MEDICAL RESEARCH FOUNDATION CLINICAL RESEARCH CONSTRUCTION

OKLAHOMA RHEUMATIC DISEASE RESEARCH CORES CENTER (OVERALL APPLICATION)

POST-TRANSLATIONAL MODIFICATIONS IN HOST DEFENSE

INTERDISCIPLINARY RESEARCH IN VASCULAR BIOLOGY

OKLAHOMA RHEUMATIC DISEASE RESEARCH CORE CENTERS

ROLE OF MUCIN-TYPE O-GLYCANS IN INTESTINAL INFLAMMATION

PROTEASE ACTIVATED RECEPTOR SIGNALING BY COAGULATION PROTEASES

MOLECULAR MECHANISMS AND GENETICS OF AUTOIMMUNITY COBRE

OKLAHOMA AUTOIMMUNITY CENTER OF EXCELLENCE

TNFAIP3 (A20) AND SUSCEPTIBILITY TO SYSTEMIC LUPUS ERYTHEMATOSUS

PROTEASE-MEDIATED VASCULAR INSTABILITY IN DEVELOPMENT AND DISEASE

AUTOIMMUNE DRIVERS AND PROTECTORS TEAM SCIENCE (ADAPTS) - PROJECT SUMMARY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS A HETEROGENEOUS, SYSTEMIC AUTOIMMUNE DISEASE THAT CAUSES SIGNIFICANT MORBIDITY AND EARLY MORTALITY, ESPECIALLY IN MINORITY POPULATIONS AND IN WOMEN OF CHILD-BEARING AGE. BY THE TIME PATIENTS REACH SLE CLASSIFICATION, THE MAJORITY EXHIBIT ORGAN/TISSUE DAMAGE AND ONGOING, AGGRESSIVE INFLAMMATORY PROCESSES. PREVENTING SLE WOULD REDUCE THE RISK OF IRREVERSIBLE ORGAN DAMAGE AND TREATMENT-RELATED TOXICITIES, BUT REQUIRES IDENTIFICATION AND VALIDATION OF DRIVERS AND RESTRAINTS OF DISEASE PROGRESSION DURING THIS PRE-/EARLY-CLINICAL TIME PERIOD. OUR RESEARCH GROUPS HAVE MADE IMPORTANT PROGRESS TOWARD DECIPHERING ASPECTS OF PRECLINICAL AUTOIMMUNITY. A FEW FINDINGS INCLUDE SHOWING: AUTOANTIBODIES OCCUR IN LINKED-SUBSETS YEARS BEFORE CLINICAL DISEASE, DEMONSTRATING HUMORAL EPITOPE SPREADING AND PERIPHERAL IMMUNE DYSREGULATION IN PRECLINICAL AUTOIMMUNE TRANSITION, DEFINING IMMUNE ENDOTYPES OF AUTOANTIBODY POSITIVE HEALTHY INDIVIDUALS, IDENTIFYING ACTIVATED B CELL AND SELECT MYELOID CELL SUBSETS PRECEDING SLE DISEASE FLARE, AND ESTABLISHING VIRAL REACTIVATION PRECEDING SLE DISEASE ONSET. THROUGH OUR NEWLY FORMED ADAPTS CONSORTIUM, WE WILL ADDRESS THESE GAPS WITH TWELVE AVAILABLE, COMPLEMENTARY PRE-DISEASE UNIQUE COLLECTIONS THAT SPAN THIS CONTINUUM FROM BENIGN AUTOIMMUNITY TO ACTIVE SLE, WHICH INCLUDES CLINICAL DATA, QUESTIONNAIRE INFORMATION, BIOSPECIMENS, AUTOANTIBODY AND OTHER EXPERIMENTAL DATA FROM NEARLY 11,000 PARTICIPANTS. A SUBSET OF THE HIGHEST-RISK INDIVIDUALS EARLIER IN THIS CONTINUUM WILL BE FOLLOWED FOR AUTOIMMUNE AND CLINICAL PROGRESSION. THESE CROSS-SECTIONAL COLLECTIONS ARE AUGMENTED WITH LONGITUDINAL TRANSITIONAL COHORTS WHICH INCLUDE OVER 400 INDIVIDUALS WHO TRANSITION TO SLE WITH PRE-DISEASE SAMPLES AND CLINICAL DATA. OUR INVESTIGATIVE TEAM MEMBERS BRING STRENGTHS IN CLINICAL INVESTIGATION, INNATE IMMUNOLOGY, CELL-FREE NUCLEIC ACID SENSING, SOLUBLE MEDIATOR CHARACTERIZATION, MULTI-OMIC ANALYSES, CELLULAR IMMUNOLOGY, IMMUNE METABOLISM, SYSTEMS IMMUNOLOGY, BIOINFORMATICS AND STRONG, PRODUCTIVE COLLABORATIONS. BUILDING ON OUR PUBLISHED AND NEW PRELIMINARY DATA, OUR ADAPTS CONSORTIUM OF CLINICAL AND BASIC SCIENTISTS WILL ASSESS POTENTIALLY INDEPENDENT OR COMPLEMENTARY MECHANISMS OF DISEASE PROGRESSION AND IMMUNE RESTRAINT BY TESTING THE ROLES OF RNA-SENSING PATHWAYS, CELL-FREE NUCLEIC ACIDS, AGE-ASSOCIATED B CELLS AND AUTOANTIBODY CHARACTERISTICS, MYELOID CELL SUBSETS, T CELL LYMPHOPENIA AND LYMPHOPENIA-INDUCED PROLIFERATION, T CELL IMMUNOMETABOLISM, AS WELL AS SELECT ENVIRONMENTAL (MICROBIOME, VIROME), GENOMIC AND INNATE AND ADAPTIVE IMMUNE RESPONSES AS DRIVERS OF SLE DISEASE. WE WILL ALSO VALIDATE AND REFINE PREDICTIVE MODELS OF CLINICAL SLE DISEASE DEVELOPMENT TO INFORM FUTURE PREVENTION TRIALS AND ASSIST THE FIELD THROUGH MOLECULAR AND CLINICAL ENDOTYPE IDENTIFICATION OF THE PRECLINICAL TIME PERIODS. DATA FROM THIS INTEGRATED RESEARCH PROGRAM WILL INFORM TARGETED IMMUNE-MEDIATED PREVENTION TRIAL DEVELOPMENT, SELECTION OF CELLULAR/MOLECULAR AND PATHWAY-DIRECTED THERAPIES, AND THE DESIGN OF LONGITUDINAL SLE NATURAL HISTORY OR PREVENTION STUDIES.

EXPANDING EXCELLENCE IN DEVELOPMENTAL BIOLOGY IN OKLAHOMA - ABSTRACT OVERALL DEVELOPMENTAL BIOLOGY RESEARCH IS FUNDAMENTAL TO PROGRESS IN DETERMINING THE MOLECULAR BASIS OF MANY DISEASES, AS WELL AS FOR THE RAPIDLY EMERGING FIELD OF REGENERATIVE MEDICINE. DURING COBRE PHASES I AND II, WE WELCOMED 12 NEW INVESTIGATORS, MANY OF WHOM USE MODEL ORGANISMS (PLANARIANS, DROSOPHILA, ZEBRAFISH, AND C. ELEGANS) WHOSE GENOMES CAN BE EFFICIENTLY MANIPULATED TO UNDERSTAND FUNDAMENTAL PHYSIOLOGICAL PROCESSES THAT ARE CONSERVED AMONG SPECIES. IN SOME CASES, FUNCTIONAL GENOMIC APPROACHES WERE USED TO MODEL HUMAN MUTATIONS TO UNDERSTAND THE BASIS OF DEVASTATING INHERITED DISEASES. ALL OUR PROJECT LEADS WERE RIGOROUSLY TRAINED IN DEVELOPMENTAL BIOLOGY, PUBLISHED IN TOP TIER JOURNALS, AND SHOWED EXCEPTIONAL PROMISE. THEIR APPOINTMENTS WERE DELIBERATELY DIVIDED AMONG FOUR OF OMRF’S RESEARCH PROGRAMS (EQUIVALENT TO ACADEMIC DEPARTMENTS) TO ENCOURAGE CROSS-FERTILIZATION AND ATTRACT ESTABLISHED INVESTIGATORS TO THE FIELD. WE NOW HAVE A CRITICAL MASS OF DEVELOPMENTAL BIOLOGISTS THAT INCLUDES COBRE-SUPPORTED INVESTIGATORS AS WELL AS MORE ESTABLISHED FACULTY. DURING PHASES I AND II WE ALSO ESTABLISHED AND EXPANDED CORES THAT PROVIDE ESSENTIAL SERVICES TO DEVELOPMENTAL BIOLOGISTS: AN IMAGING CORE AND A BIOINFORMATICS AND PATHWAYS CORE. OUR GOALS IN PHASE III ARE TO CONSOLIDATE THE PROGRESS WE HAVE MADE IN PHASES I AND II BY WELCOMING NEW DEVELOPMENTAL BIOLOGISTS INTO OUR COMMUNITY THROUGH THE SUPPORT OF PILOT PROJECTS AND TO EXPAND THE SERVICES OFFERED BY OUR CORES, WHILE AT THE SAME TIME POSITIONING THEM FOR SUSTAINABILITY WHEN COBRE FUNDING ENDS. WE WILL ACCOMPLISH OUR GOALS THROUGH THE FOLLOWING SPECIFIC AIMS. AIM 1. TO EXPAND THE BREADTH OF OUR RESEARCH PROGRAM IN DEVELOPMENTAL BIOLOGY THROUGH THE CHOICE OF PILOT PROJECT INVESTIGATORS WITH OUTSTANDING POTENTIAL TO BECOME LEADERS IN THEIR FIELDS. AIM 2. TO MENTOR THE PILOT PROJECT INVESTIGATORS USING A COMBINATION OF SENIOR SCIENTISTS AND RECENTLY GRADUATED COBRE PROJECT LEADS WITHIN EXISTING OMRF AND UNIVERSITY OF OKLAHOMA FACULTY. AIM 3. TO FORM AN EXTERNAL ADVISORY COMMITTEE OF INTERNATIONALLY RECOGNIZED DEVELOPMENTAL BIOLOGISTS TO EVALUATE THE PROGRESS OF, AND OFFER ADVICE TO, THE PILOT PROJECT INVESTIGATORS AND CORE DIRECTORS. AIM 4. TO EXPAND THE CAPABILITIES OF THE CORE FACILITIES SO THEY ARE WELL-POSITIONED TO SUPPORT THE RESEARCH OF BOTH CURRENT AND FUTURE DEVELOPMENTAL BIOLOGISTS (INCLUDING CURRENT PILOT PROJECT INVESTIGATORS) AND LAY THE GROUNDWORK FOR SUSTAINABLE OPERATION AFTER COBRE FUNDING ENDS. AIM 5. TO PROMOTE SCIENTIFIC INTERACTIONS AMONG THE PILOT PROJECT INVESTIGATORS AND FORMER PROJECT LEADS TO CREATE AN ENVIRONMENT CONDUCIVE TO INVESTIGATOR RETENTION.

MOLECULAR MECHANISMS AND GENETICS OF AUTOIMMUNITY

TRANSLATIONAL CONTROL OF MEGAKARYOCYTE AND PLATELET GENE EXPRESSION IN DISEASE

SERPIN REGULATION OF COAGULATION PROTEASES

SCIENCE IN A CULTURE OF MENTORING

CANDIDATE CAUSAL VARIANTS IN SYSTEMIC LUPUS ERYTHEMATOSUS

DISEASE AND RACE SPECIFIC SINGLE-CELL EPIGENETIC MECHANISMS IN HUMAN SLE - PROJECT SUMMARY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS A PROTOTYPIC AUTOIMMUNE DISEASE CHARACTERIZED BY DYSREGULATED INTERFERON RESPONSES AND LOSS OF SELF-TOLERANCE TO CELLULAR ANTIGENS THAT RESULT IN INFLAMMATORY PROCESSES THAT ULTIMATELY LEAD TO SYSTEMIC END-ORGAN DAMAGE. STRIKING GENETIC AND CLINICAL DIFFERENCES EXIST BETWEEN EUROPEAN AMERICAN (EA) AND AFRICAN AMERICAN (AA) SLE PATIENTS THAT RESULT IN DISPARITIES IN THEIR DIAGNOSES, MANAGEMENT AND OUTCOMES. DESPITE DECADES OF RESEARCH AND OVER 100 RISK LOCI IDENTIFIED, THE UNDERLYING MECHANISMS DRIVING THE PATHOGENESIS OF SLE AND ITS RACIAL DISPARITIES REMAIN INCOMPLETELY UNDERSTOOD. OUR LAB HAS JOINED FORCES WITH THE TEAM LED BY JASON BUENROSTRO, PHD, AT HARVARD TO EVALUATE AND UNDERSTAND HOW EPIGENETIC REGULATION IN SPECIFIC CELL TYPES CONTRIBUTES TO BOTH DISEASE- AND RACE-SPECIFIC SLE PATHOGENESIS. WE USE A SCI-ATAC-SEQ (SINGLE CELL INDEXING ASSAY FOR TRANSPOSASE-ACCESSIBLE CHROMATIN), METHOD DEVELOPED AND OPTIMIZED IN THE BUENROSTRO LABORATORY, TO ASSESS DISEASE- AND RACE-SPECIFIC DIFFERENCES IN GENOME-WIDE CHROMATIN ACCESSIBILITY (CA) IN CELL SUBTYPES FROM CRYOPRESERVED PERIPHERAL BLOOD MONONUCLEAR CELLS. INTEGRATING THESE DATA WITH GENOTYPING DATA ALLOWS US TO IDENTIFY ALLELICALLY IMBALANCED VARIANTS THAT ARE ENRICHED IN REGIONS OF CA (CAQTLS), SUGGESTING A MECHANISTIC ROLE FOR THESE VARIANTS IN REGULATING CA. OUR PRELIMINARY DATA CONDUCTED IN EA SLE AND HEALTHY CONTROLS DEMONSTRATE THAT WE ARE ABLE TO DEFINE MULTIPLE MAJOR PBMC CELL SUBSETS, AND THAT EXTENSIVE DIFFERENTIAL CA AND CAQTLS DISCRIMINATE BETWEEN SLE CASE AND CONTROL SUBJECTS. WE NOW PROPOSE AN EXPANDED STUDY THAT WILL NOT ONLY EVALUATE DISEASE-SPECIFIC CA AND CAQTL ALTERATIONS IN A MUCH LARGER SAMPLE, BUT ALSO RACE-SPECIFIC ALTERATIONS. INTEGRATING SINGLE-CELL TRANSCRIPTOMIC DATA AND CLINICAL DATA WITH CA AND CAQTL DATA WILL FACILITATE THE FUNCTIONAL INTERPRETATION OF SLE RELEVANT CA DIFFERENCES. OUR GOALS FOR THIS AR077434 RESUBMISSION ARE TO 1) DEFINE CELL TYPE-SPECIFIC DIFFERENCES IN CHROMATIN ACCESSIBILITY THAT IDENTIFY BOTH DISEASE- SPECIFIC AND RACE-SPECIFIC ALTERATIONS; 2) IDENTIFY CELL TYPE AND RACE-SPECIFIC CAQTLS THAT ASSOCIATE WITH DIFFERENCES IN CA BETWEEN SLE CASES AND CONTROLS; AND 3) DEFINE THE MECHANISTIC RELATIONSHIPS BETWEEN CA, CAQTLS, GENE EXPRESSION ARCHITECTURES, WITHIN THE CONTEXT OF CIS CO-ACCESSIBILITY NETWORKS. WE BELIEVE THIS PROJECT POSITIONS US AT THE LEADING EDGE TO DEVELOP A PRECISE EPIGENETIC ROADMAP CONNECTING GENETIC VARIATION TO DELETERIOUS CELLULAR AND CLINICAL PHENOTYPES THAT UNDERLIE THE DISEASE-SPECIFIC MECHANISMS OF SLE AND ITS REMARKABLE RACE-SPECIFIC DISPARITIES.

DEFINING THE MECHANISMS OF LYMPHATIC AND LYMPHOVENOUS VALVE DEVELOPMENT

CELLULAR REGULATION OF SELECTIN-LIGAND INTERACTIONS

SCIENCE IN A CULTURE OF MENTORING

DISCOVERY AND CHARACTERIZATION OF GENETIC RISK LOCI IN SJOGREN'S SYNDROME

PERIPHERAL BLOOD MONONUCLEAR CELL EPIGENETIC ASSOCIATIONS IN AND BIOMARKERS FOR KNEE OSTEOARTHRITIS DEVELOPMENT AND PROGRESSION

FUNCTIONAL AND MECHANISTIC CHARACTERIZATION OF LUPUS SUSCEPTIBILITY - PROJECT ABSTRACT SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS A COMPLEX AUTOIMMUNE DISEASE CHARACTERIZED BY CHRONIC INFLAMMATION AND MULTI-ORGAN INVOLVEMENT, LEADING TO SIGNIFICANT MORBIDITY AND MORTALITY. SLE DISPROPORTIONATELY AFFECTS WOMEN AND INDIVIDUALS OF ASIAN, AFRICAN, AND HISPANIC DESCENT, WITH A HIGHER PREVALENCE AND SEVERITY COMPARED TO OTHER POPULATIONS. DESPITE ADVANCEMENTS IN RESEARCH, THE MOLECULAR MECHANISMS UNDERLYING SLE REMAIN POORLY UNDERSTOOD, AND CURRENT THERAPIES ARE OFTEN INSUFFICIENT DUE TO CLINICAL HETEROGENEITY AND UNPREDICTABLE DISEASE TRAJECTORIES, UNDERSCORING THE URGENT NEED FOR NOVEL INTERVENTIONS. AUTOPHAGY, A CRITICAL CELLULAR MECHANISM FOR MAINTAINING HOMEOSTASIS, HAS EMERGED AS A KEY PLAYER IN SLE PATHOGENESIS. DYSFUNCTIONAL AUTOPHAGY DISRUPTS CELLULAR PROCESSES, INCLUDING LYSOSOMAL FUNCTION, ANTIGEN PRESENTATION, AND CYTOKINE REGULATION, WHICH ARE CENTRAL TO BOTH IMMUNE AND NON-IMMUNE CELL FUNCTIONS. WHILE B AND T CELLS HAVE LONG BEEN IMPLICATED IN SLE, RECENT EVIDENCE HIGHLIGHTS THE ROLE OF CLASSICAL MONOCYTES (CD14++CD16−) AND KIDNEY-RESIDENT PODOCYTES IN DRIVING DISEASE PROGRESSION. IN SLE, MONOCYTES EXHIBIT IMPAIRED AUTOPHAGY, RESULTING IN DEFECTIVE PHAGOCYTOSIS AND HEIGHTENED PRODUCTION OF PRO-INFLAMMATORY CYTOKINES, SUCH AS TNF-Α AND IL-6, FUELING SYSTEMIC INFLAMMATION. SIMILARLY, PODOCYTES RELY ON AUTOPHAGY FOR MAINTAINING STRUCTURAL INTEGRITY AND GLOMERULAR FILTRATION. AUTOPHAGY IMPAIRMENTS IN PODOCYTES LEAD TO CYTOSKELETAL DAMAGE, APOPTOSIS, AND PROTEINURIA, WHICH ARE HALLMARK FEATURES OF LUPUS NEPHRITIS. GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED NUMEROUS SLE SUSCEPTIBILITY LOCI; YET TRANSLATING THESE FINDINGS INTO MECHANISTIC INSIGHTS REMAINS CHALLENGING. OUR STUDY FOCUSES ON FOUR KEY GWAS LOCI LINKED TO GENES INVOLVED IN AUTOPHAGY, LYSOSOMAL FUNCTION, AND APOPTOSIS—PATHWAYS CRUCIAL FOR IMMUNE REGULATION AND TISSUE HOMEOSTASIS. WE HYPOTHESIZE THAT GENETIC VARIANTS WITHIN REGULATORY ELEMENTS OF THESE GENES DISRUPT AUTOPHAGY, EXACERBATING IMMUNE DYSREGULATION AND ORGAN-SPECIFIC DAMAGE IN SLE. AIM 1 INVOLVES FINE-MAPPING SLE SUSCEPTIBILITY LOCI THROUGH IMPUTATION-BASED ANALYSIS OF RESEQUENCING DATA FROM PATIENTS AND CONTROLS. FUNCTIONAL VARIANTS WILL BE PRIORITIZED USING ALLELE-SPECIFIC EXPRESSION, CHROMATIN PROFILING, AND 3D GENOME INTERACTION DATA, FOLLOWED BY GENOTYPING VALIDATION. AIM 2 EVALUATES THE FUNCTIONAL IMPACT OF THESE VARIANTS ON AUTOPHAGY AND CELLULAR PROCESSES USING CRISPR-BASED MODIFICATIONS IN IMMUNE (E.G., THP-1 MONOCYTES) AND NON-IMMUNE (E.G., PODOCYTE) CELL MODELS. COMPLEMENTARY STUDIES IN PRIMARY MONOCYTES FROM SLE PATIENTS AND HEALTHY CONTROLS WILL VALIDATE FINDINGS. BY ELUCIDATING THE ROLE OF AUTOPHAGY-RELATED GENETIC VARIANTS IN SLE, THIS RESEARCH AIMS TO UNCOVER NOVEL MECHANISMS OF DISEASE AND IDENTIFY THERAPEUTIC TARGETS FOR PRECISION MEDICINE APPROACHES.

SLE SUSCEPTIBILITY AND CLINICAL SIGNIFICANCE AT 2Q22-24 ACROSS MULTIPLE ETHNICITI

EPIGENOME-GUIDED CAUSAL VARIANT DISCOVERY AND MECHANISMS

UNDERSTANDING CELL DIVISION

IKAROS FAMILY GENES AND LUPUS SUSCEPTIBILITY ACROSS ETHNICALLY DIVERSE POPULATIONS

REGULATION OF CHROMOSOME COHESION DURING CELL CYCLE PROGRESSION

COMPLEMENT C5 INHIBITION AS SEPSIS THERAPY - PROJECT SUMMARY SEPSIS IS A LIFE-THREATENING ORGAN DYSFUNCTION CAUSED BY DYSREGULATED HOST RESPONSE TO INFECTION. SEPSIS IS A LEADING CAUSE OF DEATH IN INTENSIVE CARE UNITS. THE CONTACT OF PATHOGENS WITH THE INTRAVASCULAR COMPARTMENT OF THE HOST LEADS TO SYSTEMIC INFLAMMATION, WHEREIN ACTIVATION OF THE COMPLEMENT AND COAGULATION SYSTEMS PLAYS CRITICAL ROLES. THE OBJECTIVES OF THIS PROPOSAL ARE TO INVESTIGATE THE ROLE OF COMPLEMENT ACTIVATION AND ITS CROSSTALK WITH INFLAMMATION AND COAGULATION TO SEPSIS PROGRESSION AND MULTIPLE ORGAN FAILURE (MOF) AND, WHETHER BLOCKING COMPLEMENT ACTIVATION AT THE C5 LEVEL COULD PREVENT MOF AND IMPROVE THE OUTCOME OF SEPSIS. WE WILL USE CLINICALLY RELEVANT MODELS OF SEPSIS CAUSED BY TWO OF THE MOST COMMON PATHOGENS, ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS TO: (I) DETERMINE THE CONTRIBUTION OF COMPLEMENT AND COAGULATION TO PATHOGENESIS OF MICROTHROMBOSIS, VASCULAR DYSFUNCTION, ORGAN FAILURE AND DEATH; (II) TEST STAGE-SPECIFIC THERAPIES TARGETING THE COMPLEMENT AND COAGULATION CASCADES TO PROVIDE ORGAN PROTECTION AND SURVIVAL BENEFIT IN SEPSIS-INDUCED PROGRESSIVE MOF. SUCCESSFUL COMPLETION OF THESE AIMS WILL DETERMINE: (I) WHETHER TIMED COMPLEMENT INHIBITION AT THE C5 LEVEL COULD BE USED AS AN EFFECTIVE THERAPY FOR SEPSIS-INDUCED MOF; (II) IF INHIBITION OF C5A RECEPTOR-1 SIGNALING COULD ATTENUATE DISEASE PROGRESSION AND PROVIDE ORGAN PROTECTION; (III) IF COMBINATION THERAPIES EMPLOYING EARLY TREATMENT WITH AN ANTICOAGULANT AND A DELAYED TREATMENT WITH A C5 INHIBITOR WILL PROVIDE SYNERGISTIC PROTECTION IN CASES OF HIGH SEPTIC BACTEREMIA/SHOCK. ALTOGETHER, OUR PROJECT WILL COMBINE BASIC AND PRECLINICAL RESEARCH TO VERIFY NOVEL HYPOTHESES ON THE PATHOPHYSIOLOGY OF BOTH GRAM-NEGATIVE AND GRAM-POSITIVE SEPSIS, AND TEST INNOVATIVE APPROACHES, WHICH IN THE LONG-TERM MAY SAVE LIVES FROM THIS DEADLY DISEASE WITH NO SPECIFIC CURE.

RESOLVING THE MECHANISMS OF IFIH1 RISK VARIANT IN SYSTEMIC LUPUS ERYTHEMATOSUS - ABSTRACT DYSREGULATED TYPE I INTERFERON (IFN I) HAS BEEN ASSOCIATED WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE). BOTH VIRAL INFECTIONS AND DETRIMENTAL SELF-RNA SENSING HAVE BEEN IMPLICATED IN DRIVING THE DYSREGULATED INFLAMMATION ASSOCIATED WITH SLE. HOWEVER, WE LACK A COMPREHENSIVE UNDERSTANDING OF THE MECHANISMS INVOLVED IN DISTINGUISHING SELF- VERSUS NON-SELF RNA AND HOW ALTERED CELLULAR RESPONSES TO SELF-RNA MIGHT PROMOTE AUTOIMMUNITY. NUMEROUS SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) WITHIN THE INNATE IMMUNE SIGNALING PATHWAY HAVE BEEN ASSOCIATED WITH SLE PATHOGENESIS. ONE SNP, RS1990760, IS IN INTERFERON-INDUCED HELICASE C DOMAIN- CONTAINING PROTEIN 1 (IFIH1), AND RESULTS IN AN ALANINE TO THREONINE SUBSTITUTION AT AMINO ACID 946 (A946T), WHICH HAS BEEN ASSOCIATED WITH SLE AND IDENTIFIED AS A CRITICAL ALLELE TO DISEASE PATHOGENESIS. OUR PREVIOUS WORK REVEALED THAT HEALTHY INDIVIDUALS EXPRESSING THIS IFIH1 RISK VARIANT (IFIH1T946) EXHIBITED A BASAL INTERFERON- STIMULATED GENE (ISG) SIGNATURE, WHICH WAS RECAPITULATED IN AN IFIH1T946 (IFIH1R) KNOCK-IN MOUSE MODEL. IN AN INDUCED SLE MURINE MODEL, IFIH1R MICE DISPLAYED PERSISTENT ELEVATED ANTI-NUCLEAR AUTOANTIBODIES LINKING THIS TO THE LOW GRADE INTERFERONOPATHY. HOWEVER, THE CELLULAR AND MOLECULAR MECHANISMS BY WHICH IFIH1R PROMOTES SLE REMAIN UNDEFINED. HEREIN, WE WILL FOCUS ON THREE AIMS: 1) EXAMINING HOW IFIH1R ENHANCES INFLAMMATION, 2) DETERMINING IF IFIH1R PROMOTES SLE PATHOGENESIS IN A MURINE MODEL, AND 3) DETERMINING HOW HUMAN IFIH1R ALTERS THE IFN SIGNATURE AND IMMUNE CELL EFFECTOR FUNCTIONS IN HEALTHY INDIVIDUALS AND IN SLE PATIENTS. DATA GENERATED FROM THIS APPLICATION WILL GIVE UNDERSTANDING INTO THE MECHANISMS THAT DRIVE INFLAMMATION THAT PROMOTES SLE PATHOGENESIS, WHICH WILL HELP STRATIFY PATIENTS AND STEER THERAPEUTIC STRATEGIES STEMMING FROM CELLULAR PATHWAYS IN SLE PATHOGENESIS.

DOES INSULIN SENSITIVITY IMPACT THE POTENTIAL OF METFORMIN TO SLOW AGING?

DETERMINING THE CONTEXT SPECIFICITY OF METFORMIN TREATMENT ON MUSCLE MITOCHONDRIA AND HEALTHSPAN - SUMMARY METFORMIN, THE MOST WIDELY PRESCRIBED MEDICATION FOR TREATING TYPE 2 DIABETES, IS INCREASINGLY RECOGNIZED FOR HEALTHSPAN EFFECTS THAT RESEMBLE EXERCISE. THE BENEFICIAL EFFECTS OF METFORMIN, LIKE AEROBIC EXERCISE, APPEAR TO BE MEDIATED THROUGH AN ENERGETIC AND/OR REDOX STRESS MECHANISM, RAISING THE PROSPECT THAT THE TWO APPROACHES COULD EXERT ADDITIVE OR EVEN SYNERGISTIC EFFECTS. SURPRISINGLY, OUR RECENTLY PUBLISHED CLINICAL TRIAL SHOWED THAT METFORMIN INHIBITS THE BENEFICIAL EFFECTS OF AEROBIC EXERCISE TRAINING (AET) ON SKELETAL MUSCLE MITOCHONDRIAL FUNCTION AND WHOLE-BODY INSULIN SENSITIVITY. INTERESTINGLY, SUBJECTS WHO ENTERED THE STUDY WITH THE HIGHEST MITOCHONDRIAL COMPLEX I SUPPORTED OXPHOS FUNCTION AND INSULIN SENSITIVITY WERE THE MOST NEGATIVELY AFFECTED BY METFORMIN TREATMENT. HOW METFORMIN INHIBITS THE POSITIVE EFFECTS OF AET, WHY THIS EFFECT IS MOST PRONOUNCED IN THOSE WITH THE HIGHEST MITOCHONDRIAL FUNCTION, AND HOW THESE INTERACTIONS ULTIMATELY IMPACT HEALTHSPAN AND LIFESPAN ARE UNKNOWN. THE HYPOTHESIS OF THIS PROPOSAL IS THAT THE EFFECTS OF METFORMIN ON HEALTHSPAN AND LIFESPAN ARE CONTEXT SPECIFIC; BENEFICIAL IN THE CONTEXT OF LOW ENERGY DEMAND/MITOCHONDRIAL CAPACITY BUT DETRIMENTAL IN THE CONTEXT OF HIGH ENERGY DEMAND/MITOCHONDRIAL CAPACITY. TO TEST THIS HYPOTHESIS, THE STUDY WILL LEVERAGE A RAT MODEL WITH DIVERGENT SELECTION FOR INTRINSIC AEROBIC CAPACITY, REFERRED TO AS HIGH CAPACITY AND LOW CAPACITY RUNNERS (HCR/LCR). BY SELECTING FOR MAXIMAL TREADMILL RUNNING CAPACITY, LCR AND HCR RATS DIVERGED IN INTRINSIC MITOCHONDRIAL FUNCTION, LIFESPAN AND METABOLIC TRAITS THAT INCREASE OR DECREASE RISK FOR CHRONIC DISEASE. CHANGES IN MITOCHONDRIAL FUNCTION WILL BE ASSESSED USING EX VIVO RESPIROMETRY THAT MEASURES THE INTERPLAY AMONG THREE THERMODYNAMIC FORCES. FURTHER, THE PROPOSAL USES TARGETED KINETIC AND QUANTITATIVE MITOCHONDRIAL PROTEOMICS TO UNDERSTAND APPROPRIATE OR ABERRANT CELLULAR REMODELING, AND NOVEL IN VIVO IMAGING APPROACHES TO UNDERSTAND CHANGES IN MITOCHONDRIAL MORPHOLOGY AND DYNAMICS. THE SPECIFIC AIMS ARE TO: 1) ESTABLISH IF MITOCHONDRIAL CHANGES TO METFORMIN TREATMENT ARE CONTEXT SPECIFIC, 2) ESTABLISH IF THE EFFECTS OF METFORMIN ON ADAPTATIONS TO AEROBIC EXERCISE TRAINING ARE CONTEXT SPECIFIC, AND 3) DETERMINE WHETHER THE BENEFICIAL EFFECTS OF METFORMIN ON HEALTHSPAN AND LIFESPAN ARE CONTEXT SPECIFIC. IT IS EXPECTED THAT WITH METFORMIN TREATMENT, REMODELING OF MITOCHONDRIA WILL BE CONSISTENT WITH IMPROVED OUTCOMES IN LCR RATS, BUT HAVE NO EFFECT OR WILL BE DETRIMENTAL IN HCR, WITH OR WITHOUT EXERCISE TRAINING. FURTHER IT IS EXPECTED THAT METFORMIN WILL EXTEND HEALTHSPAN AND LIFESPAN IN LCR RATS, BUT NOT HCR RATS. SUCCESSFUL COMPLETION OF THESE AIMS WILL REVEAL THE IMPORTANCE OF CONTEXT SPECIFICITY ON METFORMIN ACTION AND THE MECHANISMS UNDERLYING ITS POSITIVE AND POTENTIALLY NEGATIVE IMPACTS ON HEALTHSPAN AND LIFESPAN. THIS INFORMATION IS CRITICAL GIVEN THE EVER EXPANDING OFF-TARGET USE OF METFORMIN IN HEALTHY INDIVIDUALS WITHOUT CHRONIC DISEASE AND/OR OVERT METABOLIC DYSFUNCTION. RESULTS FROM THIS PROJECT WILL HELP INFORM WHO CAN BENEFIT FROM METFORMIN TREATMENT, AND MORE IMPORTANTLY, WHO SHOULD AVOID IT.

UNDERSTANDING THE ROLES TYPE I INTERFERON AND TH17 PLAY IN NEUROMYELITIS OPTICA AND OTHER AUTOIMMUNE DISEASES.

INTERROGATING THE INTERSECTION BETWEEN DIET AND OCULAR AUTOIMMUNITY - 5-8% OF THE US POPULATION SUFFERS FROM AT LEAST ONE AUTOIMMUNE DISEASE. AN UPWARD TRAJECTORY HAS OCCURRED IN THE LAST FEW DECADES, IMPLICATING DIET, LIFESTYLE, ENVIRONMENT, AND IMPROVED DIAGNOSTICS. THE CONTRIBUTIONS OF DIET TO THIS INCREASED INCIDENCE HAVE BEEN ATTRIBUTED TO THE EXCESSIVE CONSUMPTION OF ULTRA- PROCESSED FOODS THAT DRIVE SYSTEMIC INFLAMMATION. JUST AS POOR DIETARY HABITS CAN COMPROMISE HEALTH, DISEASES CAN POTENTIALLY BE TREATED OR PREVENTED BY DIETS THAT PROMOTE AND RESTORE METABOLIC HOMEOSTASIS. CONSISTENT WITH THIS NOTION, WE HAVE DEMONSTRATED THAT A WELL-FORMULATED KETOGENIC DIET (KD), CONTAINING MEDIUM CHAIN TRIGLYCERIDES AS THE PRIMARY SOURCE OF FAT, CAN MITIGATE THE VISUAL AND MOTOR DEFICITS IN A MOUSE MODEL OF AUTOIMMUNITY CALLED MOG-EAE. THIS MODEL RECONSTITUTES MANY OF THE SIGNATURE OCULAR AND MOTOR PATHOLOGIES EXPERIENCED BY PATIENTS WITH MULTIPLE SCLEROSIS (MS) AND NEUROMYELITIS OPTICA. THE GOALS OF THIS APPLICATION ARE: (1) TO IDENTIFY THE IMMUNO-MODULATORY MECHANISMS BY WHICH A WELL-FORMULATED KD PRESERVES METABOLIC HOMEOSTASIS, GUT HEALTH, AND IMMUNE TOLERANCE, (2) TO DETERMINE IF A KD CAN SERVE AS AN ADJUVANT TO ENHANCE THE VISION-SPARING CAPACITY OF EXISTING MS TREATMENTS AND (3) TO DETERMINE IF THE KD CAN REPAIR DAMAGED NERVES BY PROMOTING REMYELINATION. OUR LEADING HYPOTHESIS IS THAT A WELL-FORMULATED KD PROMOTES IMMUNE TOLERANCE AND NEUROPROTECTION BY CREATING A SYSTEMIC ANTI-INFLAMMATORY MILIEU. SPECIFIC AIM 1 WILL IDENTIFY THE MECHANISMS LINKING GUT INTEGRITY AND PLASMA FATTY ACIDS TO OPTIC NEURITIS, A PAINFUL AND OFTEN BLINDING INFLAMMATION OF THE OPTIC NERVE EXPERIENCED BY MS PATIENTS. SPECIFIC AIM 2 WILL DETERMINE IF THE KD CAN ENHANCE THE VISION-SPARING CAPACITY OF SEVERAL CURRENT MS TREATMENTS. SPECIFIC AIM 3 WILL IDENTIFY NOVEL IMMUNOLOGICAL MECHANISMS THAT MEDIATE THE EFFICACY OF THE KD DURING AUTOIMMUNE CHALLENGE. THIS WORK COMBINES PSYCHOPHYSICAL MEASUREMENTS OF VISION, A NOVEL REPORTER MOUSE, GUT PERMEABILITY ASSAYS, HIGH RESOLUTION LIPIDOMICS, ADOPTIVE TRANSFER ASSAYS, AND BLOOD ANALYSES FOR MARKERS OF INFLAMMATION AND METABOLIC/CARDIOVASCULAR STATUS. THIS WORK IS DISTINGUISHED FROM PREVIOUS STUDIES BY COMPARING THE ANATOMIC-SPECIFIC EFFECTS OF THE KD AT EACH OF THE PRIMARY SITES OF MS LESIONS (OPTIC NERVE, SPINAL CORD, AND BRAIN). THESE STUDIES ARE DESIGNED TO OVERCOME THE SERIOUS ADVERSE EFFECTS ASSOCIATED WITH CURRENT MS TREATMENTS BY EMPOWERING PATIENTS WITH A READILY-IMPLEMENTABLE DIETARY STRATEGY TO PREVENT OR REDUCE FLARE UPS OF OPTIC NEURITIS AND OTHER DEBILITATING SEQUELAE. ADDITIONALLY, THESE FINDINGS WILL PROVIDE A FRAMEWORK TO FACILITATE INTERPRETING OUTCOMES FROM THE NUMEROUS CLINICAL TRIALS CURRENTLY TESTING THE KD ACROSS A RANGE OF DISEASES.

A NOVEL ROLE FOR OXIDIZED LIPID MEDIATORS AS EFFECTORS OF MUSCLE ATROPHY AND WEAKNESS IN AGING - ABSTRACT. SARCOPENIA, THE LOSS OF MUSCLE MASS AND FUNCTION WITH AGE, IS A UNIVERSAL PROBLEM IN THE GROWING ELDERLY POPULATION. TO DESIGN EFFECTIVE INTERVENTIONS WE NEED TO BETTER UNDERSTAND THE MECHANISM(S) RESPONSIBLE FOR INITIATION AND PROGRESSION OF MUSCLE ATROPHY AND WEAKNESS IN AGING. STUDIES FROM OUR LAB AND OTHERS HAVE SHOWN THAT LOSS OF INNERVATION IS A KEY DRIVER OF MUSCLE ATROPHY WITH AGE. THE GOAL OF THIS PROPOSAL IS TO TEST A NOVEL HYPOTHESIS THAT BIOACTIVE LIPID MEDIATORS (OXYLIPINS AND OXIDIZED PHOSPHOLIPIDS (OXPL)), ARE PRIMARY EFFECTORS FOR MUSCLE ATROPHY AND WEAKNESS. OUR HYPOTHESIS IS STRONGLY SUPPORTED BY OUR DATA SHOWING THAT DENERVATION INDUCES ACTIVATION OF PHOSPHOLIPASE A2 (CPLA2), RELEASING ARACHIDONIC ACID (AA) FROM MUSCLE MEMBRANES THAT CAN PROMOTE GENERATION OF OXIDIZED LIPIDS, EITHER NON-ENZYMATICALLY OR VIA 12/15 LIPOXYGENASE (ALOX15) DEPENDENT GENERATION. WE HAVE ALSO SHOWN THAT DENERVATION-INDUCED MUSCLE LOSS IS DECREASED WHEN AA RELEASE AND OXIDIZED LIPIDS ARE BLOCKED BY INHIBITION OF CPLA2 OR ALOX15, OR BY SCAVENGING OF LOOH USING LIPROXSTATIN-1 OR GPX4TG MICE, THUS SUPPORTING OXPL/OXYLIPINS AS A CRITICAL MECHANISTIC LINK BETWEEN DENERVATION AND MUSCLE WASTING. HOWEVER, THE MECHANISMS BY WHICH OXPL/OXYLIPINS CAUSE MUSCLE ATROPHY HAVE NOT BEEN DEFINED. BASED ON PREVIOUSLY IDENTIFIED TARGETS OF THESE LIPID MEDIATORS, WE ARE SPECIFICALLY TESTING THE HYPOTHESIS THAT OXPL/OXYLIPINS INDUCED BY DENERVATION CAUSE DAMAGE TO MEMBRANES, PROMOTE MITOCHONDRIAL CHANGES AND ACTIVATE PROTEOLYTIC AND CELL DEATH PATHWAYS TO INDUCE AGE-RELATED MUSCLE ATROPHY. IN AIM 1, WE WILL DEFINE THE EFFECT OF MODULATING OXYLIPINS ON ATROPHY RELATED TARGETS BY INHIBITING GENERATION OF OXPL/OXYLIPINS (USING CPLA2KO AND ALOX15KO MICE) AND BY ALTERING REDUCTION OF LIPID HYDROPEROXIDES (USING GPX4/TG AND MUSCLE SPECIFIC GPX4KO MICE AND TREATMENT WITH LIPROXSTATIN-1) ON MEMBRANE OXIDATIVE DAMAGE, MITOCHONDRIAL FUNCTION, MUSCLE DEGRADATIVE AND CELL DEATH PATHWAYS AND MUSCLE MASS AFTER DENERVATION. THESE EXPERIMENTS WILL IDENTIFY THE PRIMARY OXYLIPINS PRODUCED IN DENERVATED MUSCLE AND IDENTIFY THE CRITICAL TARGETS OF OXYLIPINS THAT LEAD TO MUSCLE ATROPHY AND WEAKNESS. IN AIM 2, WE WILL MEASURE THE EFFECT OF KEY OXYLIPINS IDENTIFIED IN AIM 1 IN VITRO IN C2C12 MUSCLE CELLS ON OXIDATIVE DAMAGE, MITOCHONDRIAL FUNCTION, PROTEIN DEGRADATION PATHWAYS AND MUSCLE FIBER DIAMETER. THESE EXPERIMENTS WILL PROVIDE NEW INFORMATION ON THE EFFECT OF SPECIFIC OXYLIPINS ON MUSCLE METABOLISM AND MITOCHONDRIAL FUNCTION. FINALLY, IN AIM 3, WE WILL TEST WHETHER INHIBITING OXYLIPIN GENERATION IN VIVO IN MUSCLE SPECIFIC ALOX15KO MICE OR REDUCING LEVELS OF LIPID HYDROPEROXIDES (LOOH) IN MICE WITH ELEVATED LEVELS OF GPX4 EXPRESSION CAN PROTECT AGAINST AGE-RELATED MUSCLE ATROPHY IN VIVO IN AGING MICE. WE PREDICT THAT REDUCED GENERATION OF OXPL/OXYLIPINS AND ENHANCED DETOXIFICATION OF LIPID HYDROPEROXIDES (LOOH) WILL MODIFY THE ATROPHY TARGETS OUTLINED IN AIM 1, REDUCING MUSCLE ATROPHY AND WEAKNESS IN AGING MICE. OVERALL, THESE EXPERIMENTS WILL BE THE FIRST TO INVESTIGATE THE ROLE OF OXYLIPINS IN SARCOPENIA AND THEIR POTENTIAL AS A TARGET FOR INTERVENTION IN MUSCLE LOSS AND WEAKNESS.

SEX DIVERGENCE AND CELL SPECIFICITY OF AGE-RELATED HIPPOCAMPAL DNA MODIFICATIONS

MOTRPAC: UC PRECLINICAL ANIMAL STUDY SITE

CHARACTERIZATION OF A DISTINCT NKT SUBSET AND ITS ROLE IN INFLUENZA RESPONSES

CARDIOVASCULAR RISK OF ANTIRETROVIRAL THERAPY DRUGS IN HIV - PROJECT SUMMARY ALTHOUGH ANTIRETROVIRAL THERAPY DRUGS (ART) HAVE PREVENTED HIV PROPAGATION AND INCREASED LIFE EXPECTANCY OF PEOPLE WITH HIV (PWH), THE RATE OF SUDDEN DEATH IN THIS POPULATION IS 2-4-TIMES HIGHER THAN PEOPLE WITHOUT HIV. AUTOPSIES HAVE REVEALED CARDIAC FIBROSIS IN HALF OF THIS HIV PATIENT POPULATION, A LIKELY ETIOLOGY FOR SUDDEN DEATH. THE PROTEASE INHIBITOR CLASS ART (PI-ART) IS LINKED TO CARDIOVASCULAR RISK IN PWH, AND IT IS PLAUSIBLE THAT ART CAN EXACERBATE THE RISK BY INDUCING CARDIAC FIBROSIS. OUR LONG-TERM GOALS ARE TO DETERMINE THE MECHANISM AND THE IMPACT OF ART-INDUCED CARDIAC FIBROSIS IN PWH, AND TO EXPLORE PREVENTIVE STRATEGIES. TRANSFORMING GROWTH FACTOR SS1 (TGFSS1) IS A STRONG PROFIBROTIC CYTOKINE AND PLATELETS CONTAIN ~100 TIMES MORE TGFSS1 THAN OTHER CELLS AND ARE A MAJOR SOURCE OF PLASMA TGFSS1 CONTRIBUTING TO ORGAN FIBROSIS. HIGHER PLASMA TGFSS1 LEVELS AND CARDIAC FIBROSIS ARE OBSERVED IN HIV+ INDIVIDUALS, BUT WHETHER ART FURTHER INCREASES PLASMA TGFSS1 AND CARDIAC FIBROSIS IN PWH IS NOT CLEAR. IN PILOT STUDIES, WE OBSERVED THAT NEWER ART REGIMENS, INCLUDING PI-BOOSTED DOSE OF RITONAVIR (RTV) AND TENOFOVIR, ACTIVATED PLATELETS TO RELEASE TGFSS1, WHICH CAN BE BLOCKED BY CEEFOURIN-1, A SPECIFIC INHIBITOR OF MRP4, A MEMBRANE TRANSPORTER HIGHLY EXPRESSED IN PLATELETS FROM HIV PATIENTS. INJECTION OF A PI-BOOSTED DOSE OF RTV IN TRANSGENIC TG26 HIV MICE (WHICH EXHIBIT MULTIPLE HIV-ASSOCIATED COMORBIDITIES) INCREASED CARDIAC FIBROSIS AND DIASTOLIC DYSFUNCTION ASSOCIATED WITH THE ACCUMULATION OF CD206+ CELLS EXPRESSING ASMA IN THE HEART, PRESUMABLY MACROPHAGES. THESE RESULTS LED TO OUR CENTRAL HYPOTHESIS THAT ART MAY ACTIVATE PLATELETS TO RELEASE TGFSS1 VIA MRP4, WHICH STIMULATES MACROPHAGES TO UNDERGO MESENCHYMAL TRANSITION, INDUCING CARDIAC FIBROSIS. THE OBJECTIVE OF THIS APPLICATION IS TO DETERMINE THE MECHANISM BY WHICH DIFFERENT CLASSES OF ART INDUCE PLATELET TGFSS1 RELEASE AND IDENTIFY THE CELL TYPES TO WHICH TGFSS1 SIGNALS, LEADING TO CARDIAC FIBROSIS. THE FOLLOWING SPECIFIC AIMS WILL ADDRESS THE OBJECTIVE: 1) SCREEN A PANEL OF DIFFERENT CLASSES OF ART, ALONE AND IN COMBINATION, FOR INDUCTION OF PLATELET RELEASE OF TGFSS1 AND IDENTIFY THE MECHANISMS OF THIS PROCESS; 2) DETERMINE WHETHER CONTEMPORARY ART-MEDIATED TGFSS1 RELEASE VIA MRP4 INDUCES CARDIAC FIBROSIS IN VIVO; 3) DETERMINE WHETHER TGFSS1 SIGNALING IN MACROPHAGES LEADS TO MESENCHYMAL TRANSITION AND CARDIAC FIBROSIS. OUR STUDIES WILL CLARIFY THE MECHANISM OF ART-INDUCED TGFSS1 RELEASE FROM PLATELETS AND THE CELL TYPES ON WHICH TGFSS1 SIGNALS, LEADING TO CARDIAC FIBROSIS. WE WILL USE INNOVATIVE TECHNIQUES TO EVALUATE PLATELET ACTIVATION, MEASURE PLASMA TGFSS1 LEVELS, AND ASSESS CELLULAR SIGNALING AND CARDIAC FIBROSIS IN TWO MURINE MODELS OF HIV. FURTHERMORE, OUR RESEARCH WILL EXPLORE THE TRANSLATIONAL POTENTIAL FOR MITIGATING ART-INDUCED CARDIAC FIBROSIS IN HIV MICE WITH ANTI-TGFSS1 AND ANTI-FIBROTIC AGENTS, SUCH AS GALUNISERTIB OR PIRFENIDONE. OUR STUDIES MAY ALSO ELUCIDATE WHETHER TGFSS1 COULD BE A POTENTIAL BIOMARKER OF UNDERLYING ORGAN FIBROSIS IN PWH. IT MAY ALSO LAY THE FOUNDATION FOR BETTER MECHANISTIC UNDERSTANDING AND NOVEL STRATEGIES FOR PREVENTING COMORBIDITIES IN PWH AND FIBROSIS IN OTHER INFECTIOUS DISEASES.

SPECIFICITY AND MOLECULAR DEFINITION OF PATHOGENIC LYMPHOCYTES IN SJOGREN'S SYNDROME

MOLECULAR AND CELLULAR MECHANISMS REGULATING MITOCHONDRIAL SUBPOPULATION DYNAMICS AND FUNCTION IN VIVO

EXPLORING THE THYMIC ORIGIN OF GROUP 2 INNATE LYMPHOID CELLS

DISSECTING THE INTEGRATED MECHANISMS OF PROTEIN TURNOVER TO PREVENT PROTEOSTATIC DECLINE WITH AGING - SUMMARY PROTEOSTATIC MECHANISMS FAIL WITH ADVANCING AGE, RESULTING IN THE ACCUMULATION OF DAMAGED AND DYSFUNCTIONAL PROTEINS. PROTEIN BREAKDOWN AND REPLACEMENT WITH NEWLY SYNTHESIZED PROTEINS (PROTEIN TURNOVER) IS THE PRIMARY MECHANISM TO MITIGATE ACCUMULATION OF DAMAGED PROTEINS OVER TIME. THERE ARE SEVERAL CONFLICTING FINDINGS RELATED TO PROTEIN TURNOVER, AGING, AND TREATMENTS THAT SLOW AGING, WHICH LEAVE THE FIELD WITH FUNDAMENTAL CONTRADICTIONS REGARDING PROTEIN TURNOVER AS A PROTEOSTATIC MECHANISM. THE CURRENT PROJECT SEEKS TO UNDERSTAND THE FUNDAMENTAL MECHANISMS THAT REGULATE PROTEOSTATIC MAINTENANCE SO THAT WE CAN OVERCOME A BARRIER TO TARGETING THIS PILLAR OF AGING TO SLOW AGE-RELATED DECLINE. OUR STUDIES INDICATE THAT: 1) AGING DOES NOT UNIVERSALLY DECREASE PROTEIN TURNOVER, AND IN FACT IT INCREASES THE TURNOVER OF MANY PROTEINS, 2) AGING DECEASES THE NUMBER OF PROTEINS THAT TURNOVER (FOR WHICH WE INTRODUCE THE TERM DYNAMIC POOL SIZE), 3) TREATMENTS THAT EXTEND LIFESPAN HAVE NEARLY AN EQUAL NUMBER OF PROTEINS THAT INCREASE AND DECREASE PROTEIN SYNTHESIS, WHICH CONTRADICTS THE NOTION THAT TREATMENTS THAT EXTEND LIFESPAN SLOW PROTEIN SYNTHESIS, AND 4) SOME TREATMENTS THAT INCREASE LIFESPAN DECREASE CELL PROLIFERATION, WHICH BY ITSELF DECREASES PROTEIN TURNOVER IN A NON- DETERMINANT MANNER. TO DATE, THESE STUDIES HAVE BEEN LIMITED TO TISSUE SAMPLES, WHICH HAS RESTRICTED THE UNDERSTANDING OF HOW INDIVIDUAL CELL TYPES WITHIN A TISSUE INFLUENCE OVERALL TISSUE PROTEOSTASIS. TO OVERCOME THESE UNKNOWNS, THE PROPOSED STUDIES USE MOUSE MODELS THAT ALLOW CELL-SPECIFIC ISOLATION OF PROTEINS AND NUCLEI IN SKELETAL MUSCLE AND BRAIN. THE PROJECT WILL USE BOTH DISCOVERY-BASED AND TARGETED PROTEOMICS WITH NOVEL DEUTERIUM OXIDE (D2O) LABELING TO EXAMINE CELL-TYPE-SPECIFIC INDIVIDUAL PROTEIN TURNOVER AND CELL REPLICATION. THROUGH LOSS OF PROTEOSTATIC MAINTENANCE (AGING) AND GAIN OF PROTEOSTATIC MAINTENANCE (TREATMENTS THAT SLOW AGING), WE WILL ADDRESS THE FOLLOWING SPECIFIC AIMS: TO DETERMINE CELL-TYPE SPECIFIC PROTEINS SUSCEPTIBLE TO PROTEOSTATIC DECLINE WITH AGING, TO DETERMINE HOW A TREATMENT THAT INHIBITS MTOR IMPROVES PROTEOSTASIS, AND TO DETERMINE HOW A TREATMENT THAT DOES NOT DIRECTLY INHIBIT MTOR IMPROVES PROTEOSTASIS. THE HYPOTHESES ARE THAT: THE LOSS OF PROTEOSTASIS WITH AGING RESULTS FROM CELL-TYPE SPECIFIC CHANGES IN INDIVIDUAL PROTEIN TURNOVER RATES AND DECREASES IN THE DYNAMIC PROTEIN POOL SIZE, THAT INHIBITING MTOR IMPROVES PROTEOSTATIC MAINTENANCE BY DECREASING CELL PROLIFERATION WHILE INCREASING TURNOVER AND DYNAMIC POOL SIZE OF PROTEINS THAT ARE SUSCEPTIBLE TO PROTEOSTATIC DECLINE, AND THAT A LIFESPAN-EXTENDING TREATMENT THAT DOES NOT DIRECTLY INHIBIT MTOR IMPROVES PROTEOSTATIC MAINTENANCE BY MECHANISMS THAT DO NOT INCLUDE SLOWED CELL PROLIFERATION. PROGRESS TOWARD TARGETING AGE-RELATED PROTEOSTATIC DETERIORATION HAS BEEN HINDERED BY CONTRADICTORY AND PARADOXICAL RESULTS FROM PROTEIN TURNOVER STUDIES. WE EXPECT THAT OUR APPROACHES WILL NARROW THE SCOPE OF PROTEINS SUSCEPTIBLE TO PROTEOSTATIC DECLINE, AND MORE IMPORTANTLY, WILL MAKE SIGNIFICANT ADVANCEMENTS TOWARD STRATEGIES TO TARGET THESE PROTEINS TO MAINTAIN PROTEOSTASIS WITH AGE.

A ROLE FOR MITOCHONDRIAL AMPK SIGNALING IN NEURONS - PROJECT SUMMARY AN EARLY HALLMARK OF ALZHEIMER’S DISEASE (AD) IS A LOSS OF NEURONAL CONNECTIVITY REFLECTED BY DECREASED DENDRITIC SPINE DENSITIES IN THE HIPPOCAMPUS AND CORTEX. COINCIDING WITH THIS SYNAPTIC LOSS, BOTH HUMAN ALZHEIMER’S DISEASE PATIENTS AND ALZHEIMER’S DISEASE ANIMAL MODELS DISPLAY A FRAGMENTED MITOCHONDRIAL PHENOTYPE. OUR RECENT WORK DEMONSTRATED THAT ALZHEIMER’S INDUCED MITOCHONDRIAL FRAGMENTATION OCCURS AS A RESULT OF THE OVERACTIVATION OF THE CAMKK2-AMPK-MFF PATHWAY. EXCITINGLY, PREVENTING AMPK-MEDIATED ACTIVATION OF MFF NOT ONLY BLOCKED MITOCHONDRIAL FRAGMENTATION BUT ALSO ABLATED SPINE LOSS ARGUING FOR A CASUAL LINK BETWEEN MITOCHONDRIAL LOSS AND SPINE MAINTENANCE. THUS, THE GOAL OF THIS PROPOSAL IS TO DETERMINE HOW AMPK GOVERNS MITOCHONDRIAL MORPHOLOGY AND FUNCTION TO COORDINATE SPINE MAINTENANCE IN VIVO. THIS TOPIC IS ESPECIALLY RELEVANT AS REPEATED ACTIVATION OF AMPK, BY DRUGS SUCH AS METFORMIN, IS BEING CONSIDERED AS AN INTERVENTION FOR BOTH NORMAL AGING AND ALZHEIMER’S DISEASE. INTERESTINGLY, UP TO TWELVE UNIQUE AMPK COMPLEXES CAN EXISTS, AND RECENT WORK HAS SHOWN THAT DIFFERENT COMPLEXES CAN HAVE DISTINCT ACTIVATION KINETICS, SUBSTRATES AND SUBCELLULAR LOCALIZATIONS INCLUDING AT THE MITOCHONDRIAL SURFACE (MITOAMPK). OUR OVERALL HYPOTHESES ARE THAT PROLONGED ACTIVATION OF MITOAMPK DRIVES DENDRITIC MITOCHONDRIAL FRAGMENTATION AND REMOVAL INDUCING SPINE LOSS, AND THAT TARGETED ABLATION OF AMPK AT THE MITOCHONDRIAL SURFACE IS SUFFICIENT TO RESCUE AΒ42-INDUCED SPINE LOSS. THE FOCUS OF AIM 1 IS TO DETERMINE THE MOLECULAR COMPOSITION AND CELLULAR DYNAMICS OF MITOAMPK IN WILDTYPE AND AD NEURONS BY USING BIOCHEMICAL, CRISPR/CAS9 KNOCK-IN AND KINETIC PROTEOMICS TECHNIQUES. AIM 2 WILL ESTABLISH THE ROLE OF MITOAMPK ACTIVITY FOR DENDRITIC STRUCTURE AND FUNCTION BY USING IN VIVO 2-PHOTON MICROSCOPY AND ELECTROPHYSIOLOGY TO DETERMINE MITOAMPK’S IMPACT ON DENDRITE BRANCHING, SPINE DYNAMICS, CALCIUM HANDLING AND NEURONAL ACTIVITY IN BOTH WILDTYPE AND AD MOUSE MODELS. AIM 3 WILL INTERROGATE THE CELLULAR CONSEQUENCES OF LONG-TERM AMPK ACTIVATION IN HIPPOCAMPAL NEURONS. WHILE MONITORING MITOCHONDRIA AND DENDRITE STRUCTURE AND FUNCTION, WE WILL GENETICALLY OR PHARMACOLOGICALLY ACTIVATE AMPK IN BOTH WILDTYPE AND AD MOUSE MODELS. THE PROPOSED WORK WILL PROVIDE A GREATER UNDERSTANDING OF THE MECHANISMS RESPONSIBLE FOR LOCAL AMPK ACTIVITY IN NEURONS, AND POTENTIALLY IDENTIFY A DISTINCT AMPK COMPLEX TARGETABLE IN NEURODEGENERATION.

ROLE OF ESTROGEN RECEPTOR-A IN AGING AND SEX-SPECIFIC RESPONSES TO 17A-ESTRADIOL - PROJECT SUMMARY/ABSTRACT SEVERAL PHARMACOLOGICAL COMPOUNDS HAVE SHOWN PROMISE AS INTERVENTIONAL STRATEGIES FOR EXTENDING LONGEVITY. ONE RECENTLY STUDIED COMPOUND SHOWN TO IMPROVE HEALTHSPAN AND LONGEVITY IS 17A-ESTRADIOL (17A-E2). 17A-E2 IS A DIASTEREOMER OF 17SS-ESTRADIOL (17SS-E2) WITH CONSIDERABLY LESS BINDING AFFINITY FOR CLASSICAL ESTROGEN RECEPTORS THAN 17SS-E2. THE NIA INTERVENTIONS TESTING PROGRAM HAS SHOWN THAT 17A-E2 EXTENDS LIFESPAN IN MALE, BUT NOT FEMALE, MICE AT TWO DOSES. THE MECHANISMS THAT PROMOTE SEXUALLY DIVERGENT RESPONSES TO 17A-E2 ARE POORLY UNDERSTOOD AND ARE ALIGNED WITH THE INCREASING RECOGNITION THAT AGING AND THE INCIDENCE OF SPECIFIC DISEASES OFTEN DIFFER BETWEEN THE SEXES. THIS PROJECT WILL EXPLORE POTENTIAL MECHANISMS BY WHICH 17A-E2 MODULATES AGING AND METABOLISM IN A SEX-SPECIFIC MANNER. SUCCESSFUL COMPLETION OF THIS PROJECT WILL DETERMINE: 1) IF ESTROGEN RECEPTOR A (ERA) IS REQUIRED FOR 17A-E2 TO ELICIT BENEFITS ON HEALTHSPAN, DISEASE PATHOLOGY, AND LIFESPAN, 2) IF 17A-E2 MODULATES METABOLIC PARAMETERS PREDOMINANTLY THROUGH HYPOTHALAMIC ERA-MEDIATED SIGNALING, 3) IF THE BENEFICIAL EFFECTS OF 17A-E2 ON METABOLIC PARAMETERS CAN BE MIMICKED BY ACTIVATING HYPOTHALAMIC ERA THROUGH CHEMOGENETIC APPROACHES, AND 4) IF THE ELIMINATION OF ENDOGENOUS ESTROGEN PRODUCTION RENDER FEMALE MICE RESPONSIVE TO 17A-E2-MEDIATED EFFECTS ON METABOLISM, AND IF THIS IS ERA DEPENDENT. WE HYPOTHESIZE THAT 17A- E2 SIGNALS THROUGH ERA IN MALE MICE TO ENHANCE HEALTHSPAN AND LONGEVITY AND THAT THESE EFFECTS ARE AT LEAST PARTIALLY MEDIATED THROUGH THE HYPOTHALAMUS. WE ALSO HYPOTHESIZE THAT FEMALE MICE LACKING ENDOGENOUS ESTROGENS WILL BENEFICIALLY RESPOND TO 17A-E2 IN AN ERA-DEPENDENT MANNER. AIM 1: DETERMINE IF 17A-E2 IMPROVES HEALTHSPAN AND LIFESPAN IN AN ERA-DEPENDENT MANNER. WE WILL EVALUATE THE EFFECTS OF CHRONIC 17A-E2 TREATMENT ON INDICES OF HEALTHSPAN AND LIFESPAN IN WILD-TYPE AND GLOBAL ERAKO MICE. AIM 2: DETERMINE IF 17A- E2 ELICITS METABOLIC BENEFITS THROUGH ERA IN THE HYPOTHALAMUS. WE WILL EVALUATE THE EFFECTS OF 17A-E2 TREATMENT ON METABOLIC AND HEALTHSPAN PARAMETERS IN MICE WITH A HYPOTHALAMUS-SPECIFIC DELETION OF ERA USING STEREOTAXIC DELIVERY OF ADENO-ASSOCIATED VIRUS (AAV) THAT DRIVES EXPRESSION OF CRE IN ERA-FLOX MICE. AIM 3: DETERMINE IF CHEMOGENETIC ACTIVATION OF HYPOTHALAMIC ERA-EXPRESSING CELLS CAN MIMIC THE HEALTH BENEFITS OBSERVED WITH 17A- E2 TREATMENT. WE WILL EVALUATE THE EFFECTS OF STIMULATING HYPOTHALAMIC ERA-EXPRESSING CELLS ON METABOLIC AND HEALTHSPAN OUTCOMES VIA SITE-SPECIFIC EXPRESSION OF AAV GQ-COUPLED DESIGNER RECEPTORS EXCLUSIVELY ACTIVATED BY DESIGNER DRUGS (DREADDS) IN ERA-FLOX MICE. AIM 4: DETERMINE IF THE ELIMINATION OF ENDOGENOUS ESTROGEN PRODUCTION RENDERS FEMALE MICE RESPONSIVE TO 17A-E2 TREATMENT. WE WILL EVALUATE THE EFFECTS OF 17A-E2 TREATMENT ON METABOLIC AND HEALTHSPAN PARAMETERS IN WILD-TYPE AND GLOBAL ERAKO FEMALE MICE UNDERGOING OVARIECTOMY OR 4-VINYLCYCLOHEXENE DIEPOXIDE-INDUCED OVARIAN INSUFFICIENCY. THE ULTIMATE GOAL OF THIS RESEARCH IS TO DEVELOP SEX-SPECIFIC PHARMACOLOGICAL INTERVENTIONS FOR ATTENUATING AGING AND CHRONIC DISEASES.

E2A TURNOVER AND NOTCH-CONTROLLED LYMPHOCYTE DEVELOPMENT

OKLAHOMA ACE: MOLECULAR DECONSTRUCTION OF AUTOIMMUNE DISEASE TO AID CLINICAL TRIAL SUCCESS

MECHANISMS FOR BLOOD CELL ADHESION UNDER FLOW

EARLY EVENTS IN MAMMALIAN B-CELL DIFFERENTIATION

MEIOTIC CENTROMERE BEHAVIOR IN YEAST

CENTER FOR GENOMIC REGULATION - ABSTRACT – OVERVIEW PROPER REGULATION OF GENOMIC STRUCTURE AND FUNCTION IS CENTRAL TO LIFE AND HEALTH. DYSFUNCTION OF CELL DIVISION, CHROMATIN STRUCTURE, AND GENE EXPRESSION ARE COMMON FEATURES AND CAUSES OF HUMAN DISEASE. AS SUCH, UNDERSTANDING GENOMIC REGULATION IS CENTRAL TO ELUCIDATING DISEASE PROCESSES AND DEVELOPING NEW INTERVENTIONS. TECHNOLOGICAL AND ANALYTICAL ADVANCES ARE RAPIDLY CHANGING THE RESEARCH LANDSCAPE IN GENOMICS, EPIGENOMICS, AND TRANSCRIPTOMICS. WHILE A CORE OF GENOMICS-RELATED RESEARCH HAS DEVELOPED AT THE OKLAHOMA MEDICAL RESEARCH FOUNDATION (OMRF), ADDITIONAL INVESTIGATORS AND RESEARCH RESOURCES ARE NEEDED TO BRING GENOMIC REGULATION RESEARCH AT OMRF, AND IN OKLAHOMA MORE BROADLY, TO THE POINT OF BEING A SELF-SUSTAINING COMMUNITY THAT IS COMPETITIVE IN THE FIELD. WHILE EFFORTS ARE CURRENTLY SCATTERED ACROSS INSTITUTIONS AND DISEASE-FOCUSED RESEARCH EFFORTS, THE CENTER FOR GENOMIC REGULATION (CGR) WILL BE A NEXUS FOR THE STATE OF OKLAHOMA. THE GOAL OF THE CGR IS TO BUILD A FOCUSED RESEARCH COMMUNITY THAT PERFORMS COLLABORATIVE RESEARCH AND EFFECTIVE TRAINING. WE DEFINE GENOMIC REGULATION AS STUDIES EXAMINING CHROMATIN, DNA MODIFICATIONS, GENOME STRUCTURE, AND TRANSCRIPTION. THE CGR WILL SUPPORT FOUR ACCOMPLISHED EARLY STAGE INVESTIGATOR (ESI) ASSISTANT PROFESSOR- LEVEL FACULTY AT OMRF AS RESEARCH PROJECT LEADERS (RPLS). THESE RPLS HAVE STELLAR TRAINING RECORDS AND HIGH POTENTIAL FOR SUCCESSFUL AND INDEPENDENT GENOMIC REGULATION-RELATED RESEARCH CAREERS. WITH THE STRATEGIC EMPHASIS ON GENOMICS AT OMRF AND GENOMIC REGULATION RESEARCH REPRESENTED ACROSS RESEARCH PROGRAMS AT OMRF, A STEADY POOL OF FUTURE RPLS IS ASSURED. THE CGR WILL PROVIDE MENTORSHIP AND RESEARCH RESOURCES FOR THESE EARLY CAREER INVESTIGATORS AND SUPPORT THE DEVELOPMENT OF THEIR INDEPENDENT CAREERS TO R01 OR R01- EQUIVALENT FUNDING. THE CGR WILL BE A HUB FOR ADVANCED RESEARCH ACROSS ORGAN AND DISEASE DOMAINS, INTEGRATING GENOMIC ANALYSIS TECHNOLOGIES AND DATA SCIENCE CAPABILITIES. THESE GOALS WILL BE ACCOMPLISHED THROUGH THE FOLLOWING SPECIFIC AIMS: AIM 1: SUPPORT PROMISING RESEARCH PROJECT LEADERS TO SCIENTIFIC INDEPENDENCE. AIM 2. PROMOTE NOVEL GENOMIC REGULATION RESEARCH THROUGH MENTORING THE RESEARCH PROJECT LEADERS. AIM 3: INTEGRATE EXISTING AND NEW RESOURCES THROUGH THE EPIGENOMICS AND ARTIFICIAL INTELLIGENCE CORES TO SUPPORT GENOMIC REGULATION RESEARCH. AIM 4: PROMOTE AN ENVIRONMENT THAT SUPPORTS THE SUCCESS AND DEVELOPMENT OF AN INDEPENDENT AND SUSTAINABLE CENTER IN GENOMIC REGULATION.

SIGNALING PATHWAYS THAT REGULATE SYNAPTIC TRANSMISSION

SCIENCE IN A CULTURE OF MENTORING

UNDERSTANDING EARLY EVENTS IN LUPUS AUTOIMMUNITY TO AID PREVENTION

INNATE IMMUNITY AND AUTOANTIBODIES IN THE PATHOGENESIS OF SJOGREN'S SYNDROME

MITOCHONDRIAL DYNAMICS IN RPE HOMEOSTASIS AND DISEASE

CONTRIBUTIONS OF AUTOPHAGY-RELATED GENES IN LUPUS - ABSTRACT LUPUS OR SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS A POTENTIALLY FATAL AUTOIMMUNE DISEASE WITH A SUBSTANTIAL GENETIC BASIS. SLE IS CHARACTERIZED BY ABNORMAL T- AND B-CELL RESPONSES, PRODUCTION OF NUMEROUS PATHOGENIC AUTOANTIBODIES, AND IMMUNE COMPLEX DEPOSITION, LEADING TO VARIOUS CLINICAL MANIFESTATIONS INCLUDING MULTI-ORGAN DAMAGE (E.G., KIDNEYS, SKIN). SLE HAS DISPROPORTIONATE IMPACTS BASED ON GENDER AND ETHNICITY. APPROXIMATELY 90% OF THOSE AFFECTED ARE WOMEN. IN ADDITION, SLE HAS A 3-5-FOLD HIGHER PREVALENCE IN ASIAN, AFRICAN-AMERICAN, AND HISPANIC INDIVIDUALS COMPARED TO THOSE WITH EUROPEAN ANCESTRY, AS WELL AS MORE SEVERE CLINICAL MANIFESTA- TIONS, INCLUDING ORGAN DAMAGE (E.G., NEPHRITIS). EVIDENCE FROM GENETICS, CELL BIOLOGY, AND ANIMAL MODELS SUGGESTS THAT AUTOPHAGY, A MAJOR PATHWAY FOR ORGANELLE AND PROTEIN TURNOVER, PLAYS A PIVOTAL ROLE IN SLE PATHOGENESIS. AUTOPHAGY IS A HIGHLY CONSERVED LYSOSOME-MEDIATED CATABOLIC PROCESS THAT REMOVES UNWANTED CYTOPLASMIC COMPONENTS (E.G., LONG-LIVED AND/OR MISFOLDED PROTEINS) AND DAMAGED ORGANELLES. THIS PROCESS MAINTAINS CELLULAR HOMEOSTASIS AND SURVIVAL UNDER METABOLIC STRESS. RECENT GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE LINKED AUTOIMMUNE DISEASES, INCLUDING SLE, TO AUTOPHAGY-RELATED GENES. HOWEVER, THERE IS CURRENTLY A HUGE GAP IN DEFINING CAUSAL RELATIONSHIPS BETWEEN ASSOCIATED VARIANTS AND MOLECULAR MECHANISMS UNDERLYING SLE. THIS LACK OF KNOWLEDGE ABOUT PATHOGENIC EFFECTS UNDERLYING GWAS SIGNALS HINDERS TRANSLATING GWAS DISCOVERIES INTO DIAGNOSIS AND TREATMENT. TO ADDRESS THIS ISSUE, WE SELECTED SIX HIGHLY RANKED, WELL-REPLICATED LOCI; FOUR OF THEM WERE INITIALLY DISCOVERED IN ASIAN POPULATIONS, TWO WE PREVIOUSLY REPORTED AS NOVEL LOCI FOR SLE SUSCEPTIBILITY. WE HAVE ASSEMBLED A RESEARCH TEAM WITH THE EXPERTISE AND RESOURCES TO DISCOVER AND MECHANISTICALLY CHARACTERIZE FUNCTIONAL VARIANTS LINKED TO SLE. WE HYPOTHESIZE THAT AT EACH GWAS LOCUS, BOTH COMMON AND RARE VARIANTS CONTRIBUTE TO SLE RISK BY AFFECTING EXPRESSION OF AUTOPHAGY GENES THROUGH TISSUE-SPECIFIC REGULATORY ELEMENTS. AIM 1 WILL IDENTIFY SLE-PREDISPOSING VARIANTS USING A COMPREHENSIVE IMPUTATION-BASED ANALYSIS OF COMBINED NOVEL RESEQUENCING DATA FROM SLE PATIENTS AND CONTROLS IN ADDITION TO OUR PREVIOUS FINE-MAPPING OF FOUR ETHNICALLY DIVERSE POPULATIONS. FUNCTIONAL VARIANTS WILL BE PRIORITIZED BY ALLELE-SPECIFIC EXPRESSION, OPEN CHROMATIN, AND 3D INTERACTION DATA FROM IMMUNE CELLS. WE WILL ELUCIDATE GENETIC AND CLINICAL HETEROGENEITY OF SLE BY ASSESSING ASSOCIATION OF IDENTIFIED VARIANTS WITH SLE AND ITS CLINICAL MANIFESTATIONS AND AUTOANTIBODY PROFILES. ASSOCIATED VARIANTS, ESPECIALLY IMPUTED AND RARE VARIANTS, WILL BE VALIDATED THROUGH FOLLOW-UP GENOTYPING. AIM 2 WILL DEFINE THE MECHANISTIC AND FUNCTIONAL CONSEQUENCES OF SLE-PREDISPOSING VARIANTS USING APPROPRIATE FUNCTIONAL ASSAYS, INCLUDING CRISPR-BASED GENE KNOCK-OUT AND BASE-EDITING IN CELL LINES (JURKAT, TOLEDO, PODOCYTE, HEK293) AND PRIMARY IMMUNE CELLS (T- AND B-CELLS) RELEVANT TO SLE. THUS, WE WILL UNCOVER FUNCTIONAL VARIANTS UNDERLYING SLE ASSOCIATION SIGNALS, AND ILLUMINATE MECHANISMS BY WHICH THESE VARIANTS CONTRIBUTE TO LUPUS AND ITS CLINICAL MANIFESTATIONS. THROUGH THIS APPROACH, OUR ANALYSIS SHOULD YIELD NOVEL TARGETS FOR POTENTIAL CLINICAL INTERVENTIONS.

SEX CHROMOSOMAL REGULATION OF HIPPOCAMPAL MICROGLIAL ACTIVATION WITH ALZHEIMER'S DISEASE AND AGING - ABSTRACT SEX AND AGE ARE THE PRIMARY RISK FACTORS FOR ALZHEIMER’S DISEASE (AD), THE MOST COMMON FORM OF DEMENTIA. AFTER DECADES OF FAILED CLINICAL TRIALS FOR THE TREATMENT OF ALZHEIMER’S DISEASE (AD), THERE IS AN URGENT NEED FOR CREATIVE APPROACHES TO UNCOVER NEW THERAPEUTIC TARGETS. WHILE WOMEN EXPERIENCE A GREATER PREVALENCE, MORE SEVERE NEUROPATHOLOGY, AND GREATER COGNITIVE DECLINE WITH AD, MEN DIAGNOSED WITH AD PROGRESS MORE QUICKLY TO DEATH. HOWEVER, LITTLE IS KNOWN ABOUT THE MECHANISMS (WHETHER HORMONAL OR SEX CHROMOSOMAL) DRIVING THE SEX-BIASED RESPONSE TO AD PATHOLOGY WITH BRAIN AGING. OUR LONG-TERM GOAL IS TO IDENTIFY THE UNDERLYING MECHANISMS GOVERNING THE SEX-BIASED RESPONSE TO AD. RECENT GWAS STUDIES HAVE IDENTIFIED SEVERAL AD RISK LOCI IN GENES EXCLUSIVELY EXPRESSED BY MICROGLIA, SHIFTING THE FIELD TO EXPLORE POTENTIAL CAUSATIVE ROLES OF MICROGLIA IN AD. AS WELL, MICROGLIA SHOW PROFOUND PHENOTYPIC SEX DIFFERENCES WITH AGING AND AD. WE HYPOTHESIZE THAT SEX DIFFERENCES IN MICROGLIAL RESPONSIVITY CONTRIBUTE MECHANISTICALLY TO THE SEX-BIASED DISEASE PROGRESSION SEEN IN AD. ALTHOUGH THE ONSET OF AD CORRELATES TO THE MENOPAUSAL TRANSITION IN WOMEN, HORMONE REPLACEMENT THERAPIES (HRT) HAVE GENERATED MIXED RESULTS. THE FORMERLY UNDER-APPRECIATED ROLE OF SEX CHROMOSOMAL CONTRIBUTIONS HAS RECENTLY COME TO THE FOREFRONT IN AD RESEARCH, WITH A SPECIAL EMPHASIS ON X-ENCODED HISTONE MODIFIERS. THE OBJECTIVE OF THIS STUDY IS TO DETERMINE IF SEX CHROMOSOME COMPLEMENT (XX V. XY), INDEPENDENT OF SEX HORMONES, ALTERS PATHOLOGICAL PROGRESSION AND MICROGLIAL ACTIVATIONAL PROFILES IN AD AND TEST THE HYPOTHESIS THAT X-ENCODED LYSINE-SPECIFIC DEMETHYLASE KDM6A CONTRIBUTES TO THE SEXUALLY DIVERGENT MICROGLIAL RESPONSE TO AD. OUR SPECIFIC AIMS WILL TEST THE FOLLOWING HYPOTHESES: (AIM 1) SEX CHROMOSOME COMPLEMENT ALTERS SURVIVAL AND PATHOLOGICAL PROGRESSION (PLAQUES/TANGLES, MICROGLIOSIS) OF AD; (AIM 2) SEX CHROMOSOMALLY REGULATED DIFFERENCES IN HETEROGENEOUS MICROGLIAL CELL RESPONSES TO AGING AND AD ARE DRIVEN, IN PART, BY ALTERATIONS IN HISTONE MODIFICATIONS (H3K27ME3); (AIM 3) MICROGLIAL X-ENCODED KDM6A EXPRESSION IS SUFFICIENT TO CAUSE SEXUALLY- DIVERGENT MICROGLIAL RESPONSE TO AD THROUGH GENOME-WIDE, TARGETED REMOVAL OF REPRESSIVE H3K27ME3. THE PAIRED PHENOTYPIC AND MULTI-OMIC DATA GENERATED IN THESE STUDIES WILL FACILITATE THE IDENTIFICATION OF SEX- DIFFERENTIALLY REGULATED GENOMIC PROGRAMS THAT CONFER PROTECTION OR RISK TO THE PROGRESSION OF AD IN BOTH SEXES IN ORDER TO PRIORITIZE TARGETS FOR SMALL MOLECULE OR EPIGENOME EDITING FOR THERAPEUTIC INTERVENTION IN AD. THE RESEARCH PLAN IS INNOVATIVE BECAUSE WE INVESTIGATE SEX DIFFERENCES IN AD THROUGH THE LENS OF SEX CHROMOSOMES AND UTILIZE GROUND-BREAKING TRANSCRIPTOMIC, EPIGENOMIC, AND ANALYTICAL TECHNIQUES TO GAIN A PREVIOUSLY UNATTAINABLE RESOLUTION OF MICROGLIA HETEROGENEITY.

ROLE OF ATAD3A IN LYSOSOMAL HOMEOSTASIS AND NEUROGENESIS - PROJECT SUMMARY/ABSTRACT ATAD3A (ATP FAMILY AAA-DOMAIN CONTAINING PROTEIN 3A) IS A MITOCHONDRIAL MEMBRANE PROTEIN THAT IS IMPLICATED IN MITOCHONDRIAL MEMBRANE DYNAMICS. WE DISCOVERED THAT DOMINANT MUTATIONS IN ATAD3A CAUSE A HUMAN NEUROLOGICAL SYNDROME CHARACTERIZED BY EARLY-ONSET PERIPHERAL NEUROPATHY, OPTIC ATROPHY AND MILD BRAIN MALFORMATION. PATIENTS WITH LOSS OF FUNCTION MUTATIONS IN ATAD3A CONTINUE TO BE IDENTIFIED, PRESENTING WITH SEVERE NEURODEVELOPMENTAL DEFECTS, SUPPORTING THE IMPORTANCE OF THIS PROTEIN IN HUMAN HEALTH. HOWEVER, THE ROOT CAUSE OF THIS SYNDROME AT THE CELLULAR AND MOLECULAR LEVELS, AS WELL AS STRATEGIES TO AMELIORATE THE SYMPTOMS REMAIN UNSOLVED ISSUES. OUR LONG-TERM GOAL IS TO DETERMINE THE ROLES OF ATAD3A IN DEVELOPMENT AND IN METABOLIC HOMEOSTASIS AS THE BASIS FOR THERAPIES TO TREAT PATIENTS SUFFERING FROM ATAD3A-ASSOCIATED DISEASES. THE OBJECTIVE OF OUR PROPOSAL IS TO UNCOVER THE MECHANISMS BY WHICH ATAD3A CONTROLS NUTRITION SENSING (I.E. MTORC1), LYSOSOMAL BIOGENESIS AND NEURONAL DEVELOPMENT USING DROSOPHILA AND ATAD3A PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS. OUR CENTRAL HYPOTHESIS IS THAT ATAD3A PLAYS A KEY ROLE IN MTORC1 SIGNALING AND LYSOSOMAL BIOGENESIS THROUGH RAG GTPASE MODULATION, AND THAT ATAD3A-DEPENDENT NUTRITION SENSING AND LYSOSOMAL HOMEOSTASIS ARE REQUIRED FOR PROPER NEUROGENESIS AND DEVELOPMENT BASED ON THE FOLLOWING COMPELLING EVIDENCE. BRIEFLY, USING IP-MASS SPEC AND CO-IP, WE IDENTIFIED ENDOGENOUS BINDING PARTNERS OF ATAD3A, INCLUDING THE LYSOSOMAL PROTEINS RAGD, A GTPASE REQUIRED FOR ACTIVATING MTORC1, AND MIT-TFE PROTEINS, TRANSCRIPTIONAL FACTORS FOR LYSOSOMAL BIOGENESIS. WE FOUND THAT ATAD3A FORMS A COMPLEX WITH ACTIVE RAG GTPASES AND MIT-TFE PROTEINS. THIS FINDING HELPED EXPLAIN OUR DISCOVERY THAT DROSOPHILA BEARING A DOMINANT NEGATIVE ATAD3A MUTATION (R528W) EXHIBIT DEFECTS IN NUTRITION SENSING (IMPLICATING RAG/MTORC1), AND ABERRANTLY ELEVATED LYSOSOMAL CONTENT IN DEVELOPING NEURONS (IMPLICATING MITF). IN DROSOPHILA, WE FOUND THAT ATAD3A NULL MUTATIONS CAUSED EMBRYONIC LETHALITY WITH ABNORMAL PATTERNING AND MORPHOLOGY OF CENTRAL AND PERIPHERAL NEURONS. IN ADDITION, WE FOUND THAT THE SIZES OF BRAIN ORGANOIDS DERIVED FROM THE PATIENT IPSCS ARE SIGNIFICANTLY SMALLER THAN THOSE DERIVED FROM ISOGENIC CONTROLS. WE WILL TEST OUR CENTRAL HYPOTHESIS BY PERFORMING THE FOLLOWING SPECIFIC AIMS: (1) TO DETERMINE HOW ATAD3A REGULATES MTORC1 SIGNALING; (2) TO DETERMINE HOW ATAD3A MUTATIONS LEAD TO ABNORMAL LYSOSOMAL BIOGENESIS IN NEURONS; (3) TO DETERMINE HOW ATAD3A LOSS CAUSES NEUROGENESIS DEFECTS. THESE STUDIES WILL CHARACTERIZE A NOVEL AXIS OF MITOCHONDRIA-LYSOSOMAL-MTORC1 SIGNALING THAT SHOULD REVEAL NOVEL MOLECULAR INSIGHTS INTO THE CELLULAR DEFECTS IN PATIENT NEURONS THAT UNDERLIE ATAD3A-ASSOCIATED NEUROLOGICAL DISEASES. WE ANTICIPATE THE IDENTIFICATION OF POTENTIAL THERAPEUTIC TARGETS FOR NEUROLOGICAL DISEASES ASSOCIATED NOT ONLY WITH ATAD3A MUTATIONS, BUT ALSO WITH DEFECTS IN MITOCHONDRIAL AND LYSOSOMAL HOMEOSTASIS.

PATHOGENIC B CELL AND T HELPER CELL INTERACTIONS IN NEUROMYELITIS OPTICA

DISCOVERY AND CHARACTERIZATION OF NOVEL SEPSIS PROTEOME BIOMARKERS - ABSTRACT THIS PROJECT WILL USE MASS SPECTROMETRY-BASED PROTEOMICS FOR BIOMARKER DISCOVERY AND VALIDATION AND WILL PERFORM EARLY EVALUATION OF STRONG CANDIDATE BIOMARKERS AND BIOMARKER SIGNATURES THAT MIGHT FORM THE FOUNDATION FOR NEW CLINICAL BLOOD TESTS. THESE BIOMARKERS CAN BE USED AS DIAGNOSTIC TOOLS FOR EARLY DETECTION OF SEPSIS AND/OR FACILITATE THE CLINICAL DEVELOPMENT OF SEPSIS THERAPEUTICS. OBJECTIVES: THE FOCUS OF THIS APPLICATION IS ON THE IDENTIFICATION AND INITIAL BIOLOGICAL, ANALYTICAL AND CLINICAL EVALUATION OF BIOMARKERS AND BIOMARKER SIGNATURES FOR SEPSIS THAT WILL BE READY FOR DEFINITIVE ANALYTICAL AND CLINICAL VALIDATION. CENTRAL HYPOTHESIS: DISCOVERY PROTEOMICS USING BLOOD SAMPLES FROM WELL-STANDARDIZED NON-HUMAN PRIMATE SEPSIS MODELS FOLLOWED BY VALIDATION IN CLINICAL SAMPLES HAS THE POTENTIAL TO REVEAL NEW BIOMARKER AND BIOMARKER PANELS FOR EARLY DETECTION, PROGNOSIS AND THERAPY MONITORING OF SEPSIS PATIENTS. RATIONALE: SEPSIS IS A COMPLEX, HETEROGENEOUS DISEASE THAT FREQUENTLY IS COMPLICATED WITH COMORBIDITIES (E.G., CANCER, DIABETES, CARDIOVASCULAR DISEASE) THAT CAN ALTER THE HOST RESPONSES TO PATHOGENS, THUS MAKING IT DIFFICULT TO DETERMINE THE SPECTRUM OF SEPSIS-SPECIFIC BIOMARKERS BY DISCOVERY PROTEOMICS IN CLINICAL PLASMA SAMPLES. WE PROPOSE TO USE UNBIASED PROTEOMICS, COUPLED WITH ROBUST BIOINFORMATICS TO DISCOVER NOVEL PREDICTIVE BIOMARKERS. WE ARE IN A UNIQUE POSITION TO ACHIEVE THESE GOALS, HAVING ACCESS TO WELL-CHARACTERIZED PLASMA SAMPLES FROM HISTORICAL TIME-COURSE EXPERIMENTS USING BABOON MODELS OF GRAM-NEGATIVE OR GRAM-POSITIVE BACTEREMIA – COUPLED WITH ACCESS TO A NIGMS NATIONAL PROTEOMICS RESOURCE EQUIPPED WITH WORLD-CLASS INSTRUMENTATION AND EXPERTISE. FURTHER, QUANTITATIVE PROTEOMICS AND IMMUNOASSAYS WILL BE USED FOR QUALIFICATION AND VALIDATION OF THE NOVEL BIOMARKERS IN HUMAN PLASMA FROM SEPSIS PATIENTS. SPECIFIC AIM 1: IDENTIFICATION AND ORGAN MAPPING OF EARLY STAGE BIOMARKERS DURING THE TIME COURSE OF EXPERIMENTAL SEPSIS IN BABOONS. SPECIFIC AIM 2: VALIDATION AND PERFORMANCE TESTING OF BIOMARKER CANDIDATES IN CLINICAL SAMPLES. IMPACT: OUR SHORT-TERM GOALS ARE TO IDENTIFY BIOMARKERS AND BIOMARKER SIGNATURES FOR SEPSIS IN BABOONS AND PERFORM THE INITIAL BIOLOGICAL, ANALYTICAL AND CLINICAL EVALUATION IN HUMANS USING CAREFULLY STANDARDIZED SAMPLES AND DATASETS. THE LONG-TERM GOAL OF OUR PROJECT IS TO DELIVER NOVEL CANDIDATE BIOMARKERS THAT ARE READY FOR DEFINITIVE ANALYTICAL AND CLINICAL VALIDATION STUDIES. THESE BIOMARKERS WILL ALLOW EARLY DIAGNOSIS AND MONITORING OF SEPSIS PROGRESSION AND WILL HELP PATIENT STRATIFICATION FOR TARGETED THERAPIES.

NOVEL LYMPHATIC GENES THAT REGULATE HEART VALVE DEVELOPMENT AND DISEASE - PROJECT SUMMARY/ABSTRACT VALVES ARE PRESENT WITHIN HEARTS, VEINS AND LYMPHATIC VESSELS TO REGULATE THE FLOW OF BLOOD AND LYMPH. DEFECTS IN THE DEVELOPMENT OR FUNCTIONING OF VALVES COULD LEAD TO DISEASES SUCH AS LYMPHEDEMA AND DEGENERATIVE HEART VALVE DISEASE. VENOUS AND LYMPHATIC (VASCULAR) VALVES ARE COMPOSED OF TWO LAYERS OF ENDOTHELIAL CELLS WITH A THIN LAYER OF COLLAGEN-RICH EXTRA CELLULAR MATRIX (ECM). FEW IF ANY INTERSTITIAL CELLS ARE OBSERVED WITHIN VASCULAR VALVES. IN CONTRAST, CARDIAC VALVES ARE MADE OF TWO MAJOR CELL TYPES: NUMEROUS ECM PRODUCING VALVULAR INTERSTITIAL CELLS (VICS), WHICH ARE SURROUNDED BY A SINGLE LAYER OF VALVULAR ENDOTHELIAL CELLS (VECS). DESPITE SIGNIFICANT DIFFERENCES IN THEIR STRUCTURE AND THE MECHANICAL FORCE THAT THEY EXPERIENCE, CARDIAC AND VASCULAR VALVES SHARE INTERESTING SIMILARITIES. THE HOMEOBOX TRANSCRIPTION FACTOR PROX1 IS NECESSARY FOR THE DEVELOPMENT OF VASCULAR VALVES. PROX1 IS ALSO EXPRESSED IN A SUBSET OF VECS ON THE DOWNSTREAM SIDE OF HEART VALVES. WE HAVE NOW DETERMINED THAT THE DELETION OF PROX1 FROM VECS RESULTS IN THE ACCUMULATION OF PROTEOGLYCANS AND THE CONSEQUENT THICKENING OF HEART VALVES. THUS, PROX1 IN VECS REGULATES THE DEVELOPMENT AND FUNCTIONING OF VICS THROUGH YET UNKNOWN MECHANISMS. BUILDING ON OUR PRELIMINARY DATA WE WILL TEST OUR CENTRAL HYPOTHESIS THAT A PREVIOUSLY UNKNOWN PROX1AFOXC2APDGF-B SIGNALING PATHWAY FROM DOWNSTREAM VECS REGULATES VIC IDENTITY AND, IN TURN, THE ECM COMPOSITION OF VALVES. ABNORMALLY HIGH SYNTHESIS OF PROTEOGLYCANS IS OBSERVED IN SEVERAL VALVE DISORDERS. VALVE DEFECTS ARE OBSERVED IN ~8% IN INDIVIDUALS >65 YEARS OLD AND ~13% IN THOSE WHO ARE >75. PHARMACOLOGICAL APPROACHES TO TREAT THESE VALVE DEFECTS DO NOT EXIST. HENCE, THE OUTCOMES OF OUR PROPOSED RESEARCH ARE EXPECTED TO BE SIGNIFICANT BECAUSE THEY WILL SUBSTANTIALLY ADVANCE OUR INSIGHT INTO THE MOLECULAR AND CELLULAR MECHANISMS OF VEC-VIC CROSSTALK DURING CARDIAC VALVE DEVELOPMENT AND SHED LIGHT ON POTENTIAL THERAPEUTIC STRATEGIES.

HEALTH DISPARITIES AND GENETIC ARCHITECTURE OF LUPUS IN AFRICAN AMERICANS

A 3D HUMAN TISSUE-ENGINEERED LUNG MODEL TO STUDY IMMUNE RESPONSES TO RESPIRATORY SYNCYTIAL VIRUS

FUNCTIONAL EVALUATION OF ESTABLISHED SJOGREN'S SYNDROME IMMUNE RESPONSE LOCI

EFFECTS OF DIETARY RESTRICTION ON AGE-RELATED NEUROPHYSIOLOGICAL ADAPTATIONS: FROM BEHAVIOR TO SINGLE DOPAMINERGIC NEURONS

THE ROLE OF TGF-BETA ACTIVATION AND SIGNALING IN AORTIC STENOSIS PROGRESSION

PDGFRB SIGNALING IN PROGRESSIVE SKIN DISEASE - PROJECT SUMMARY/ABSTRACT SOLVING THE PATHOGENESIS OF RARE GENETIC DISEASES LEADS TO GREATER UNDERSTANDING OF BASIC PHYSIOLOGY AND GENE FUNCTION. IT WAS RECENTLY DISCOVERED THAT GAIN-OF-FUNCTION MUTATIONS IN HUMAN PDGFRB, ENCODING PLATELET-DERIVED GROWTH FACTOR RECEPTOR SS (PDGFRSS), UNEXPECTEDLY ASSOCIATE WITH SYNDROMIC DISEASES INVOLVING THE SKIN. THE PHENOTYPES INVOLVE DERMAL ATROPHY RESEMBLING ACCELERATED AGING OR, PARADOXICALLY, DERMAL THICKENING AND FIBROSIS WITH KELOID-LIKE SCARRING. THESE OBSERVATIONS RAISE IMPORTANT QUESTIONS ABOUT FUNCTIONS OF PDGFRSS IN THE SKIN, INCLUDING THE TARGET CELL TYPES AND SIGNALING PATHWAYS RESPONSIBLE FOR DISEASE. WHETHER DIFFERENT HUMAN PDGFRB MUTATIONS CAUSE DISTINCT DISEASE PHENOTYPES IS UNCLEAR BECAUSE OF LIMITED NUMBERS OF PATIENTS TO DATE AND LACK OF APPROPRIATE GENETIC MODELS. ADDRESSING THESE QUESTIONS WILL SHED NEW LIGHT ON THE ROLE OF PDGFRSS IN THE SKIN. THE OBJECTIVE IS TO IDENTIFY PDGFRSS-REGULATED MECHANISMS CONTROLLING DERMAL CELLS AND THEIR CONTRIBUTION TO SKIN DISEASE AND PHYSIOLOGY. THE CENTRAL HYPOTHESIS IS THAT PDGFRSS IN DERMAL FIBROBLASTS CONTROLS THE BALANCE OF DOWNSTREAM EFFECTORS INCLUDING STATS AND AKT, WHICH ARE CRITICAL FOR THE MAINTENANCE OF HEALTHY SKIN AND ARE IMBALANCED IN DISEASE. AIM 1 WILL USE INDUCIBLE CRE/LOX APPROACHES IN MICE TO INDUCE PDGFRSS ACTIVATING MUTATIONS IN FIBROBLASTS OR DERMAL ADIPOCYTES, WITH COMBINATORIAL DELETION OF STAT FAMILY MEMBERS AND STAT TARGET GENES TO UNCOVER NEW DOWNSTREAM MEDIATORS OF DERMAL FIBROSIS. AIM 2 WILL CHARACTERIZE A NEW MOUSE MODEL WITH A MUTATION FOUND IN KOSAKI OVERGROWTH SYNDROME, AND TEST THE HYPOTHESIS THAT AKT ACTIVATION IS REQUIRED FOR OVERGROWTH WITH PROGRESSIVE LOSS OF DERMAL ADIPOSE TISSUE. AIM 3 WILL CHARACTERIZE A NEW MOUSE MODEL WITH A PENTTINEN PROGEROID SYNDROME MUTATION, AND TEST THE HYPOTHESIS THAT STAT1 IS A CENTRAL MEDIATOR OF THE PROGEROID SKIN PHENOTYPE AND ANTI-GROWTH CHARACTERISTICS OF MUTANT FIBROBLASTS. THIS PROJECT DRAWS ON THE PI’S EXPERTISE IN PDGF SIGNALING TO RESOLVE THE PARADOXICAL CONSEQUENCES OF ELEVATED PDGFRSS SIGNALING IN SKIN DISEASE. THE RESULTS OF THIS PROJECT WILL DEVELOP GENETIC MODELS OF TWO HUMAN DISEASES, BY DISSECTING THE CELL TYPE-SPECIFIC ROLE OF PDGFRSS SIGNALING IN DERMAL FIBROSIS AND DERMAL ADIPOSE TISSUE ATROPHY, AND BY ESTABLISHING THE CONCEPTUAL FRAMEWORK OF PDGFRSS-STAT SIGNALING IN THE SKIN. THIS INFORMATION MAY POINT TO NOVEL THERAPEUTIC STRATEGIES FOR FIBROSIS AND AGING IN CONTEXTS BEYOND THESE RARE GENETIC DISEASES.

COMPLEMENT INHIBITION AS SEPSIS THERAPY

REGULATION OF VENOUS AND LYMPHATIC IDENTITY BY CHROMATIN-REMODELING

CHROMOSOME MOVEMENT IN PROMETAPHASE

MECHANISMS OF APP ECTODOMAIN FUNCTION - PROJECT SUMMARY CELL MEMBRANE PROTEINS REGULATE A MULTITUDE OF CELLULAR PROCESSES THAT ARE VITAL FOR ORGANISMAL HEALTH. THE EXTRACELLULAR DOMAIN IS CRITICAL FOR LIGAND BINDING AND SIGNAL TRANSDUCTION OF THE FULL-LENGTH RECEPTOR. IN ADDITION, APPROXIMATELY 2% OF CELL SURFACE PROTEINS UNDERGO ECTODOMAIN SHEDDING TO RELEASE THEIR EXTRACELLULAR DOMAIN. ECTODOMAIN SHEDDING MAY TERMINATE THE FUNCTION OF THE FULL-LENGTH RECEPTOR AND/OR LEAD TO THE GENERATION OF EITHER A SOLUBLE DECOY RECEPTOR OR AN ACTIVE LIGAND. THE AMYLOID PRECURSOR PROTEIN (APP), A TYPE 1 MEMBRANE PROTEIN CENTRAL TO THE ETIOLOGY OF ALZHEIMER’S DISEASE, IS ONE SUCH CELL SURFACE PROTEIN THAT UNDERGOES ECTODOMAIN SHEDDING TO GENERATE VARIOUS SOLUBLE APP (SAPP) FRAGMENTS. DESPITE THE KNOWN EFFECTS OF THE APP ECTODOMAIN ON CELLULAR AND COGNITIVE FUNCTIONS AND THE POTENTIAL APPLICATIONS TO DISEASE, THE PRECISE MOLECULAR MECHANISMS MEDIATING THE FUNCTION OF APP ECTODOMAIN REMAINS LARGELY ENIGMATIC DUE TO THE FOCUS OF THE FIELD ON THE AMYLOID- SS REGION OF APP. THUS, THE GOAL OF THE LAB IS TO ELUCIDATE THE NORMAL PHYSIOLOGICAL FUNCTIONS OF THE EXTRACELLULAR DOMAIN OF THE AMYLOID PRECURSOR PROTEIN. OUR APPROACHES ARE DESIGNED TO ADDRESS THE MECHANISTIC GAPS IN KNOWLEDGE BY LEVERAGING OUR RECENT DISCOVERY THAT SAPP FUNCTIONS AS A LIGAND FOR THE GABA TYPE B RECEPTOR (GABABR) AND OUR RECENT IDENTIFICATION OF PHOSPHOGLYCERATE MUTASE FAMILY MEMBER 5 (PGAM5), A MITOCHONDRIAL SERINE/THREONINE PROTEIN PHOSPHATASE, AS A NOVEL CANDIDATE INTERACTOR OF THE APP ECTODOMAIN. ONE LINE OF RESEARCH WILL ELUCIDATE THE CELLULAR RESPONSES OF NON-NEURONAL CELLS TO SAPP AND GABA B RECEPTOR MODULATION, IDENTIFY SIGNALING PATHWAYS DOWNSTREAM OF THE SAPP AND GABA B RECEPTOR INTERACTION, AND DETERMINE MECHANISMS INVOLVED IN THE POSITIVE REGULATION OF GABA B RECEPTOR SIGNALING BY SAPP. A SECOND LINE OF RESEARCH WILL INVESTIGATE THE CELLULAR CONDITIONS AND SUBCELLULAR COMPARTMENTS IN WHICH APP AND PGAM5 COME INTO CONTACT, THE MOLECULAR BASIS OF THE APP AND PGAM5 INTERACTION (I.E. BINDING AFFINITIES, CRITICAL BINDING DOMAINS), AND THE CONSEQUENCES OF PGAM5 ON APP PHOSPHORYLATION AND PROTEOLYTIC PROCESSING. THE WORK IN THESE STUDIES WILL GENERATE FUNDAMENTAL KNOWLEDGE OF THE MOLECULAR MECHANISMS UNDERLYING THE PROTEIN-PROTEIN INTERACTIONS OF THE APP ECTODOMAIN AND THE CELLULAR CONSEQUENCES OF THESE INTERACTIONS ACROSS DIFFERENT CELL TYPES.

FUNCTIONAL MECHANISMS OF CAUSAL VARIANTS IN SYSTEMIC LUPUS ERYTHEMATOSUS

MAJOR HISTOCOMPATIBILITY COMPLEX I REGULATION OF MICROGLIAL FUNCTION IN AGING AND ALZHEIMER'S DISEASE - ABSTRACT: SYSTEMIC AND CNS STERILE INFLAMMATION (INFLAMMAGING) ARE PROPOSED DRIVERS OF BRAIN AGING AND NEURODEGENERATIVE DISEASES (E.G., ALZHEIMER’S DISEASE, AD). MICROGLIA, THE RESIDENT CNS MACROPHAGES, ALTER THEIR PHENOTYPE WITH AGING AND AD, BUT HOW THIS REACTIVITY CONTRIBUTES TO, OR PREVENTS, AD DEVELOPMENT AND PROGRESSION IS UNCLEAR. AN OVERARCHING VIEW IN THE FIELD IS THAT MICROGLIOSIS INITIALLY PROTECTS AGAINST ACCUMULATING DAMAGE IN THE AGED BRAIN, BUT WITH TIME AND AD NEUROPATHOLOGY, PERSISTENT HYPERACTIVATION BECOMES DELETERIOUS AND PROMOTES NEURODEGENERATION. IN OUR STUDIES, WE HAVE IDENTIFIED A RELATIVELY UNEXPLORED ASPECT OF NEUROINFLAMMATION WITH AGING AND AD CHARACTERIZED BY UPREGULATION OF THE MAJOR HISTOCOMPATIBILITY COMPLEX I (MHC-I). MICROGLIAL MHC-I IS INDUCED IN HUMANS, MICE, AND OTHER SPECIES WITH AGING, AND OCCURS IN AD PATIENTS AND AD MOUSE MODELS. WE HAVE ALSO DETERMINED THAT ANTIGEN-INDEPENDENT MHC-I RECEPTORS, LEUKOCYTE IMMUNOGLOBULIN-LIKE RECEPTOR SUBFAMILY RECEPTORS (LILR) AND PAIRED IMMUNOGLOBULIN-LIKE TYPE 2 RECEPTORS (PILR), ARE ALSO PRESENT AND INDUCED IN MICROGLIA WITH AGING AND AD. THESE RECEPTORS ARE ALMOST EXCLUSIVELY RESTRICTED TO MICROGLIA IN THE CNS AND CONTAIN EITHER IMMUNORECEPTOR TYROSINE-BASED ACTIVATION OR INHIBITION MOTIFS (PRO- INFLAMMATORY ITAMS AND ANTI-INFLAMMATORY ITIMS, RESPECTIVELY) THAT REGULATE SYK ACTIVITY, A CENTRAL MODULATOR OF MICROGLIAL PHENOTYPE. THUS, IN THE ABSENCE OF, OR IN ADDITION TO, CANONICAL SIGNALING TO T CELL RECEPTORS ON T CELLS, MHC-I COULD CELL-AUTONOMOUSLY REGULATE MICROGLIAL PHENOTYPE THROUGH LILRS/PILRS AND THIS SYK. WE HYPOTHESIZE THAT MHC-I IS A CELL-AUTONOMOUS REGULATORY MECHANISM FOR MICROGLIA THAT PROMOTES A PRO-INFLAMMATORY PHENOTYPE AND ALTERS MICROGLIA FUNCTION. THIS PROINFLAMMATORY MICROGLIAL PHENOTYPE MAY ALSO HELP RECRUIT INFILTRATING T CELLS TO THE BRAIN PARENCHYMA HERE MHC-I COULD SIGNAL DIRECTLY TO T CELLS. USING A NOVEL, TEMPORALLY CONTROLLED AND MICROGLIA-SPECIFIC MHC-I KNOCKOUT MODEL WE WILL: 1) DETERMINE IF MHC-I EXPRESSION REGULATES MICROGLIAL REACTIVITY AND PHAGOCYTIC PHENOTYPES WITH AGING, 2) DETERMINE IF MHC-I SUPPRESSION EXACERBATES PRODROMAL AD PHENOTYPES WHILE SLOWING PROGRESSION AFTER SYMPTOM ONSET, AND 3) DETERMINE IF T CELL INFILTRATION INTO THE BRAIN AND PHENOTYPE WITH AGING AND IN MODELS OF AD IS DEPENDENT ON MICROGLIAL MHC-I. VALIDATION OF MHC-I AS A NOVEL REGULATOR OF MICROGLIAL FUNCTION WILL OPEN NEW RESEARCH AVENUES INTO CONTROLLING MICROGLIAL FUNCTION AND REGULATING T CELL INFILTRATION AND FUNCTION.

AGING AND OXIDATIVE STRESS INFLUENCE SALIVARY GLAND DISEASE IN SJOGREN'S SYNDROME - SJÖGREN’S SYNDROME (SS) IS A CHRONIC AND DEBILITATING SYSTEMIC AUTOIMMUNE DISORDER AFFLICTING MULTIPLE ORGAN SYSTEMS. THE TARGETING OF THE EXOCRINE SALIVARY AND LACRIMAL GLANDS BY AN AUTOIMMUNE AND INFLAMMATORY RESPONSE LEADS TO ORGAN DYSFUNCTION CAUSING REDUCED FLUID SECRETION, WHICH MANIFESTS INTO THE DRY MOUTH AND DRY EYE SYMPTOMS OF THE DISORDER. ALTHOUGH A WIDE AGE RANGE IS REPORTED IN PATIENTS AT DIAGNOSIS, IT IS WELL KNOWN THAT MOST SS PATIENTS ARE OLDER. WHY PHENOTYPIC TRAITS ARE MORE PROMINENT IN OLDER PATIENTS IS UNKNOWN. AGING ORGANS SHOW HEIGHTENED OXIDATIVE STRESS, WHICH IS OFTEN ASSOCIATED WITH DECLINING ORGAN FUNCTION. ALTHOUGH,. SS PATIENTS SHOW EVIDENCE OF INCREASED OXIDATIVE STRESS MARKERS; WHETHER ELEVATED OXIDATIVE STRESS IS CAUSATIVE OR THE OUTCOME OF AN INFLAMMATORY RESPONSE IS UNCLEAR AND CHALLENGING TO INVESTIGATE IN PATIENTS. HENCE BASED ON PUBLISHED LITERATURE AND OUR PRELIMINARY DATA, THIS PROPOSAL WILL TEST THE OVERALL HYPOTHESIS THAT THE COMBINED EFFECTS OF AUTOIMMUNITY AND AGING-ASSOCIATED OXIDATIVE STRESS CONTRIBUTE TO SALIVARY GLAND DISEASE AND DYSFUNCTION IN SS. AIM 1 OF THIS PROPOSAL WILL SPECIFICALLY ADDRESS THE HYPOTHESIS THAT AGING-ASSOCIATED OXIDATIVE STRESS MAKES SALIVARY GLANDS MORE SUSCEPTIBLE TO IMMUNE-MEDIATED DAMAGE. AND IN AIM 2, BY USING A NOVEL MOUSE MODEL SYSTEM, WE WILL TEST THE HYPOTHESIS THAT OXIDATIVE STRESS PER SE IN SALIVARY GLAND EPITHELIAL CELLS IS INSUFFICIENT TO CAUSE SS. THE PRIMARY GOAL OF THIS PROPOSAL IS TO UNDERSTAND THE MECHANISMS BEHIND KEY CLINICAL OBSERVATIONS IN SS PATIENTS: PROMINENCE OF CLINICAL SYMPTOMS AT AN OLDER AGE AND THE POSSIBLE ROLE OF ELEVATED OXIDATIVE STRESS IN THE DISEASE PROCESS. UNDERSTANDING BASIC MECHANISMS LINKING AGING WITH ORGAN DYSFUNCTION IN SS WILL BE ESSENTIAL IN DEVELOPING NOVEL MODALITIES TO TREAT THE DISEASE.

AFRICAN AMERICAN SARCOIDOSIS GENETICS RESOURCE

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION

CELLULAR SENESCENCE AND EPIGENOMIC REMODELING IN OVARIAN AGING

THE REGULATION OF INFLAMMATORY RESPONSES BY CD73

MECHANISMS REGULATING DNA REPLICATION IN THE DEVELOPING VERTEBRATE EMBRYO

PDGF-REGULATED CELL FATE AND DERMAL FIBROSIS

PDGF-REGULATED STEM CELLS AND BONE DISEASE

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION - THIS PROJECT INCLUDES THE RENOVATIONS OF UNDERUTILIZED SPACE AND INSTALLATION OF NEW EQUIPMENT CRITICAL TO EXPAND RESEARCH AND EDUCATION EFFORTS TO IMPROVE THE EVALUATION AND CARE OF NATIVE AMERICAN AND RURAL PATIENTS WITH ARTHRITIS. OSTEOARTHRITIS (OA), THE MOST COMMON ARTHRITIS, IS A CHRONIC, DEBILITATING DISEASE CHARACTERIZED BY PROGRESSIVE LOSS OF JOINT FUNCTION LEADING TO SIGNIFICANT PAIN, MOBILITY LOSS, AND FUNCTIONAL LIMITATION. AN UNRECOGNIZED PANDEMIC, OA IS THE LEADING CAUSE OF CHRONIC DISABILITY IN THE US. UNFORTUNATELY, BIOMARKERS DO NOT EXIST FOR EITHER DIAGNOSIS OR PROGNOSIS. FURTHERMORE, NO DISEASE-MODIFYING THERAPIES EXIST FOR OA, DUE PARTLY TO OUR LACK OF INSIGHT INTO THE UNDERLYING CAUSES OF THE DISEASE AND PROGRESSION, AND LACK OF MARKERS FOR PATIENTS AT HIGHEST RISK OF SEVERE DISEASE/RAPID PROGRESSION. IN THIS PROPOSAL, OMRF REQUESTS SUPPORT FOR THE CREATION OF THE NATIVE AMERICAN AND RURAL ARTHRITIS RESEARCH AND TRAINING CENTER. THE GOAL OF THE CENTER IS TO ADDRESS ARTHRITIS OUTCOME DISPARITIES AND IMPROVE THE LIVES OF NA AND RURAL PATIENTS WITH ARTHRITIS BY (A) INCREASING TRAINING OF TRIBAL HEALTHCARE PROVIDERS IN RHEUMATOLOGIC CONDITIONS DISPROPORTIONATELY AFFECTING NA PATIENTS (B) EXPANDING CLINICAL RESEARCH INFRASTRUCTURE AT OMRF FOCUSED ON NA ARTHRITIS, AND (C) BUILDING A NEW BIOMEDICAL DATA SCIENCE CENTER AT OMRF TO FACILITATE NA-FOCUSED RESEARCH PROJECTS AND PROVIDE TRAINING OPPORTUNITIES IN BIG DATA FOR TRIBAL STUDENTS. THIS CENTER WILL PROVIDE OPTIONS FOR CLINICAL CARE FOR MEMBERS OF ALL TRIBES AND FOR TRIBAL MEMBERS FROM OUTSIDE THE OKLAHOMA TRIBAL JURISDICTIONS, ALONG WITH PATIENTS FROM MEDICALLY UNDERSERVED AREAS WHO MEET HRSA DEFINITION AS RURAL INHABITANTS. STUDENTS, IN-HOUSE STAFF, AND TRIBAL AND RURAL CLINIC STAFF WILL BE PROVIDED WITH CUTTING-EDGE TELEHEALTH RHEUMATOLOGY TRAINING OPPORTUNITIES, AS WELL AS WITH TRAINING IN CLINICAL RESEARCH AND INTEGRATION WITH TRIBAL- AND COMMUNITY-LED RESEARCH PROJECTS, PR OVIDING TRIBAL CLINICIANS WITH THE SKILLS NECESSARY TO PROVIDE EFFECTIVE ARTHRITIS CARE AND TO INCREASE FEDERAL RESEARCH FUNDING TO THE TRIBES AND OUR RURAL COMMUNITIES. TO ADVANCE ARTHRITIS RESEARCH IN NA PATIENTS, RENOVATIONS AND UPGRADES OF FACILITIES WILL BE PERFORMED TO ALLOW FOR THE TREATMENT OF MOBILITY CHALLENGED PATIENTS, INCLUDING THOSE WHO ARE SIGNIFICANTLY OVERWEIGHT. IMPLEMENTATION OF A DIGITAL RADIOLOGY RESEARCH FACILITY AND ASSOCIATED SCIENTIFIC INFRASTRUCTURE FOCUSED ON ARTHRITIS WILL PROVIDE FREE IMAGING OPPORTUNITIES TO TRIBAL AND RURAL CITIZENS, ALLOWING FOR EARLIER DETECTION OF ARTHRITIS PROGRESSION AND NEED FOR MORE AGGRESSIVE INTERVENTIONS. THIS WILL ALLOW US TO DEVELOP THE NEW NATIVE AMERICAN AND RURAL OSTEOARTHRITIS COHORT, BASED AT OMRF, WHICH WILL FOCUS ON IDENTIFYING AND VALIDATING CUTTING-EDGE EARLY DIAGNOSTIC BIOMARKERS FOR OA. TO ENSURE THAT SCIENTISTS ENGAGED IN NA-FOCUSED PROJECTS ARE ABLE TO MEET THE RIGOROUS DATA STORAGE AND PROCESSING DEMANDS OF CUTTING-EDGE CLINICAL RESEARCH PROJECTS TODAY AND IN THE FUTURE, AS WELL AS TO PROVIDE UNIQUE TRAINING OPPORTUNITIES FOR TRIBAL AND RURAL CITIZENS, WE WILL CREATE THE STATE’S FIRST CENTER FOR BIOMEDICAL DATA SCIENCES. THIS CENTER WILL SERVE AS A HUB FOR DATA ANALYTICS EXPERTS WHO WILL WORK TO DEVELOP AND DEPLOY INNOVATIVE DATA SCIENCE SOLUTIONS TO NA PROJECTS. THE CENTER WILL ENHANCE RESEARCH PRODUCTIVITY, WHICH IN TURN WILL PROVIDE INSIGHTS THAT LEAD TO NEW DIAGNOSTICS AND THERAPEUTICS FOR A VARIETY OF DISEASES DISPROPORTIONATELY AFFECTING NA PATIENTS. THIS PROPOSAL WILL PROVIDE RESOURCES FOR THE RENOVATIONS OF SPACE FOR THIS CENTER, ALONG WITH NECESSARY COMPUTING INFRASTRUCTURE AND UPGRADES. ADDRESS: 825 N.E. 13TH STREET, OKLAHOMA CITY, OK, USA 73104-5005; PROJECT DIRECTOR NAME: TIM HASSEN, VP & CFO; WEBSITE: OMRF.ORG ; TOTAL GRANT PROGRAM FUNDS REQUESTED: $1,910,000

CHONDROCYTE METABOLIC STRESS IN THE DEVELOPMENT OF OSTEOARTHRITIS

ROLES OF SARM1 NAD HYDROLASE IN METABOLIC CARDIOMYOPATHY - NAD+ DEPLETION IS OBSERVED IN DISEASES INCLUDING HEART FAILURE AND METABOLIC CARDIOMYOPATHY. MAINTAINING NAD+ HOMEOSTASIS BY ACTIVATING NAD+ SYNTHESIS PATHWAYS HAS SHOWN GREAT PROMISE TO TREAT DISEASES. WE HERE EXPLORE THAT INHIBITING NAD+ CONSUMPTION WILL PREVENT NAD+ DEPLETION AND AMELIORATE HEART DISEASE. NAD+ HYDROLASES CATALYZE NAD+ DEGRADATION TO FORM NICOTINAMIDE AND ADPR. SARM1 IS AN INTRACELLULAR NAD+ HYDROLASE THAT IS KNOWN AS AN EXECUTER OF AXONAL DEGENERATION AND PROMOTES MITOCHONDRIAL DYSFUNCTION. RECENT STUDIES SHOWED THAT SARM1 IS ACTIVATED BY INCREASED NMN/NAD+ RATIO AND SARM1 PHOSPHORYLATION. HOWEVER, THE ROLE OF SARM1 IN METABOLIC CARDIOMYOPATHY HAS NEVER BEEN REPORTED, AND HOW SARM1 IS ACTIVATED TO CAUSE MITOCHONDRIAL DYSFUNCTION IS NOT KNOWN. THE OVERALL OBJECTIVE OF THIS PROJECT IS TO INVESTIGATE HOW SARM1 PROMOTES NAD+ DECLINE AND REGULATES MITOPHAGY DURING METABOLIC CARDIOMYOPATHY. FURTHER UNDERSTANDING OF SARM1 IN THE HEART WILL LEAD TO A NEW TARGET FOR THERAPEUTIC DEVELOPMENT OF HEART DISEASE. IN OUR PILOT DATA, WE SHOWED THAT SARM1 DEFICIENCY IN SARM1-KO MICE AND IN NEWLY DEVELOPED CARDIOMYOCYTE-SPECIFIC SARM1-KO MICE (SARM1-CKO) PROTECTED AGAINST METABOLIC STRESS-INDUCED CARDIOMYOPATHY, PROVIDING THE FIRST EVIDENCE ON THE ROLE OF SARM1 IN CAUSING DYSFUNCTION OF DIABETIC HEARTS. PILOT DATA FURTHER SUGGESTED THAT NAD+ METABOLIC AND JNK1 PHOSPHORYLATION SIGNALING MAY ACTIVATE SARM1, AND SARM1 PROMOTES MITOPHAGY IN DIABETIC HEARTS. BASED ON THESE RESULTS, WE PROPOSE TO DISSECT THE PATHOGENIC MECHANISMS OF SARM1 IN METABOLIC CARDIOMYOPATHY WITH THE THREE AIMS. SPECIFIC AIM 1 WILL USE SARM1-CKO MICE TO DETERMINE THE IMPACT OF CARDIOMYOCYTE SARM1 DEFICIENCY ON TYPE 1 DIABETES (T1D)- INDUCED AND DIET-INDUCED OBESITY (DIO)-INDUCED CARDIOMYOPATHY. MULTI-OMICS ANALYSES TARGETING KNOWN MECHANISTIC PATHWAYS OF DIABETIC HEARTS WILL BE PERFORMED TO IDENTIFY HOW CARDIOMYOCYTE SARM1 DEFICIENCY PROTECTS HEARTS AGAINST THE TWO TYPES OF METABOLIC STRESSES. SPECIFIC AIM 2 WILL UNDERSTAND HOW METABOLIC STRESS ACTIVATES SARM1 TO CAUSE COMPARTMENTAL NAD+ DECLINE. WE WILL MANIPULATE NAD+ METABOLIC AND JNK1- SARM1 PHOSPHORYLATION SIGNALING TO ESTABLISH HOW THE TWO PATHWAYS ACTIVATE SARM1 TO CAUSE NAD+ DECLINE IN MITOCHONDRIA OR CYTOSOL USING COMPARTMENT-SPECIFIC NAD+ BIOSENSORS. SPECIFIC AIM 3 WILL DETERMINE HOW SARM1 PROMOTES MITOPHAGY IN DIABETIC HEARTS. MITOPHAGY FLUX IMPACTED BY METABOLIC STRESS AND SARM1 DEFICIENCY WILL BE MEASURED BY MITO-KEIMA MICE. HOW SARM1 PROMOTES MITOPHAGY VIA TRADITIONAL PINK1- PARKIN AND/OR ALTERNATIVE ULK1-RAB9 PATHWAYS WILL BE EXAMINED IN DIABETIC HEARTS. THIS PROJECT WILL ESTABLISH SARM1 AS A NEW TARGET TO PROMOTE NAD+ DECLINE MITOCHONDRIAL DYSFUNCTION AND METABOLIC CARDIOMYOPATHY. THE RESULTS FROM THIS PROJECT WILL IDENTIFY NEW THERAPEUTIC TARGETS LINKED TO SARM1-DEPENDENT MECHANISMS.

THE ROLE OF MITOCHONDRIAL REGULATION IN CELL LINEAGE SPECIFICATION AND FUNCTION - ABSTRACT THE ROLE OF MITOCHONDRIAL REGULATION IN CELL LINEAGE SPECIFICATION AND FUNCTION DURING CELL LINEAGE SPECIFICATION, METABOLIC REPROGRAMMING, TOGETHER WITH TRANSCRIPTIONAL AND EPIGENETIC MODIFICATIONS IN THE PROGENITOR CELLS ARE INTEGRAL PARTS OF CELLULAR ADAPTATIONS TO THE CELL FATE INSTRUCTIVE CUES. WHEN THIS PROCESS GOES AWRY, DEVELOPMENTAL DEFECTS AS WELL AS MALIGNANCIES WILL OCCUR. COMMON IN ALL THE CELLULAR SYSTEMS, MITOCHONDRIA ARE BIOENERGETIC, BIOSYNTHETIC AND SIGNALING ORGANELLES CENTRAL TO METABOLIC REMODELING. UNDERSTANDING MECHANISMS OF MITOCHONDRIAL FUNCTION DURING LINEAGE SPECIFICATION WILL BE CRITICAL FOR THERAPEUTIC NORMALIZATION OF DERAILED CELLULAR METABOLISM AND FUNCTIONS IN PATHOGENESIS. THOUGH MITOCHONDRIAL REGULATION IS STILL UNCLEAR, OUR PUBLISHED AND PRELIMINARY WORK REVEALED CRITICAL ROLES OF MITOCHONDRIAL METABOLISM AND MITOCHONDRIAL DYNAMICS DURING CELL LINEAGE SPECIFICATION. FURTHERMORE, DIFFERENTIATED CELLS CONTINUE TO RECEIVE LOW-LEVEL, CONSTITUTIVE SIGNALING IN THE BASAL STATE (“TONIC SIGNALS”), IMPORTANT FOR CELL SURVIVAL OR FUNCTIONS. OUR RESEARCH SUGGESTED THAT MITOCHONDRIAL REGULATION IS ACTIVELY MAINTAINED IN MATURE DIFFERENTIATED CELLS DOWNSTREAM OF TONIC SIGNALS AND REVEALED POTENTIAL NEW REGULATORS FOR THIS PROCESS. THE OVERARCHING THEME OF OUR RESEARCH IS TO UNRAVEL THE DIVERSE MITOCHONDRIA MEDIATED MECHANISMS REGULATING CELL LINEAGE DIFFERENTIATION AND HOMEOSTATIC FUNCTIONS. IN PROJECT 1, WE WILL USE HIGH RESOLUTION IMAGING TO CHARACTERIZE DISTINCT MITOCHONDRIAL MORPHOLOGY, AND DYNAMIC EVENTS IN LIVE CELLS. USING GENETIC TOOLS, AND AN ORGAN CULTURE SYSTEM TO MODEL CELL LINEAGE SPECIFICATION, WE WILL DECIPHER THE ROLE OF MITOCHONDRIAL DYNAMICS IN METABOLIC REMODELING DURING DIFFERENTIATION. IN PROJECT 2, USING A SOPHISTICATED SYSTEM OF TRANSFERRING CELLS INTO ENVIRONMENTS WITH DEFINED LEVELS OF TONIC SIGNALS, WE WILL ASSESS THE ALTERATIONS IN MITOCHONDRIAL DYNAMICS AND FUNCTIONS IN THE PRESENCE AND ABSENCE OF TONIC SIGNALS. THROUGH PHARMACOLOGICAL MEANS OR GENETIC MODIFICATIONS SUCH AS CRISPR/CA9 IN DIFFERENTIATED CELLS, WE WILL DETERMINE THE CAUSAL RELATIONSHIP BETWEEN MITOCHONDRIAL REGULATION AND MAINTENANCE OF CELLULAR FUNCTIONS. UPON COMPLETION OF OUR WORK, WE WILL HAVE UNCOVERED THE VERSATILE MECHANISMS BY WHICH MITOCHONDRIA INFLUENCE LINEAGE SPECIFICATION AND HOMEOSTATIC FUNCTIONS.

MECHANISMS OF LUPUS DISEASE TRANSITION AND HYDROXYCHLOROQUINE IMMUNE MODULATION

GENE MAPPING IN WOMEN WITH SYSTEMIC LUPUS ERYTHEMATOSUS

MITOCHONDRIAL NAD+ METABOLISM IN CARDIAC AGING - PROJECT SUMMARY/ABSTRACT NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD+) IS A REDOX COFACTOR FOR ENERGY METABOLISM AND A CO-SUBSTRATE OF SIRTUINS FOR PROTEIN DEACETYLATION. CELLULAR NAD+ LEVELS DECLINE WITH AGE IN THE HEART AND OTHER ORGANS. PREVIOUS STUDIES HAVE SHOWN THAT CELLULAR NAD+ REPLETION OR BOOSTING IS PROTECTIVE AGAINST AGING AND AGE-RELATED DISEASES. NAD+ IS COMPARTMENTALIZED AT SUBCELLULAR LEVELS AND THE PROPER DISTRIBUTION OF NAD+ INTO MITOCHONDRIA IS CRUCIAL FOR NAD+-DEPENDENT METABOLIC AND SIGNALING FUNCTION IN MITOCHONDRIA. SLC25A51 WAS RECENTLY IDENTIFIED AS THE PRIMARY MAMMALIAN MITOCHONDRIAL NAD+ (MTNAD+) TRANSPORTER THAT IMPORTS NAD+ FROM CYTOSOL INTO MITOCHONDRIA. UNLIKE THE ESTABLISHED ROLES OF NAD+ SYNTHESIS AND CONSUMPTION, THERE IS A CRITICAL KNOWLEDGE GAP IN THE ROLE OF MTNAD+ TRANSPORT BY SLC25A51 IN AGING AND HEART FUNCTION. DESPITE THE IMPORTANT ROLES OF MTNAD+ IN MITOCHONDRIAL ENERGY METABOLISM AND PROTEIN DEACETYLATION, HOW MTNAD+ LEVELS CHANGE WITH AGE AND IMPACT CARDIAC AGING REMAIN UNKNOWN. OUR PRELIMINARY STUDY DISCOVERED THAT OLD MURINE HEARTS HAD REDUCED MTNAD+ LEVELS AND LOWER SLC25A51 EXPRESSION COMPARED TO YOUNG HEARTS. THE OBJECTIVE OF THIS STUDY IS TO DETERMINE THE MECHANISTIC ROLE OF SLC25A51 IN CARDIAC AGING USING OUR NEWLY DEVELOPED GENETIC TOOLS TO OVEREXPRESS OR KNOCKDOWN SLC25A51 IN THE HEART. MULTIPLE PRECLINICAL STUDIES AND CLINICAL TRIALS HAVE EXAMINED OR ARE INVESTIGATING THE THERAPEUTIC POTENTIAL OF VARIOUS NAD+ BOOSTING STRATEGIES IN DIFFERENT DISEASES. IMPORTANTLY, THE EXISTING NAD+ BOOSTING STRATEGIES DO NOT DIRECTLY BOOST MTNAD+ LEVELS, WHICH MAY LIMIT THEIR EFFICACY. OUR PRELIMINARY DATA SHOWED THAT SUPPLEMENTATION WITH AN NAD+ PRECURSOR NMN SIGNIFICANTLY ELEVATED TOTAL NAD+ LEVELS IN OLD MURINE HEARTS BUT DID NOT EFFECTIVELY INCREASE MTNAD+ LEVELS. THIS HIGHLIGHTS THE POTENTIAL OF ENHANCING MTNAD+ TRANSPORT AS A NOVEL APPROACH TO IMPROVE THE BENEFITS OF NAD+ BOOSTING STRATEGIES. WE HYPOTHESIZE THAT AN AGE-RELATED DECLINE IN SLC25A51 FUNCTION REDUCES MTNAD+ LEVELS, LEADING TO COMPROMISED MITOCHONDRIAL METABOLISM AND CARDIAC DYSFUNCTION. WE ALSO PROPOSE THAT ENHANCING SLC25A51-MEDIATED MTNAD+ TRANSPORT WILL IMPROVE THE PROTECTIVE EFFECTS OF NAD+ BOOSTING STRATEGY IN THE OLD HEART. IN THIS STUDY, WE WILL 1) DETERMINE IF SLC25A51 DEFICIENCY LOWERS MTNAD+ LEVELS AND ACCELERATES CARDIAC AGING; 2) DETERMINE IF CARDIAC-SPECIFIC SLC25A51 OVEREXPRESSION ENHANCES MTNAD+ LEVELS AND CARDIAC FUNCTION IN OLD MICE; AND 3) TEST THE HYPOTHESIS THAT LEVELS OF SLC25A51 EXPRESSION REGULATE THE EFFICACY OF NAD+ PRECURSOR TO IMPROVE CARDIAC FUNCTION. THE FINDINGS OF THIS STUDY WILL ESTABLISH THE ROLES OF SLC25A51 IN CARDIAC AGING AND OPEN NEW AVENUES TOWARDS DEVELOPING NEW INTERVENTIONS TO ENHANCE MTNAD+ METABOLISM OR TO MAXIMIZE THE EFFICACY OF NAD+ BOOSTING STRATEGIES.

MECHANISMS OF COHESIN REGULATION IN VERTEBRATES - ABSTRACT IN VERTEBRATE CELLS THE COHESIN PROTEIN COMPLEX PLAYS CRITICAL ROLES IN NUCLEAR STRUCTURE AND FUNCTION. IT TETHERS TOGETHER THE IDENTICAL PRODUCTS OF DNA REPLICATION, CALLED SISTER CHROMATIDS, UNTIL CELL DIVISION AND IT ALSO MEDIATES INTRA-CHROMOSOMAL BRIDGING INTERACTIONS, FORMING CHROMOSOME LOOPS AND DOMAINS. WHILE COHESION BETWEEN SISTER CHROMATIDS IS CRITICAL FOR ACCURATE CHROMOSOME SEGREGATION AND CERTAIN KINDS OF DNA REPAIR, THE COMPACTION OF CHROMOSOMES INTO LOOPS AND DOMAINS IS ESSENTIAL FOR PROPER TRANSCRIPTION AND NORMAL DEVELOPMENT. HOW THESE DIFFERENT KINDS OF COHESION DIFFER AT THE MOLECULAR LEVEL, THE MECHANISMS THAT ENSURE EACH OUTCOME, AND THE AMOUNT OF OVERLAP BETWEEN THEM ARE NOT WELL UNDERSTOOD. USING MOLECULAR GENETIC APPROACHES, WE ARE TESTING THE IMPACTS OF SPECIFIC INTERACTIONS, IN MULTIPLE EXPERIMENTAL SYSTEMS, ON COHESIN REGULATION. IN THIS APPLICATION WE ARE REQUESTING SUPPLEMENTAL FUNDS TO PURCHASE CHROMATOGRAPHY EQUIPMENT THAT WILL ENHANCE OUR EFFICIENCY IN PURIFICATION AND CHARACTERIZATION OF PROTEINS TO COMPLETE THE PROJECTS DESCRIBED IN THE PARENT PROPOSAL.

PODOPLANIN-MEDIATED PLATELET ACTIVATION AND VASCULAR INTEGRITY IN THE DEVELOPING BRAIN

COMMUNITY PROJECT FUNDING/CONGRESSIONALLY DIRECTED SPENDING - CONSTRUCTION

METABOLIC MECHANISMS CONTROLLING LYMPHATIC VESSEL FORMATION - PROJECT SUMMARY/ABSTRACT LYMPHATIC VESSELS PLAY CRITICAL ROLES IN REGULATING TISSUE FLUID DRAINAGE, DIETARY FAT ABSORPTION, AND REVERSE CHOLESTEROL TRANSPORT. FULFILLING THESE IMPORTANT PHYSIOLOGICAL FUNCTIONS REQUIRES PROPER DEVELOPMENT OF THE LYMPHATIC VASCULATURE, WHICH IS MAINLY DRIVEN BY VASCULAR ENDOTHELIAL GROWTH FACTOR C (VEGF-C). VEGF-C SIGNALING ALSO STIMULATES PATHOLOGICAL LYMPHANGIOGENESIS IN TUMORS AND ORGAN TRANSPLANTATION, WHICH MAY, IN TURN, PROMOTE CANCER METASTASIS AND ALLOGRAFT REJECTION RESPECTIVELY. THEREFORE, ELUCIDATING THE MECHANISMS BY WHICH VEGF-C SIGNALING DRIVES LYMPHANGIOGENESIS WILL NOT ONLY ENHANCE THE FUNDAMENTAL UNDERSTANDING OF PHYSIOLOGICAL PROCESSES REGULATED BY LYMPHATICS, BUT ALSO FACILITATE THE DEVELOPMENT OF NOVEL ANTI- LYMPHANGIOGENIC STRATEGIES FOR DISEASE TREATMENTS. WE PREVIOUSLY DISCOVERED THAT LYMPHATIC ENDOTHELIAL CELLS (LECS), EVEN WHEN GROWN IN AN OXYGEN-RICH ENVIRONMENT, PREFERENTIALLY CONVERT GLUCOSE TO LACTATE. THIS UNIQUE METABOLIC FEATURE IS TERMED THE WARBURG EFFECT. DESPITE THIS FINDING, IT REMAINS UNCLEAR WHETHER AND HOW THE WARBURG EFFECT IS REGULATED BY VEGF-C SIGNALING FOR PROMOTING DEVELOPMENTAL AND PATHOLOGICAL LYMPHANGIOGENESIS. LACTATE DEHYDROGENASE A (LDHA) CATALYZES THE REDUCTION OF PYRUVATE TO LACTATE AND THE REGENERATION OF OXIDIZED NICOTINAMIDE ADENINE DINUCLEOTIDE (NAD+) FROM ITS REDUCED FORM NADH. OUR PRELIMINARY STUDIES SUGGEST THAT LDHA MEDIATES THE WARBURG EFFECT IN LECS. WE ALSO FOUND THAT GENETIC ABLATION OF LDHA IN MICE IMPAIRS LEC PROLIFERATION AND MIGRATION DURING LYMPHATIC VASCULAR DEVELOPMENT. MOREOVER, VEGF-C ENHANCES LDHA TRANSCRIPTION AND LACTATE GENERATION IN LECS. THESE DATA COLLECTIVELY SUPPORT OUR CENTRAL HYPOTHESIS THAT LDHA COUPLES VEGF-C SIGNALING WITH CELLULAR METABOLISM TO DRIVE LYMPHANGIOGENESIS, WHICH WILL BE TESTED THROUGH TWO SPECIFIC AIMS. AIM 1 WILL USE GENETIC MOUSE MODELS AND CULTURED LECS TO DETERMINE THE MOLECULAR MECHANISMS BY WHICH VEGF-C SIGNALING INDUCES LDHA EXPRESSION DURING LYMPHATIC VESSEL FORMATION. AIM 2 WILL COMBINE SEVERAL ADVANCED ANALYTICAL TOOLS TO ELUCIDATE THE MECHANISMS BY WHICH LDHA CONTROLS CELLULAR METABOLISM TO PROMOTE LYMPHANGIOGENESIS. TAKEN TOGETHER, OUR PROPOSED STUDIES WILL IDENTIFY LDHA AS A NOVEL MECHANISTIC LINK BETWEEN VEGF-C SIGNALING AND METABOLIC PROCESSES CRITICAL FOR LYMPHATIC VESSEL FORMATION. OUR WORK MAY ALSO SUGGEST AN INNOVATIVE STRATEGY, I.E., TARGETING THE WARBURG EFFECT VIA LDHA INHIBITION, FOR SUPPRESSING VEGF-C-INDUCED PATHOLOGICAL LYMPHANGIOGENESIS.

INCREASING GLYCOLYSIS IN THE DIABETIC HEART IS CARDIOPROTECTIVE AND IMPROVES GLUCOSE TOLERANCE - THE GOAL OF THIS PROPOSAL IS TO IDENTIFY WHETHER INCREASING CARDIAC GLYCOLYSIS AT ITS RATE-LIMITING STEP CAN MITIGATE DIABETIC CARDIOMYOPATHY (DC) AND IMPROVE WHOLE BODY GLUCOSE TOLERANCE. DC IS A MAJOR PUBLIC HEALTH ISSUE THAT ARISES IN BOTH TYPE 1 AND TYPE 2 DIABETES AND IS MEDIATED BY NUMEROUS FACTORS. CHIEF AMONGST THEM IS THE LOSS OF METABOLIC FLEXIBILITY, WHICH IS THE CAPACITY OF THE HEART TO TAKE UP AND METABOLIZE AVAILABLE CIRCULATING NUTRIENTS. THE HEALTHY HEART PRIMARILY USES FATTY ACIDS, BUT IT CAN SHIFT TO GLUCOSE METABOLISM IN RESPONSE TO FEEDING. HOWEVER, WITH DIABETES THE HEART RELIES ALMOST EXCLUSIVELY ON FATTY ACID OXIDATION AND IF CHRONIC, THIS LEADS TO MITOCHONDRIAL DYSFUNCTION, OXIDATIVE STRESS, AND ULTIMATELY DC. WHILE RESTORING PROPER CARDIAC METABOLISM HAS THERAPEUTIC POTENTIAL, THERE ARE CURRENTLY NO TREATMENTS TO NORMALIZE METABOLIC INFLEXIBILITY. WE POSIT THAT INCREASING GLYCOLYSIS CAN NORMALIZE METABOLIC INFLEXIBILITY AND MITIGATE DC. WE HAVE BEEN TESTING THIS HYPOTHESIS USING MICE THAT HAVE ENHANCED CARDIAC GLUCOSE METABOLISM (GLYCOHI MICE) VIA THE EXPRESSION OF A CONSTITUTIVELY ACTIVE FORM OF THE GLYCOLYTIC REGULATOR, PHOSPHOFRUCTOKINASE-2 (PFK-2). WE FOUND THAT: GLYCOHI MICE ARE RESISTANT TO DIET-INDUCED CARDIAC DIASTOLIC DYSFUNCTION; GLYCOHI HEART MITOCHONDRIA HAVE AN ENHANCED CAPACITY TO USE PYRUVATE, INDICATIVE OF INCREASED METABOLIC FLEXIBILITY; AND FEMALE GLYCOHI MICE HAVE IMPROVED SYSTEMIC GLUCOSE TOLERANCE AND ARE RESISTANT TO HFD EFFECTS. THIS SUPPORTS OUR HYPOTHESIS THAT INCREASING CARDIAC PFK-2 ACTIVITY CAN MITIGATE DC AND HAVE BENEFICIAL EFFECTS ON WHOLE BODY GLUCOSE REGULATION. OUR FIRST AIM IS TO TEST THE HYPOTHESIS THAT INCREASING CARDIAC GLYCOLYSIS IMPROVES METABOLIC FLEXIBILITY IN RESPONSE TO HFD OR TYPE 1 DIABETES. CONTROL AND GLYCOHI MICE WILL BE SUBJECTED TO HFD OR INDUCED WITH TYPE 1 DIABETES. WE WILL DETERMINE CARDIAC FUNCTION AND METABOLIC PROFILE BY BOTH PROTEOMICS AND METABOLOMICS. METABOLIC FLEXIBILITY WILL BE MEASURED IN ADULT CARDIOMYOCYTES USING A RADIOLABELED ASSAY. AIM 2 WILL TEST THE HYPOTHESIS THAT INCREASING CARDIAC GLYCOLYSIS SUSTAINS MITOCHONDRIAL FUNCTION UNDER DIABETIC CONDITIONS. WE WILL INTERROGATE MITOCHONDRIAL FUNCTION IN DIABETIC (T1D AND T2D) CONTROL GLYCOHI, AND PFK-2 KNOCKOUT MICE. WE WILL ALSO DETERMINE HOW THE INCREASE IN GLYCOLYSIS IS ABLE TO SUSTAIN PYRUVATE DEHYDROGENASE ACTIVITY. AIM 3 WILL DETERMINE THE MECHANISMS BY WHICH INCREASING CARDIAC GLYCOLYSIS IMPROVES WHOLE BODY GLUCOSE TOLERANCE IN DIABETIC GLYCOHI MICE. WE WILL DISCERN BETWEEN INCREASED ENERGY EXPENDITURE, USING METABOLIC CAGES, AND INCREASED INSULIN SENSITIVITY IN HEART, SKELETAL MUSCLE, AND ADIPOSE TISSUE. WE WILL ALSO TEST THE HYPOTHESIS THAT THE EFFECTS ARE MEDIATED THROUGH CHANGES IN ADIPOCYTE DIFFERENTIATION AND BIOENERGETICS. THE OCCURRENCE OF DIABETES CONTINUES TO INCREASE, AND HEART DISEASE AND HEART FAILURE ARE LEADING CAUSES OF DEATH IN THIS POPULATION. IT IS NOT KNOWN WHETHER INCREASING CARDIAC GLYCOLYSIS HAS THERAPEUTIC POTENTIAL IN MITIGATING DC. THESE RESULTS WILL BE AN IMPETUS FOR FUTURE STUDIES THAT EXAMINE THE THERAPEUTIC POTENTIAL OF TARGETING PFK-2 TO NORMALIZE CARDIAC METABOLIC FLEXIBILITY AND GLUCOSE HOMEOSTASIS.

SITE-1 PROTEASE-MEDIATED LIPID METABOLISM IN LYMPHATIC VASCULAR DEVELOPMENT

PLATELET CLEC-2 IN ARTERIAL THROMBOSIS

EPIGENETIC CONTROL OF MEIOTIC RECOMBINATION IN MAMMALS.

THE ROLE OF DENDRODENDRITIC DOPAMINE NEUROTRANSMISSION IN METHAMPHETAMINE ABUSE

IRF4-MEDIATED REGULATION OF LUNG DENDRITIC CELLS DURING VIRAL INFECTION

PLATELET CLEC-2 REGULATION OF VASCULAR INTEGRITY DURING INFLAMMATION

PKA SIGNALING AND METABOLIC INFLEXIBILITY IN THE DIABETIC HEART

THE MECHANISM OF SHEAR-INDUCED RELEASE AND ACTIVATION OF TGF-BETA1

IOS EDGE: RAPID AND EFFICIENT GENE EDITING OF AMPHIBIANS THROUGH NUCLEAR TRANSFER FROM ENGINEERED CELL LINES

SJOGREN'S SYNDROME PATHOGENIC AUTOANTIBODIES

DEFINING THE REGULATORY LANDSCAPE OF LUPUS WITH CVD COMORBIDITY - PROJECT SUMMARY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS AN INCURABLE AUTOIMMUNE DISEASE FEATURING LARGE CLINICAL HETEROGENEITY AND MULTI-ORGAN DYSFUNCTION, INCLUDING CARDIOVASCULAR DISEASES (CVD). UNDERSTANDING THIS HETEROGENEITY IS VITAL FOR ACCURATE DIAGNOSIS, IDENTIFYING THERAPEUTIC TARGETS, PROVIDING PERSONALIZED TREATMENT, AND IMPROVING PATIENT CARE. LUPUS HAS A SUBSTANTIAL GENETIC COMPONENT, AND STUDYING IMMUNE CELL-SPECIFIC GENETIC ARCHITECTURE REVEALS PATHOGENIC MECHANISMS AND THERAPEUTIC TARGETS FOR SLE. GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED NUMEROUS SLE-PREDISPOSING LOCI, TYPICALLY DEFINED BY BLOCKS OF CORRELATED SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS). GWAS LOCI ARE ENRICHED IN REGULATORY ELEMENTS (RES) AND CAN INFLUENCE TARGET GENE EXPRESSION THROUGH REGULATORY SNPS (RSNPS). IT IS CHALLENGING BUT CRITICAL TO IDENTIFY THE UNDERLYING CAUSAL VARIANTS, TARGET GENES, AND CELL TYPES INVOLVED IN SLE PATHOGENESIS. MONOCYTE-DERIVED MACROPHAGES ARE PIVOTAL IN ATHEROSCLEROSIS AND SLE, AS THEIR DYSFUNCTION IMPAIRS PHAGOCYTOSIS AND CLEARANCE OF DEAD CELLS, CONTRIBUTING TO BOTH DISEASES. YET, THE IMPACT OF SLE AND CVD-ASSOCIATED VARIANTS ON MACROPHAGE BIOLOGY REMAINS UNCLEAR. OUR CENTRAL HYPOTHESIS IS THAT SOME SLE RSNPS ALTER CIS-REGULATORY ELEMENTS (CRES) CONTROLLING TARGET GENES INVOLVED IN MACROPHAGE BIOLOGY, FUELING SLE AND CVD. TO TEST THIS HYPOTHESIS, WE WILL PERFORM SYSTEMATIC, HIGH- THROUGHPUT, UNBIASED, LARGE-SCALE STUDIES IN MACROPHAGES TO DETERMINE THE REGULATORY EFFECTS OF 9,931 SNPS WITHIN 182 SLE RISK LOCI USING INDUCED PLURIPOTENT STEM CELLS (IPSCS) FROM SLE PATIENTS AND HEALTHY DONORS. WE WILL DIFFERENTIATE IPSC-DERIVED MACROPHAGE PROGENITORS (MONOCYTES) INTO UNPOLARIZED (M0), PRO-INFLAMMATORY (M1), AND ANTI-INFLAMMATORY (REGULATORY, M2) PHENOTYPES, RECAPITULATING THE NATURAL MOLECULAR AND FUNCTIONAL PHENOTYPES OF PRIMARY MACROPHAGES. THROUGH OUR STUDIES, WE AIM TO IDENTIFY DYSFUNCTIONS ARISING FROM GENETIC DIFFERENCES, AND UNCOVER REGULATORY ELEMENTS AND TARGET GENES IN MACROPHAGES DISRUPTED BY SLE RISK ALLELES. AIM 1 WILL UTILIZE THE HIGH-THROUGHPUT TECHNIQUE, “SELF-TRANSCRIBING ACTIVE REGULATORY REGION-SEQUENCING” (STARR- SEQ) TO SCREEN THOUSANDS OF SNP-CONTAINING REGIONS FOR THEIR REGULATORY POTENTIAL IN MACROPHAGES. AIM 2 WILL EMPLOY NEXT-GENERATION (NG)-CAPTURE-C TO DISCOVER SNP-SPECIFIC CIS INTERACTIONS WITH ENDOGENOUS, COGNATE TARGET GENES IN MACROPHAGES. AIM 3 WILL APPLY CRISPR-CAS9 APPROACHES TO UNCOVER THE ALLELE-SPECIFIC FUNCTIONAL CONSEQUENCES OF THE TOP 10 SELECTED RSNPS ON THEIR TARGET GENE(S) IN IPSC-DERIVED MACROPHAGES. ALSO, TO DETERMINE THE POTENTIAL ASSOCIATION OF SLE-ASSOCIATED RISK ALLELES WITH CVD RISK, WE WILL ANALYZE TARGET GENE EFFECTS IN PRIMARY MONOCYTES/MACROPHAGES FROM SLE PATIENTS WITH OR WITHOUT ATHEROSCLEROSIS, AS WELL AS HEALTHY CONTROLS. THROUGH THIS ANALYSIS, WE AIM TO IDENTIFY GENETIC FACTORS CONTRIBUTING TO BOTH SLE AND CVD, GAINING VALUABLE INSIGHTS INTO THEIR INTERPLAY. THIS ENHANCED KNOWLEDGE MAY INFORM TARGETED THERAPIES OR DRUG REPURPOSING STRATEGIES TO ADDRESS MACROPHAGE DYSFUNCTION IN TREATING SLE AND CVD.

BETA SECRETASE INHIBITION FOR TREATING ALZHEIMER'S DISEASE

HEMATOPOIETIC STEM CELL SENESCENCE

COAGULATION AND COMPLEMENT ACTIVATION IN SEPSIS

PRE-CLINICAL STUDY OF COMBINATION ORAL TREATMENT FOR ACUTE ACOUSTIC TRAUMA (AAT)

CONTROL OF REDOX REGULATORS BY THE UBIQUITIN SYSTEM

RETINAL PIGMENTED EPITHELIUM EPIGENOME DYSREGULATION WITH AGING AND MODULATION BY DIET - ABSTRACT/SUMMARY AGING IS THE PRINCIPAL RISK FACTOR FOR AGE-RELATED MACULAR DEGENERATION (AMD), A NEURODEGENERATIVE DISEASE CHARACTERIZED BY THE IRREVERSIBLE LOSS OF VISION. CLINICAL AND MOUSE STUDIES INDICATE THAT CONSUMPTION OF DIETS WITH HIGHER DIETARY GLYCEMIC INDICES INCREASE AMD RISK. ATROPHY OF THE RETINAL PIGMENTED EPITHELIUM (RPE) LAYER IS AN AMD HALLMARK THAT PRECEDES PHOTORECEPTOR CELL LOSS. HOWEVER, THE MECHANISMS UNDERLYING RPE IMPAIRMENT WITH AGING AND EXACERBATION BY POOR DIET ARE UNCLEAR. THUS, THERAPEUTIC APPROACHES TO MAINTAIN RPE FUNCTION WITH AGING AND PREVENT AMD ARE YET TO BE DEVELOPED. EPIGENETIC PROCESSES (DNA MODIFICATIONS AND CHROMATIN ACCESSIBILITY) IN THE RPE MAY PLAY A CENTRAL MECHANISTIC ROLE IN THE PATHOGENESIS AND PROGRESSION OF AMD. DNA MODIFICATIONS, [CYTOSINE BASE METHYLATION AND HYDROXYMETHYLATION (MC AND HMC RESPECTIVELY)], ARE FUNDAMENTAL REGULATORS OF DNA ACCESSIBILITY AND GENE REGULATION/EXPRESSION. A BARRIER TO PROGRESS IN UNDERSTANDING THE ROLE OF EPIGENETIC MECHANISMS IN RPE AGING AND DNA MODIFICATIONS IN PARTICULAR, HAS BEEN THE LACK OF QUANTITATIVELY ACCURATE, GENOME-WIDE DATA IN THIS SPECIFIC CELL TYPE. WITHOUT THE KNOWLEDGE OF THE SPECIFIC GENOMIC LOCATIONS OF ALTERED MODIFICATIONS/ACCESSIBILITY WITH AGING IT IS IMPOSSIBLE TO DESIGN MECHANISTIC STUDIES THAT UNRAVEL THE FUNCTIONAL EFFECTS OF EPIGENETIC RECONFIGURATION. THEREFORE, THE CRITICAL NEXT STEP FOR THE FIELD IS TO GENERATE THESE GENOME-WIDE MAPS OF MC AND HMC IN CG AND CH CONTEXTS AND GENOMIC ACCESSIBILITY IN THE PRIMARY CELLULAR SITE OF AMD PATHOGENESIS, THE RPE, FROM BOTH SEXES ACROSS THE LIFESPAN. TO ADDRESS THIS CRITICAL ISSUE, WE HAVE DEVELOPED A CELL-TYPE SPECIFIC, TAMOXIFEN-INDUCIBLE CRE, TRANSGENIC NUTRAP MODEL TO ALLOW ISOLATION OF NUCLEIC ACIDS (DNA & RNA), SPECIFICALLY FROM RPE CELLS. IN AIM 1, CHANGES IN MC/HMC AND CHROMATIN ACCESSIBILITY PATTERNS WITH AGING WILL BE EXAMINED BY WHOLE GENOME OXIDATIVE BISULFITE SEQUENCING (WGOXBS) AND ATAC-SEQ IN RPE. IN AIM 2, THE RPE-SPECIFIC DIFFERENTIAL CHANGES IN THE TRANSLATOME WILL BE IDENTIFIED AS A FUNCTION OF AGING. IN PRIOR STUDIES WE HAVE DETERMINED THAT AGE-RELATED DNA MODIFICATION CHANGES CAN BE PREVENTED BY CALORIC RESTRICTION. IN AIM 3, WE WILL INTERROGATE THE POTENTIAL OF WESTERN AND KETOGENIC DIETARY PATTERNS, IN COMBINATION WITH IMPAIRED OXIDATIVE STRESS RESOLUTION PATHWAYS, TO EXACERBATE OR AMELIORATE CHANGES IN THE RPE EPIGENOME AND GENE EXPRESSION PROFILES. PAIRED EPIGENOMIC AND TRANSCRIPTOMIC DATA FROM THE SAME ANIMALS WILL BE USED TO: 1) ASSESS AGING WITH ‘EPIGENETIC CLOCKS’ IN RPE, 2) DETERMINE THE ROLE OF ALTERED MODIFICATION PATTERNS IN AGE- AND DIETARY/OXIDATIVE STRESS- RELATED CHANGES IN GENE EXPRESSION, 3) DETERMINE ENRICHMENT OF DIFFERENTIAL MODIFICATIONS/ACCESSIBILITY IN REGULATORY REGIONS OF THE GENOME, AND 4) IDENTIFY AND REFINE GENOMIC LOCI FOR EPIGENOME EDITING. THESE STUDIES WILL DETERMINE CRITICAL GENOMIC REGIONS WITH ALTERED DNA MODIFICATION PATTERNS THAT CAN BE MANIPULATED IN FUTURE INTERVENTIONAL STUDIES. THE ULTIMATE GOAL OF THE RESEARCH IS TO DEVELOP CLINICAL INTERVENTIONS THAT TARGET THE RPE EPIGENOME TO MAINTAIN VISUAL FUNCTION WITH AGING AND PREVENT AMD.

IDENTIFICATION OF TYROSINE-SULFATED PROTEINS IN THE MALE GENITAL TRACT

MECHANICS AND REGULATION OF CHROMOSOME DYNAMICS IN MEIOTIC PROPHASE

A GENE KNOCKOUT RESOURCE FOR C. ELEGANS

DEVELOPMENT OF A LACO/LACI BASED FLUORESCENCE REPORTER-OPERATOR SYSTEM TO STUDY CHROMOSOME DYNAMICS AND DOUBLE-STRAND BREAK REPAIR IN MOUSE MEIOSIS. - SUMMARY FERTILIZATION OF ANEUPLOID GAMETES OFTEN LEADS TO PREGNANCY LOSS OR DISORDERS SUCH AS DOWN’S SYNDROME. ANEUPLOIDY OCCURS IN 10-30% OF HUMAN GAMETES AND TYPICALLY ARISES FROM CHROMOSOME SEGREGATION ERRORS DURING MEIOSIS. MEIOTIC EVENTS SUCH AS HOMOLOGOUS CHROMOSOME PAIRING, DOUBLE STRAND BREAKS (DSBS), RECOMBINATION AND THE FORMATION OF CROSSOVERS, ENSURE PROPER CHROMOSOME SEGREGATION. NEVERTHELESS, THE MECHANISMS THAT REGULATE THESE EVENTS ARE INCOMPLETELY UNDERSTOOD. CONSEQUENTLY, THERE ARE CURRENTLY FEW TO NO STRATEGIES TO PREDICT OR PREVENT ANEUPLOIDY DURING GAMETOGENESIS. TO OVERCOME SOME LIMITATIONS IN THE FIELD, WE ESTABLISHED A FLUORESCENCE REPORTER-OPERATOR SYSTEM (FROS) BASED ON THE LAC OPERATOR-LAC REPRESSOR (LACO-LACR) PARADIGM, WHICH ENABLES PROTEIN TARGETING TO DISTINCT GENOMIC REGIONS. OUR LONG-TERM GOAL IS TO ELUCIDATE THE MECHANISMS THAT REGULATE CHROMOSOME SEGREGATION DURING MEIOSIS AND THUS ENSURE THE FORMATION OF GAMETES WITH A NORMAL KARYOTYPE. AIM 1 USE FROS TO DISSECT RAPID CHROMOSOME MOTIONS IN MOUSE SPERMATOCYTES AT PROPHASE I. WE WILL ALSO COMBINE FROS WITH LONG-TERM 3D MEASUREMENTS OF CHROMOSOME MOTIONS IN SEMINIFEROUS TUBULES TO DIRECTLY TEST WHETHER HOMOLOGOUS CHROMOSOME PAIRING ARISES FROM INCREASINGLY PRODUCTIVE INTERACTIONS (REELING IN), VERSUS REITERATIVE ROUNDS OF TRANSIENT INTERACTIONS (CATCH AND RELEASE). ADDITIONALLY, WE WILL ANALYZE MUTANT SPERMATOCYTES BY FROS TO DETERMINE HOW KEY PLAYERS, INCLUDING COMPONENTS OF THE LINKER OF NUCLEOSKELETON AND CYTOSKELETON (LINC) COMPLEX AND THE SYNAPTONEMAL COMPLEX, AFFECT HOMOLOGOUS AND NON-HOMOLOGOUS INTERACTIONS AT SPECIFIC LOCI. DYNEIN AND MICROTUBULES INTERACT WITH THE LINC COMPLEX AND ARE KNOWN TO CONTRIBUTE TO RPMS. AIM 2 WILL IDENTIFY ADDITIONAL MOTORS AND CYTOSKELETON COMPONENTS THAT SUPPORT THESE MOVEMENTS, BUILDING ON OUR PRELIMINARY DATA UNCOVERING KINESINS AS CANDIDATE MOLECULAR MOTORS INVOLVED IN GENERATING RPMS. WE WILL EXTEND THESE FINDINGS WITH UNBIASED PROTEOMIC APPROACHES, FUNCTIONAL ASSAYS AND FROS TO DECIPHER THE DYNAMIC FORCES THAT GOVERN HOMOLOG PAIRING. AIM 3 WILL INVESTIGATE THE MECHANISM UNDERLYING THE ASSOCIATION BETWEEN ANKRD31 AND PRO-DSB FACTORS AT RECOMBINATION HOTSPOTS. USING FROS, WE WILL TARGET ANKRD31-GFP-LACI TO THE LACO REGION IN SPERMATOCYTES, WHICH WE PREDICT WILL RECRUIT PRO-DSB FACTORS AND RECOMBINATION PROTEINS. WE WILL ALSO ANALYZE THE EFFECT OF ANKRD31-GFP-LACI ON DOWNSTREAM RECOMBINATION INTERMEDIATES AT LACO SITES. THE PROPOSED RESEARCH WILL FOR THE FIRST TIME DISCERN HOW CHROMOSOME CONTEXT AFFECTS THE MECHANISMS UNDERLYING CHROMOSOME SEGREGATION AND THUS ADVANCE NEW KNOWLEDGE OF FUNDAMENTAL MEIOTIC PROCESSES AND THE CAUSES OF ANEUPLOIDY.

THE ROLE OF COHESION FATIGUE IN CHROMOSOME INSTABILITY

??TCR-DEPENDENT AND INDEPENDENT DIFFERENTIATION OF INNATE LYMPHOID CELLS - ABSTRACT INNATE LYMPHOID CELLS (ILCS) RESIDE IN TISSUES AND ACT AS SENTINELS TO A VARIETY OF ENVIRONMENTAL CUES FROM NUTRIENTS, NEUROPEPTIDES, MICROBIOTA TO PATHOGENS. THEY ARE HETEROGENEOUS POPULATIONS WITH DIVERSE FUNCTIONS AND DEVELOPMENTAL ORIGINS. WE HAVE PREVIOUSLY SUGGESTED THAT IN ADDITION TO THE BONE MARROW, THE THYMUS CAN CONTRIBUTE TO ILC POOLS IN PERIPHERAL TISSUES BASED ON FINDINGS THAT LUNG ILC2S CONTAIN REARRANGED T CELL RECEPTOR GENES, LIKELY DUE TO THE DIVERGENCE OF DEVELOPING THYMOCYTES INTO THE ILC FATE. CONSISTENTLY, T CELL PRECURSORS FROM THE THYMUS CAN DIFFERENTIATE INTO ILC2S IN VITRO. SINGLE CELL RNA SEQUENCING STUDIES DETECTED A HETEROGENEOUS POPULATION OF ILC PRECURSORS IN THE BLOOD, WHICH IS GREATLY DIMINISHED IN ATHYMIC NUDE MICE, SUGGESTING THE INVOLVEMENT OF THE THYMUS. THESE CELLS POSSESS CD3 PROTEINS IN THE CYTOPLASM BUT NOT ON THE SURFACE, THUS MAKING INTRACELLULAR CD3 (ICCD3) A MARKER FOR THYMUS-DERIVED ILCS. INDEED, ICCD3+ ILC SUBSETS HAVE BEEN DETECTED IN THE LUNG AND SMALL INTESTINE. INTERESTINGLY, THE PRODUCTION OF ICCD3+ ILC1S AND TO A LESSER EXTENT, ILC3S DEPENDS ON TCRD BUT THAT OF ICCD3+ ILC2S DOES NOT. THIS PROMPTED US TO PROPOSE THAT ILC DIFFERENTIATION IN THE THYMUS INVOLVES TCRD-DEPENDENT AND INDEPENDENT MECHANISMS. THE PURPOSE OF THIS APPLICATION IS TO STUDY HOW THYMUS-DERIVED ILCS ARE GENERATED AND TO INVESTIGATE THE FUNCTIONS OF THESE CELLS. THERE ARE THREE AIMS. AIM1 WILL TEST THE HYPOTHESIS THAT DEVELOPING T CELLS WITH NON-PRODUCTIVE TCR GENE REARRANGEMENT CONTRIBUTES TO MULTIPOTENT ILC PRECURSORS AND IMMATURE D T CELLS GIVES RISE TO ILC1- OR ILC3- BIASED PRECURSORS AFTER DOWN-REGULATION OF THEIR TCRS. WE WILL EMPLOY APPROACHES SUCH AS IN VITRO DIFFERENTIATION CULTURES AND ANALYSES OF TCR GENE REARRANGEMENT EVENTS. AIM2 WILL CHARACTERIZE ICCD3+ ILCS IN THE SMALL INTESTINE BY EXAMINING THE AGE-DEPENDENT DIFFERENTIATION OF ILC1S AND ILC2 IN RELATIONSHIP TO T CELL DEVELOPMENT AND CHANGES IN GUT MICROBIOTA, AND BY COMPARING THE TYPE 1 RESPONSES OF ICCD3+ ILC1S TO THOSE OF CONVENTIONAL ILC1S AND NK CELLS AS WELL AS D T CELLS. AIM3 WILL INVESTIGATE THE INTERPLAY OF TYPE 2 AND TYPE 3 IMMUNE RESPONSES AND COMPARE THE BEHAVIORS OF ICCD3+ ILC2S TO THEIR ICCD3- COUNTERPARTS. TAKEN TOGETHER, THESE STUDIES MAY MAKE CONCEPTUAL ADVANCEMENT IN DEVELOPMENTAL IMMUNOLOGY AND SHED LIGHT ON THE CONTRIBUTION OF THYMUS-DERIVED ILCS TO AGE-RELATED DIFFERENCES IN IMMUNITY.

CENTROMERE BEHAVIORS THAT PROMOTE PROPER CHROMOSOME SEGREGATION IN MEIOSIS AND MITOSIS - PROJECT SUMMARY IN BOTH MITOSIS AND MEIOSIS, SUCCESS DEPENDS ON ACCURATELY PULLING PARTNER CHROMOSOMES AWAY FROM ONE ANOTHER ON A SPINDLE COMPOSED OF MICROTUBULES. THE MICROTUBULES ATTACH TO THE CHROMOSOMES AT THEIR KINETOCHORES. WHEN THE PARTNERS ERRANTLY MOVE TO THE SAME POLE A CHROMOSOME IMBALANCE IS CREATED, PRODUCING CELLS WITH TOO MANY OR TOO FEW CHROMOSOMES (ANEUPLOIDY). MEIOTIC ERRORS PRODUCE ANEUPLOID GAMETES, THE MAJOR CAUSE OF BIRTH DEFECTS AND INFERTILITY, WHILE ANEUPLOIDY IN SOMATIC CELLS IS ASSOCIATED WITH TUMOR PROGRESSION. THE GOAL OF THIS PROJECT IS TO ELUCIDATE MECHANISMS THAT ALLOW CHROMOSOMES TO CORRECTLY ATTACH TO MICROTUBULES SO THEY SEGREGATE APPROPRIATELY IN MEIOSIS AND MITOSIS. THREE CONCURRENT PROJECTS WILL ADDRESS THIS GOAL. EXPERIMENTS IN PROJECT 1 WILL ELUCIDATE THE MECHANISMS USED TO PARTITION CHROMOSOMES IN MEIOSIS I. WHEN CELLS ENTER MEIOSIS, HOMOLOGOUS CHROMOSOMES ARE NOT CONNECTED TO EACH OTHER, BUT THEY BECOME LINKED BY CROSSOVERS (CHIASMA). THESE LINKAGES HELP HOMOLOGOUS CHROMOSOMES TO MOVE AWAY FROM EACH OTHER IN MEIOSIS I. THE LINKAGES TRANSMIT TENSION BETWEEN THE HOMOLOGOUS CENTROMERES WHEN THEY BECOME ATTACHED CORRECTLY TO MICROTUBULES FROM OPPOSITE SIDES OF THE SPINDLE. TENSION STABILIZES THESE ATTACHMENTS. A SECOND KIND OF CONNECTION CALLED CENTROMERE PAIRING ALLOWS THE CENTROMERES OF MODEL CHROMOSOMES TO BECOME LINKED. PROJECT 1 WILL EXPLORE THE BASIS BY WHICH CENTROMERES BECOME CONNECTED BY CENTROMERE PAIRING AND DETERMINE HOW OFTEN NATURAL CHROMOSOMES RELY ON CENTROMERE PAIRING TO ENSURE THEY SEGREGATE CORRECTLY AT MEIOSIS I. PROJECT 2 WILL DEFINE THE BIOPHYSICAL PROPERTIES OF CONNECTIONS MADE BY CENTROMERE PAIRING AND CHIASMATA IN TRANSMITTING TENSION BETWEEN CENTROMERES. PROJECT 2 WILL ALSO INVESTIGATE WHETHER CHIASMATA IN DIFFERENT CHROMOSOMAL POSITIONS HAVE DIFFERENT BIOPHYSICAL PROPERTIES THAT EXPLAIN THEIR DIFFERENT ABILITIES TO ENSURE PROPER SEGREGATION. A FINAL PROJECT FOCUSES MORE ON MITOSIS. CELLS WITH ABNORMALLY HIGH CHROMOSOME NUMBERS, FOR EXAMPLE MANY TUMOR CELL LINES AND POLYPLOID CELLS, RELY ON HIGH LEVEL EXPRESSION OF THE CELL CYCLE KINASE MPS1 FOR THEIR SURVIVAL. THE SAME IS TRUE FOR MEIOTIC CELLS. MPS1 PLAYS MANY ROLES, BUT THIS MPS1-ADDICTION OF CERTAIN VULNERABLE CELL TYPES APPEARS LINKED TO A ROLE FOR MPS1 IN ATTACHING CHROMOSOMES CORRECTLY TO MICROTUBULES IN MITOSIS AND MEIOSIS. THE EXPERIMENTS IN PROJECT 3 WILL TEST THE HYPOTHESIS THAT MPS1 MEDIATES A FORM OF CHROMOSOME MOVEMENT CALLED CHROMOSOME GLIDING THAT MIGHT HELP CHROMOSOMES MOVE TO FAVORABLE POSITIONS ON PROMETAPHASE SPINDLES. FINALLY, GENETIC SCREENS IN MPS1- ADDICTED MAMMALIAN AND YEAST CELLS WILL BE USED TO IDENTIFY THE VULNERABLE MPS1 PATHWAYS. THESE PROJECTS WILL BE COLLABORATIVE AND SYNERGISTIC AND REVEAL UNIQUE INSIGHTS INTO CENTROMERE BEHAVIOR IN MEIOSIS AND CHROMOSOME MICROTUBULE INTERACTIONS IN MITOSIS. THEY WILL INCLUDE EMERGING TECHNOLOGIES IN BUDDING YEAST AND COLLABORATIVE EFFORTS THAT EXPLOIT OTHER MODEL SYSTEMS TO ADVANCE OUR KNOWLEDGE OF CHROMOSOME BIOLOGY.

COMPREHENSIVE CANDIDATE PATHWAY ANALYSIS IN SLE

DEVELOPMENT OF SCALABLE METHODS FOR RAPID PHENOTYPING AND FUNCTIONAL TESTING OF VARIANTS - PROJECT SUMMARY ADVANCES IN SEQUENCING HAVE GREATLY IMPROVED OUR CAPACITY TO DISCOVER THE GENETIC CAUSES OF HUMAN DISEASES. MANY GENOME-WIDE ASSOCIATION STUDIES (GWAS) AND EXOME SEQUENCING PROJECTS HAVE UNCOVERED THOUSANDS OF CANDIDATE GENES LINKED TO HUMAN DISEASES. HOWEVER, HOW THESE GENES CONTRIBUTE TO DISEASE PATHOLOGY IS STILL UNCLEAR. RESEARCHERS USE ZEBRAFISH TO VALIDATE THESE CANDIDATE GENES AND IDENTIFY PATHOLOGICALLY RELEVANT GENES TO UNDERSTAND THE PATHOPHYSIOLOGY BETTER. CRISPR/CAS9 TECHNOLOGY HAS SIGNIFICANTLY IMPACTED FUNCTIONAL STUDIES IN ZEBRAFISH, ENABLING THE GENERATION OF LOSS-OF-FUNCTION ALLELES. GENE KNOCKOUT GENERATION IS EFFICIENT, BUT POINT MUTATIONS REMAIN CHALLENGING TO CREATE IN A HIGH-THROUGHPUT MANNER. UNDERSTANDING DISEASE PATHOPHYSIOLOGY THROUGH KNOCKOUT ALLELES IS INFORMATIVE BUT HYPOMORPHIC, AND GAIN-OF-FUNCTION ALLELES REQUIRE ADVANCED METHODS LIKE CRISPR BASE EDITORS. THESE TOOLS ARE UNDERUTILIZED DUE TO LIMITED TARGETING COVERAGE AND LOW EFFICIENCY, BUT EXPANDING THEIR COVERAGE WOULD BENEFIT THE RESEARCH COMMUNITY. IN THE FIRST AIM, WE WILL USE A KNOCKOUT LIBRARY OF 80 MUTANTS TO DEVELOP A HIGH-THROUGHPUT PHENOTYPING PIPELINE. THEN IN THE SECOND AIM, WE WILL OPTIMIZE AND EXPAND THE TARGETING COVERAGE OF CRISPR-BASED BASE EDITORS THAT CAN INDUCE SINGLE NUCLEOTIDE CHANGES IN THE GENOME IN A TARGETED MANNER. WE WILL THEN USE THE CANDIDATE GENES AND PHENOTYPE DATA FROM THE AIM 1 TO TEST THESE BASE EDITORS ON A LARGER NUMBER OF LOCI. THESE AIMS WILL GENERATE 80 DISEASE MODELS AND ROBUST BASE EDITING TOOLS TESTED ON A DIVERSE SET OF GENES. COMPLETING THESE AIMS WILL RESULT IN 80 DISEASE MODELS AND IMPROVED BASE EDITING TOOLS THAT WILL BE ACCESSIBLE TO THE RESEARCH COMMUNITY VIA THE ZEBRAFISH INTERNATIONAL RESOURCE CENTER (ZIRC).

INFLUENCE OF NEUROMELANIN ON SINGLE DOPAMINERGIC NEURONS IN PARKINSON'S DISEASE. - DOPAMINE NEURONS IN THE SUBSTANTIA NIGRA PARS COMPACTA (SNC) ARE EXQUISITELY SUSCEPTIBLE TO DEGENERATION AND ARE RESPONSIBLE FOR THE CARDINAL MOTOR SYMPTOMS OF PARKINSON’S DISEASE (PD). ALL CURRENTLY AVAILABLE TREATMENTS FOR PD SUCH AS L-DOPA FOCUS ON SYMPTOMS, AND NONE OF THE PROMISING RESULTS FROM PRECLINICAL STUDIES HAVE TRANSLATED TO A SINGLE DISEASE-MODIFYING THERAPEUTIC AGENT. IN HUMANS, CATECHOLAMINERGIC NEURONS, INCLUDING DOPAMINE NEURONS IN THE SNC, SPECIFICALLY EXPRESS THE DARK BROWN PIGMENT NEUROMELANIN AND ARE AMONG THE MOST SENSITIVE NEURONS TO DEGENERATION IN PD. NEUROMELANIN ACCUMULATION IS NOT OBSERVED IN RATS AND MICE, THUS THE CELLULAR AND SYSTEMIC EFFECTS OF NEUROMELANIN ACCUMULATION IN DOPAMINE NEURONS ARE NOT CURRENTLY UNDERSTOOD. WE HAVE RECENTLY DEVELOPED AND VALIDATED THE FIRST CRE-DEPENDENT ADENO-ASSOCIATED VIRUS (AAV-DIO-HTYR) THAT CAN BE USED TO EXPRESS THE HUMAN TYROSINASE ENZYME SPECIFICALLY IN DOPAMINE NEURONS OF DOPAMINE TRANSPORTER (DAT)-CRE MICE. PRELIMINARY RESULTS INDICATE THAT FOLLOWING VIRUS INJECTION, DOPAMINE NEURONS PROGRESSIVELY ACCUMULATE NEUROMELANIN, PRODUCING DISRUPTED CELLULAR PHYSIOLOGY, PD-LIKE MOTOR IMPAIRMENT, AND ULTIMATELY CATASTROPHIC DOPAMINE NEURON CELL DEATH. THIS PROJECT WILL LEVERAGE THE EXPERTISE OF THREE ESTABLISHED LABS WITH DEEP EXPERIENCE STUDYING NEURODEGENERATIVE DISEASE, CATECHOLAMINERGIC NEURONS, ELECTROPHYSIOLOGY, AND NEUROINFLAMMATION TO EXPLORE NEUROMELANIN ACCUMULATION IN THIS NOVEL MOUSE MODEL. THE HYPOTHESIS TO BE TESTED IS THAT PROGRESSIVE NEUROMELANIN ACCUMULATION DISRUPTS CELLULAR PHYSIOLOGY, NEURITIC MORPHOLOGY, BEHAVIOR, AND ULTIMATELY CAUSES NEUROINFLAMMATION AND CELL DEATH. EXPERIMENTS IN AIM 1 WILL DELINEATE THE ANATOMICAL AND BEHAVIORAL CONSEQUENCES OF NEUROMELANIN ACCUMULATION IN DOPAMINE NEURONS FROM MICE THAT HAVE AND HAVE NOT BEEN EXPOSED TO L-DOPA. EXPERIMENTS IN AIM 2 WILL USE PATCH CLAMP ELECTROPHYSIOLOGY TO DETERMINE THE EFFECTS OF NEUROMELANIN ON NEURONAL EXCITABILITY IN THE SNC AND THE ADJACENT VENTRAL TEGMENTAL AREA (VTA), WHICH IS SOMEWHAT PROTECTED IN PD. RESULTS WILL BE ASSESSED BY TIME POINT AND LEVEL OF NEUROMELANIN ACCUMULATION IN SINGLE NEURONS. EXPERIMENTS IN AIM 3 WILL USE PATCH-SEQUENCING TO CORRELATE ELECTROPHYSIOLOGICAL FINDINGS WITH MOLECULAR PHENOTYPES IN SINGLE DOPAMINE NEURONS FOLLOWING NEUROMELANIN ACCUMULATION AND L-DOPA TREATMENT. ASTROCYTE AND MICROGLIA WILL BE ALSO TESTED FOR INFLAMMATORY AND DISEASE-RELATED PHENOTYPES IN THE MIDBRAIN AND ALSO THE STRIATUM, THE MAIN TERMINAL FIELD OF DOPAMINE NEURONS. RESULTS WILL BE COMPARED TO OTHER MODELS AND POSTMORTEM DATA FROM PD PATIENTS. THESE STUDIES WILL BE THE FIRST TO EXPLORE THE DISTINCT FACTORS INTRODUCED BY NEUROMELANIN ACCUMULATION AND L-DOPA TREATMENT IN MOUSE DOPAMINE NEURONS. RESULTS WILL FILL A KEY KNOWLEDGE GAP IN THE PD FIELD AND INFORM ONGOING AND FUTURE STUDIES INTO PD PATHOGENESIS THAT COULD BE TRANSFORMATIVE.

EQUIPMENT SUPPLEMENT FOR CENTROMERE INTERACTIONS AND MEIOTIC CHROMOSOME SEGREGATION IN YEAST - PROJECT SUMMARY HOMOLOGOUS CHROMOSOMES ENTER MEIOSIS UNCONNECTED TO EACH OTHER, BUT THEY BECOME LINKED BY CROSSOVERS (EXCHANGES). THESE LINKAGES HELP THE HOMOLOGOUS CHROMOSOMES TO MOVE AWAY FROM EACH OTHER IN MEIOSIS I. THE LINKAGES TRANSMIT TENSION BETWEEN THE HOMOLOGOUS CENTROMERES WHEN THEY BECOME ATTACHED TO MICROTUBULES FROM OPPOSITE SIDES OF THE SPINDLE (BI-ORIENTED). TENSION STABILIZES THESE MICROTUBULE ATTACHMENTS. ACCORDINGLY, FAILURES IN CROSSING-OVER CAN RESULT IN MEIOTIC ERRORS AND ANEUPLOID GAMETES. THE PAIRING OF HOMOLOGOUS CENTROMERES (CEN-PAIRING) IN MEIOTIC PROPHASE, IS A RECENTLY DISCOVERED, CONSERVED PHENOMENON. IN YEAST, CEN-PAIRING HELPS THE PARTNER CHROMOSOMES, LATER, IN ANAPHASE I, TO SEGREGATE PROPERLY, EVEN IF THEY HAVE FAILED TO EXPERIENCE A CROSSOVER. THE MECHANISM BY WHICH CEN-PAIRING PROMOTES SEGREGATION IS UNKNOWN. WHILE CROSSOVER FORMATION HAS BEEN INVESTIGATED FOR DECADES, VERY LITTLE IS KNOWN ABOUT HOW CEN-PAIRING PROMOTES MEIOTIC CHROMOSOME SEGREGATION. THIS IS THE FOCUS OF OUR PROPOSAL. IN AIM 1 WE TEST THE HYPOTHESIS THAT THE CEN-PAIRING ALLOWS THE FORMATION OF CENTROMERE-CENTROMERE CONNECTIONS THAT IMPROVE THE SEGREGATION FIDELITY OF MEIOTIC CHROMOSOME PARTNERS. WE WILL USE LIVE CELL IMAGING TO MONITOR THE MEIOTIC BEHAVIOR OF CHROMOSOME PAIRS HAVE OR HAVE NOT UNDERGONE CEN-PAIRING. IN AIM 2 WE WILL TEST THE HYPOTHESIS THAT CONNECTIONS FORMED DURING CEN-PAIRING ARE MEDIATED BY COHESIN PROTEINS. AIM 3 WILL EXPLORE MECHANISM BY WHICH CENTROMERE-CENTROMERE CONNECTIONS HELP PARTNER CHROMOSOMES BECOME BI-ORIENTED ON THE SPINDLE. WE WILL DIRECTLY MEASURE THE SPRING-LIKE PROPERTIES OF THE CENTROMERE-TO-CENTROMERE CONNECTIONS USING BIO-PHYSICAL APPROACHES AND TEST WHETHER PARTNERS WITH A CENTROMERE-CENTROMERE CONNECTION CAN USE THE TENSION-SENSING SIGNALING PATHWAYS. IN ADDITION, THESE EXPERIMENTS WILL TEST THE HYPOTHESIS THAT CENTROMERE-CENTROMERE CONNECTIONS ARE IMPORTANT TO IMPROVE THE SEGREGATION FIDELITY OF CHROMOSOME PAIRS LINKED BY CROSSOVERS THAT ALONE WOULD BE INEFFECTIVE IN CREATING A TENSION-TRANSMITTING BRIDGE AS THE PAIR ATTACHES TO MICROTUBULES. TOGETHER, THE EXPERIMENTS IN THIS PROJECT WILL ELUCIDATE THE REASONS THAT SOME CROSSOVERS DO, AND SOME DON’T, EFFECTIVELY ENSURE HIGH FIDELITY CHROMOSOME SEGREGATION IN MEIOSIS AND WILL EXPLAIN HOW THE RECENTLY DESCRIBED PROCESS OF CENTROMERE PAIRING CAN MEDIATE THE FORMATION OF A CENTROMERE- TO-CENTROMERE CONNECTION BETWEEN HOMOLOGS THAT AUGMENTS THE FUNCTIONALITY OF CROSSOVERS, OR CAN ACT ALONE WHEN CROSSOVERS FAIL.

T CELL TOLERANCE & AUTOIMMUNITY TO NUCLEAR ANTIGEN LA

EFFECTS OF TOBACCO SMOKING ON PROGNOSIS AND IMMUNE FUNCTION IN MULTIPLE SCLEROSIS - PROJECT SUMMARY MULTIPLE SCLEROSIS (MS) IS A CHRONIC AUTOIMMUNE DISORDER CHARACTERIZED BY CENTRAL NERVOUS SYSTEM (CNS) INFLAMMATION AND NEURONAL DEMYELINATION. DESPITE THERAPEUTIC ADVANCEMENTS, THE CLINICAL HETEROGENEITY AMONG MS PATIENTS PRESENTS CHALLENGES IN ACCURATELY PREDICTING AND MONITORING DISEASE PROGRESSION. WHILE PREVIOUS STUDIES HAVE LINKED POOR PROGNOSIS IN MS PATIENTS TO ELEVATED TH17 AND B CELL ACTIVITIES, THE UNDERLYING REASONS FOR THE ACTIVATION OF THESE PATHWAYS REMAIN UNCLEAR. IN OUR EXPLORATORY PROTEOMIC STUDY FOCUSED ON RELAPSING-REMITTING MS (RRMS) PATIENTS, WE DISCOVERED THAT CORRELATION BETWEEN POOR PROGNOSIS AND HEIGHTENED TH17 AND B CELL ACTIVATION PATHWAYS IS INFLUENCED BY TOBACCO SMOKING. BUILDING ON OUR PRELIMINARY DATA AND EXISTING RESEARCH, OUR CENTRAL HYPOTHESIS SUGGESTS THAT CIGARETTE SMOKE DIRECTLY INDUCES THE RELEASE OF DAMAGE-ASSOCIATED MOLECULAR PATTERNS (DAMPS), LEADING TO INCREASED INFLAMMATORY TH17 AND B CELL ACTIVITIES, THEREBY EXACERBATING MS PROGNOSIS. WE FURTHER PROPOSE THAT TOBACCO SMOKE, THROUGH EXOSOME RELEASE OF DAMPS, AMPLIFIES THE INFLAMMATORY FUNCTION OF B CELLS AND TH17 CELLS. NOTABLY, OUR PRELIMINARY DATA INDICATE THAT B CELL DEPLETION THERAPY MITIGATES THE ADVERSE EFFECTS OF SMOKING. THIS PROPOSAL HAS THREE SPECIFIC AIMS. IN AIM 1, WE PLAN TO CONFIRM OUR HYPOTHESIS THAT TOBACCO SMOKING ELEVATES TH17 AND B CELL ACTIVITY BY ASSESSING IMMUNE POPULATIONS IN THE BLOOD OF SMOKERS AND NON-SMOKERS USING FLOW CYTOMETRY AND SINGLE-CELL TRANSCRIPTOMICS. AIM 2 FOCUSES ON DETERMINING THE IMMUNOLOGICAL EFFECTS OF B CELL DEPLETION AND NON-B CELL DEPLETION THERAPY ON PATIENTS WHO ARE SMOKERS AND NON-SMOKERS. IN AIM 3, WE PLAN TO IDENTIFY MOLECULAR DIFFERENCES IN EXOSOMES FROM SMOKERS AND NON-SMOKERS AND INVESTIGATE WHETHER SMOKER EXOSOMES ENHANCE THE INFLAMMATORY FUNCTION OF TH17 CELLS AND B CELLS. SUCCESSFUL COMPLETION OF THIS PROJECT HOLDS THE POTENTIAL TO ADDRESS CRITICAL KNOWLEDGE GAPS IN MS RESEARCH. FIRST, IT AIMS TO PROVIDE MECHANISTIC INSIGHTS INTO THE LINK BETWEEN SMOKING, MS DEVELOPMENT, AND WORSENED PROGNOSIS. SECOND, BY IDENTIFYING POTENTIAL BIOMARKERS IN CONJUNCTION WITH ENVIRONMENTAL FACTORS, THIS RESEARCH COULD CONTRIBUTE TO IMPROVED CLINICAL MANAGEMENT AND PROGNOSIS PREDICTION IN MS.

GENETIC ASSOCIATION IN AMERICAN BLACKS WITH LUPUS

VALIDATION OF EXTRACELLULAR HISTONES AS BIOMARKER AND THERAPEUTIC TARGET IN SEPSI

IDENTIFICATION OF SUSCEPTIBILITY GENES FOR SLE OF AMERINDIAN ORIGIN IN HISPANICS

ELUCIDATING THE IMPACT OF IFIH1 DURING SYSTEMIC LUPUS ERYTHEMATOSUS

PREDICTING SYSTEMIC AUTOIMMUNITY THROUGH FOLLOW-UP OF BLOOD RELATIVES

IMPROVING RECRUITMENT OF MINORITY PATIENTS TO LUPUS TRIALS THROUGH AN EDUCATIONAL PROGRAM WITH NEW CONTENT AND METHODS

MODULATION OF MITOCHONDRIAL FUNCTION BY PRO-OXIDANTS

REGULATION OF GENE TRANSCRIPTION AND DNA REPLICATION BY CHROMATIN READERS FROM THE BET FAMILY - PROJECT SUMMARY PRECISE AND COORDINATED CONTROL OF GENE TRANSCRIPTION AND DNA REPLICATION IS NECESSARY FOR CELL GROWTH, PROLIFERATION, AND DIFFERENTIATION. DYSREGULATION OF TRANSCRIPTION AND DNA REPLICATION CAN RESULT IN TRANSCRIPTION- REPLICATION CONFLICTS AND GENOME INSTABILITY, OFTEN LEADING TO CANCER AND OTHER HUMAN DISEASES. DESPITE SIGNIFICANT PROGRESS IN UNDERSTANDING HOW TRANSCRIPTION AND DNA REPLICATION ARE REGULATED, THE MECHANISMS RESPONSIBLE FOR COORDINATED CONTROL OF THESE PROCESSES ARE LARGELY UNKNOWN. OUR RESEARCH PROGRAM AIMS TO ADDRESS THIS KNOWLEDGE GAP BY COMBINING OUR PRIOR WORK ON TRANSCRIPTION WITH A NEW LINE OF INVESTIGATION INTO DNA REPLICATION. WE WILL INITIALLY FOCUS ON ELUCIDATING THE ROLES OF CHROMATIN READERS FROM THE BROMODOMAIN AND EXTRA-TERMINAL DOMAIN (BET) FAMILY, WHICH ARE KNOWN TO BE IMPORTANT REGULATORS OF TRANSCRIPTION. BET PROTEINS HAVE ADDITIONALLY BEEN IMPLICATED IN DNA REPLICATION AND THE RESPONSE TO REPLICATION STRESS, BUT THE MECHANISTIC ROLES BET PROTEINS PLAY IN THESE PROCESSES HAVE NOT BEEN DETERMINED. OUR PRELIMINARY FINDINGS REVEALED THAT BET PROTEINS ARE INVOLVED IN DNA REPLICATION INITIATION. BUILDING ON THIS FOUNDATION, WE ARE WELL-POSITIONED TO EXPLORE THE FUNDAMENTAL MECHANISMS THAT REGULATE AND COORDINATE TRANSCRIPTION AND DNA REPLICATION. WE WILL FOCUS ON THREE BROAD RESEARCH AREAS: 1) THE ROLES OF BET PROTEINS IN REGULATING A TEMPORAL PROGRAM OF ORIGIN ACTIVATION. THE TEMPORAL PROGRAM OF ORIGIN ACTIVATION IS IMPORTANT FOR AVOIDING TRANSCRIPTION-REPLICATION CONFLICTS. OUR PRELIMINARY RESULTS ILLUSTRATED THAT BET PROTEINS FACILITATE THE RECRUITMENT OF A FACTOR REQUIRED FOR ORIGIN ACTIVATION. WE WILL BUILD ON THESE RESULTS TO CHARACTERIZE THE MECHANISMS OF BET PROTEINS IN ORIGIN ACTIVATION AND IN ORCHESTRATING THE TIMING OF ORIGIN FIRING. 2) THE ROLES OF BET PROTEINS IN MANAGING TRANSCRIPTION-REPLICATION CONFLICTS. PRIOR STUDIES AND OUR PRELIMINARY DATA SUGGEST THAT BET PROTEINS ARE IMPORTANT FOR AVOIDING TRANSCRIPTION-REPLICATION CONFLICTS AND MITIGATING THEIR CONSEQUENCES. HERE, WE WILL CHARACTERIZE THE ROLES OF BET PROTEINS IN COORDINATING TRANSCRIPTION AND DNA REPLICATION AND RESOLVING TRANSCRIPTION-REPLICATION CONFLICTS. 3) CELL CYCLE-DEPENDENT INTERACTIONS INVOLVING BET PROTEINS. BASED ON CURRENT UNDERSTANDING, THE COMPLEX ROLES OF BET PROTEINS ARE MEDIATED THROUGH INTERACTIONS WITH OTHER FACTORS, BUT THE ENSEMBLE OF BET PROTEIN PARTNERS REMAINS TO BE DETERMINED. PRIOR STUDIES AND OUR PRELIMINARY DATA SUGGEST THAT THE COMPOSITION OF FACTORS INTERACTING WITH BET PROTEINS MAY DEPEND ON THE CELL CYCLE STAGE AND REPLICATION STRESS. THUS, WE WILL DEFINE THE BET PROTEIN INTERACTION NETWORK AND DETERMINE IF AND HOW THIS NETWORK CHANGES DURING CELL CYCLE PROGRESSION AND IN RESPONSE TO REPLICATION STRESS. WE WILL USE A COMBINATION OF GENOMIC, PROTEOMIC, BIOCHEMICAL, AND COMPUTATIONAL APPROACHES TO ADDRESS THESE RESEARCH AREAS. OUR STUDIES WILL ADVANCE CURRENT MODELS OF REGULATORY DYNAMICS IN THE CELL NUCLEUS AND FACILITATE THE LONG-TERM GROWTH OF OUR RESEARCH PROGRAM TOWARD A COMPREHENSIVE UNDERSTANDING OF THE FUNDAMENTAL MECHANISMS THAT MAINTAIN THE FLOW OF GENETIC INFORMATION AND SAFEGUARD GENOME INTEGRITY.

EPIGENETIC REGULATION OF ENDOTHELIAL CELL NECROPTOSIS IMPACTS VASCULAR INTEGRITY

AN INTEGRATIVE STUDY OF CIRCULATING LEUKOCYTE COMPOSITION, EPIGENETIC PATTERNS, AND FUNCTIONAL CONSEQUENCES IN KNEE OSTEOARTHRITIS.

INTRAARTICULAR MICROBIAL DNA AS A NOVEL MEDIATOR OF OSTEOARTHRITIS

OPTIMIZED ORAL TREATMENT OF ACUTE ACOUSTIC TRAUMA (AAT)

FUNCTION AND REGULATION OF DNA REPLICATION ORIGIN FIRING FACTORS - ABSTRACT DNA REPLICATION IS A HIGHLY DYNAMIC AND DEEPLY REGULATED PROCESS DURING WHICH THOUSANDS OF REPLICATION ORIGINS WILL FIRE. THE EFFICIENCY OF THESE ORIGIN FIRING EVENTS IS HIGHLY VARIABLE ACROSS GENOMIC REGIONS AND CHANGES THROUGHOUT S-PHASE. THIS VARIABILITY IN ORIGIN FIRING EFFICIENCY CARRIES PROFOUND IMPLICATIONS, AS ITS DEREGULATION LEADS TO GENOME INSTABILITY AND CANCER. DESPITE ITS CRITICAL IMPORTANCE, OUR CURRENT UNDERSTANDING OF THE FUNDAMENTAL MECHANISMS GOVERNING THESE VARIABLE ORIGIN FIRING EFFICIENCIES AT DIFFERENT GENOMIC LOCATIONS AND TIMES DURING S PHASE REMAINS SURPRISINGLY LIMITED. THIS PROPOSAL’S OVERALL OBJECTIVE IS TO ADDRESS CRITICAL GAPS IN UNDERSTANDING THE REGULATION OF ORIGIN FIRING EFFICIENCY, FOCUSING ON THE RECRUITMENT OF TWO ESSENTIAL ORIGIN FIRING FACTORS CALLED TICRR/TRESLIN AND MTBP, TO REPLICATION ORIGINS THROUGHOUT THE CELL CYCLE. THE RESEARCH PROPOSAL SEEKS TO COMPLETE THREE SPECIFIC AIMS: 1) DETERMINE THE MECHANISMS FOR REPLICATION ORIGIN SELECTION BY TICRR AND MTBP; 2) DEFINE ROLES FOR TRANSCRIPTION FACTORS AND CHROMATIN MODIFIED IN DETERMINING WHERE REPLICATION FACTORS BIND AND REPLICATION ORIGINS FIRE; 3) UNCOVER HOW TICRR/TRESLIN AND MUVB-MYBL2 FUNCTIONALLY INTERACT TO COORDINATE MITOTIC ENTRY WITH THE COMPLETION OF DNA REPLICATION. THE APPLICANT HAS CREATED A SERIES OF HUMAN CELL LINES THAT WILL SERVE AS IDEAL MODELS FOR ELUCIDATING THE MECHANISM OF ACTION FOR SPECIFIC REPLICATION INITIATION PROTEINS THROUGH APPROACHES INCLUDING FLOW CYTOMETRY-BASED ASSAYS, CUT&RUN, OPTICAL REPLICATION MAPPING, CHIP-QPCR AND SLAM-SEQ. IN AIM 1, THE APPLICANT WILL EXAMINE THE SPECIFIC CONTRIBUTIONS OF TICRR AND MTBP TO ORIGIN SITE SPECIFICATION AS CELLS PROGRESS FROM G1 TO EARLY AND LATE S-PHASE. AIM 2 WILL TEST WHETHER ORIGIN SITE SPECIFICATION DEPENDS ON PHYSICAL INTERACTIONS, WHICH THE APPLICANT DISCOVERED, BETWEEN TICRR AND A TRANSCRIPTION FACTOR AND HISTONE ACETYLATION READER PROTEINS. AIM 3 WILL EXAMINE THE COORDINATION OF GENE ACTIVATION OF G2/M GENES WITH THE COMPLETION OF DNA REPLICATION USING BOTH CULTURED HUMAN CELLS AND ZEBRAFISH EMBRYOS. THIS COORDINATION IS CRUCIAL FOR CELL CYCLE PROGRESSION, YET IT REMAINS UNDER-RESEARCHED, HIGHLIGHTING ITS POTENTIAL IMPACT. THE OVERALL RESEARCH IN THIS PROPOSAL IS INNOVATIVE BECAUSE IT EXAMINES A MECHANISM BY WHICH CELLS ACTIVELY CONTROL THE ACTIVATION OF REPLICATION ORIGINS AND COORDINATE THE COMPLETION OF DNA REPLICATION WITH MITOTIC ENTRY. SUCCESSFUL ACHIEVEMENT OF THE AIMS PRESENTED IN THIS GRANT WILL BE SIGNIFICANT BECAUSE IT WILL HELP ANSWER LONG-STANDING, FUNDAMENTAL QUESTIONS ABOUT HOW THE GENOME IS ACCURATELY AND COMPLETELY REPLICATED. IMPORTANTLY, THE STUDIES ARE FOCUSED ON DELINEATING MECHANISMS THAT MAY BE MANIPULATED FOR TREATING DISEASES SUCH AS CANCER.

COMPREHENSIVE CANDIDATE PATHWAY ANALYSIS IN SLE

UNDERSTANDING SARCOIDOSIS SUSCEPTIBILITY AND DISEASE BURDEN THROUGH MULTI OMICS - ABSTRACT SARCOIDOSIS IS A SYSTEMIC GRANULOMATOUS DISEASE WITH STRIKING HETEROGENEITY OF CLINICAL COURSE AND INCREASING MORTALITY AND MORBIDITY RATES. DIAGNOSIS IS DIFFICULT AND PREDICTING DISEASE OUTCOMES IS VIRTUALLY IMPOSSIBLE. WHILE IT IS KNOWN THAT SARCOIDOSIS LIKELY INVOLVES HOST GENETIC SUSCEPTIBILITY AND A DYSREGULATED IMMUNE RESPONSE TO ANY NUMBER OF ENVIRONMENTAL FACTORS, THE MECHANISMS BY WHICH GRANULOMAS FORM AND THE DETERMINANTS OF SEVERITY AND DISEASE MANIFESTATIONS REMAIN ELUSIVE. OUR TEAM HAS BEEN AT THE FOREFRONT OF GENETICS AND TRANSCRIPTOMICS IN SARCOIDOSIS, INCLUDING THE IDENTIFICATION OF GENES FOR SUSCEPTIBILITY, SEVERITY, ANCESTRY- AND ORGAN-SPECIFIC EFFECTS, THE FIRST AND ONLY SINGLE-CELL RNA SEQUENCING STUDY OF SARCOIDOSIS AND THE FIRST GWAS OF PULMONARY FIBROSIS IN PATIENTS OF AFRICAN ANCESTRY (AA). THESE FINDINGS PROVIDE THE FOUNDATION FOR FURTHER DISSECTION OF CELLULAR MECHANISMS THAT LEAD TO SYSTEMIC IMMUNE DYSREGULATION AND TISSUE-SPECIFIC INFLAMMATORY RESPONSE. OUR CURRENT PROPOSAL WILL ADVANCE THE FIELD BY FILLING CRITICAL GAPS IN SARCOIDOSIS RESEARCH, INCLUDING THE LACK OF MECHANISM AND BIOMARKERS FOR SARCOIDOSIS AND ITS PATIENT-SPECIFIC DISEASE BURDEN, POOR CONSENSUS OF EXISTING GENETIC AND GENOMIC STUDIES, AND THE ABSENCE OF MECHANISTIC CONNECTIONS BETWEEN IMMUNE PROFILES IN THE PERIPHERY AND AFFECTED ORGANS. WE WILL EXPLOIT THE CLINICAL, IMMUNOLOGICAL, GENOMIC, AND BIOINFORMATIC EXPERTISE OF OUR TEAM TO APPLY AN INNOVATIVE, AND INTEGRATIVE MULTI-OMIC APPROACH TO FIL THESE GAPS. SPECIFICALLY, IN AIM 1, WE WILL USE HIGH-THROUGPUT PLASMA PROTEOMICS FROM OVER 1000 SARCOIDOSIS PATIENTS OF AA AND EUROPEAN ANCESTRY (EA), 800 HEALTHY AND 275 DISEASED CONTROLS TO A) DISTINGUISH SARCOIDOSIS PATIENTS FROM HEALTHY AND OTHER IMMUNE-MEDIATED DISEASE CONTROLS, B) TO DISTINGUISH PATIENTS WITH HIGH DISEASE BURDEN FROM THOSE WITH LOW DISEASE BURDEN, AND C) ELUCIDATE ANCESTRY AND DISEASE COURSE SPECIFIC MECHANISMS AND BIOMARKERS. IN AIM 2, WE WILL IDENTIFY CANDIDATE GENES FOR SUSCEPTIBILITY TO AND DISEASE BURDEN OF SARCOIDOSIS BY A) CONDUCTING A LARGE TWAS OF OVER 2,000 EA AND AA PATIENTS COMPARED TO 3900 HEALTHY CONTROLS, AND B) REPLICATION AND CHARACTERIZATION OF THESE EFFECTS VIA SCRNASEQ OF AN INDEPENDENT COHORT, AND C) PRIORITIZE TRANSCRIPTOMIC EFFECTS VIA COLOCALIZATION OF GENETIC AND QTL EFFECTS. FINALLY, IN AIM 3, WE WILL A) IDENTIFY CANDIDATE GENES THAT ARE DIFFERENTIALLY EXPRESSED IN GRANULOMAS VIA SPATIAL TRANSCRIPTOMICS AND WHOSE PROTEIN PRODUCTS ARE DYSREGULATED IN BRONCHOALVEOLAR LAVAGE (BAL) AND B) IDENTIFY GENES BEST SUITED FOR FUNCTIONAL AND DRUG TARGET STUDIES BASED ON INTEGRATING THE DATA COLLECTED ACROSS MULTIPLE BIOLOGICAL SYSTEMS AND DATA TYPES. THE SUCCESS OF OUR WORK WILL BE FACILITATED BY THE EXTENSIVE GENETIC, GENOMIC, AND CLINICAL DATA AVAILABLE TO US FROM OUR OWN COHORTS AND THE TOPMED CONSORTIUM (OF WHICH WE ARE MEMBERS) AND ITS RIGOR SUPPORTED BY STRONG PRELIMINARY DATA INDICATIVE OF NOVEL THERAPIES FOR AA AND HIGH DISEASE BURDEN PATIENTS.

A NOVEL APPROACH TO UNDERSTAND A MECHANISM OF PROTEOSTATIC DECLINE WITH AGING

FUNCTIONAL DISSECTION OF BET CHROMATIN READERS IN TRANSCRIPTION -THE PRECISE CONTROL OF GENE TRANSCRIPTION IS NECESSARY FOR THE NORMAL DEVELOPMENT OF CELLS. MISTAKES IN THIS PROCESS CAN HAVE DETRIMENTAL CONSEQUENCES. IN EUKARYOTIC ORGANISMS, FROM SIMPLE UNICELLULAR YEAST TO HUMANS, DNA IS STORED AS CHROMATIN, WHICH CONSISTS OF A DNA STRAND WRAPPED AROUND PROTEINS CALLED HISTONES. MANY PROTEINS THAT REGULATE TRANSCRIPTION CAN ?READ? CHROMATIN THROUGH SPECIFIC MODIFICATIONS ON HISTONES, AND THIS ACTIVITY HELPS GUIDE THEIR FUNCTION. PROTEINS FROM THE BROMODOMAIN AND EXTRA-TERMINAL DOMAIN (BET) FAMILY ARE CHROMATIN READERS THAT REGULATE THE TRANSCRIPTION OF MOST GENES IN EUKARYOTES. DESPITE THE IMPORTANCE OF BET PROTEINS IN TRANSCRIPTION, THE SPECIFIC ROLES OF THESE PROTEINS ARE NOT FULLY UNDERSTOOD. THE PROJECT WILL ADDRESS THIS GAP IN KNOWLEDGE BY INVESTIGATING THE ROLES OF BET PROTEINS IN BAKER?S YEAST, WHICH IS AN EXCELLENT MODEL FOR UNDERSTANDING PROCESSES IN HUMAN CELLS. THE PROJECT WILL HAVE IMPORTANT SOCIETAL IMPACTS BY PROVIDING EXPERIENCE IN ORIGINAL, HYPOTHESIS-DRIVEN RESEARCH FOR A BROADLY DIVERSE GROUP OF YOUNG SCIENTISTS, FROM DIFFERENT BACKGROUNDS AND AT DIFFERENT LEVELS OF EDUCATION, FROM HIGH SCHOOL TO COLLEGE STUDENTS. THERE ARE IMPORTANT GAPS IN UNDERSTANDING THE MAJOR STEPS OF GENE TRANSCRIPTION DUE TO LIMITED INSIGHT INTO THE BIOLOGY OF BET PROTEINS. FOR EXAMPLE, OTHER THAN THE BROMODOMAINS, WHICH TARGET BET PROTEINS TO ACETYLATED CHROMATIN, THE FUNCTIONS OF CONSERVED DOMAINS OF BET PROTEINS ARE LARGELY UNKNOWN. FURTHER, RECENT FINDINGS INDICATE THAT THERE ARE MECHANISMS OF BET PROTEIN RECRUITMENT THAT DO NOT INVOLVE BROMODOMAINS. THE OVERALL OBJECTIVE OF THE PROJECT IS TO DETERMINE THE CONTRIBUTIONS OF ALL CONSERVED DOMAINS OF BET PROTEINS TO TRANSCRIPTION IN YEAST. THE OBJECTIVE WILL BE ACCOMPLISHED BY DEFINING THE CONTRIBUTIONS OF CONSERVED BET PROTEIN DOMAINS TO MRNA SYNTHESIS, BET PROTEIN RECRUITMENT TO CHROMATIN, AND FORMATION OF THE TRANSCRIPTION PRE-INITIATION COMPLEX AT GENE PROMOTERS. THE PROJECT WILL LEVERAGE POWERFUL YEAST GENETIC TOOLS AND MODERN GENOMIC TECHNOLOGIES TO FACILITATE HIGH-THROUGHPUT INVESTIGATION. THE OUTCOME OF THE PROJECT WILL BE THE FIRST SYSTEMATIC CHARACTERIZATION OF THE MECHANISMS BY WHICH BET PROTEINS REGULATE TRANSCRIPTION IN A SINGLE MODEL SYSTEM. THUS, THE PROJECT WILL EXPAND THE MODEL OF TRANSCRIPTIONAL REGULATION IN YEAST, AND DUE TO THE CONSERVATION OF BET PROTEINS, IT WILL DIRECT FUTURE STUDIES IN DIVERSE EUKARYOTES. 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.- SUBAWARDS ARE NOT PLANNED FOR THIS AWARD.

ATP-DEPENDENT CHROMATIN-REMODELING COMPLEXES AND VASCULAR DEVELOPMENT

MOLECULAR MECHANISMS OF RAPAMYCIN - INDUCED REVERSAL OF CARDIAC AGING

MOLECULAR STRATIFICATION OF MULTIPLE SCLEROSIS AND ASSOCIATED NEURO-AUTOIMMUNE DI

MPS1 AND REGULATION OF KINETOCHORE-MICROTUBULE INTERACTIONS IN MEIOSIS

FREE RADICALS AND REDOX SIGNALING TRIGGERED BY LIPOPOLYSCCHARIDE IN MACROPHAGES

DISSECTING INFLAMMATORY AND IMMUNE MECHANISMS DRIVING OVARIAN TISSUE AGING - PROJECT SUMMARY/ABSTRACT AGE-RELATED OVARIAN DYSFUNCTION IS ACCOMPANIED BY SUSTAINED, LOW-GRADE INFLAMMATION AND MALADAPTIVE IMMUNE ACTIVATION—CHARACTERIZED BY IMMUNE CELL ACCUMULATION, FIBROSIS, AND MULTINUCLEATED GIANT CELL (MNGC) FORMATION. THESE PATHOLOGICAL FEATURES ARISE BEFORE REPRODUCTIVE SENESCENCE AND PRECEDE INFLAMMATORY REMODELING SEEN DURING AGING IN OTHER TISSUES. YET, THE CELLULAR DRIVERS, SIGNALING MECHANISMS, AND INTERCELLULAR COMMUNICATION UNDERLYING THESE INFLAMMATORY RESPONSES IN OVARIAN TISSUE REMAIN LARGELY UNKNOWN. THIS PROPOSAL FOCUSES ON HOW INNATE AND ADAPTIVE IMMUNE COMPONENTS CONTRIBUTE TO CHRONIC, LOW-GRADE INFLAMMATION AND STRUCTURAL TISSUE REMODELING IN THE OVARY. WE AIM TO DEFINE HOW IMMUNE-TISSUE CROSSTALK PROMOTES FIBROSIS, IMMUNE CELL ACCUMULATION AND ACTIVATION, AND OTHER AGING HALLMARKS. OUR PRELIMINARY DATA IDENTIFIED PROINFLAMMATORY PROGRAMS THAT ARE ACTIVATED IN A CELL-TYPE-SPECIFIC MANNER DURING EARLY OVARIAN AGING, IMPLICATING GRANULOSA CELLS, TISSUE-RESIDENT MACROPHAGES (TRMS), AND TYPE 17 T CELLS IN A CASCADE OF NON-RESOLVING INFLAMMATORY SIGNALING. OUR CENTRAL HYPOTHESIS IS THAT GRANULOSA CELL–INTRINSIC NF-ΚB ACTIVATION INITIATES A FEED- FORWARD INFLAMMATORY LOOP WITH TRMS AND TYPE 17 T CELLS, RESULTING IN TISSUE FIBROSIS, MNGC FORMATION, AND IMMUNE-MEDIATED OVARIAN DYSFUNCTION. WE WILL TEST THIS HYPOTHESIS THROUGH THE FOLLOWING MECHANISTIC AIMS: AIM 1: DETERMINE WHETHER NF-ΚB SIGNALING IN GRANULOSA CELLS ORCHESTRATES PARACRINE IMMUNE ACTIVATION. WE PREDICT THAT NF-ΚB ACTIVATION INITIATES PROINFLAMMATORY SIGNALING THAT PROMOTES THE RECRUITMENT AND ACTIVATION OF TRMS AND TYPE 17 T CELLS. CONDITIONAL ABLATION OF NF-ΚB IN GRANULOSA CELLS WILL ALLOW US TO ISOLATE ITS ROLE IN INITIATING IMMUNE CROSSTALK AND DOWNSTREAM FIBROTIC PATHOLOGY. AIM 2: DEFINE THE FUNCTIONAL ROLE OF TRMS IN SUSTAINING OVARIAN INFLAMMATION. WE HYPOTHESIZE THAT OVARIAN TRMS ACT AS CRITICAL AMPLIFIERS OF AGE-RELATED INFLAMMATORY RESPONSES AND TISSUE REMODELING. USING TARGETED MACROPHAGE DEPLETION STRATEGIES, WE WILL ASSESS WHETHER TRMS ARE REQUIRED FOR T CELL EXPANSION, FIBROTIC SIGNALING, AND MNGC FORMATION. AIM 3: CHARACTERIZE TYPE 17 T CELLS IN THE AGING OVARY AND THEIR CONTRIBUTION TO FIBROTIC REMODELING. TYPE 17 T CELLS HAVE BEEN IMPLICATED IN CHRONIC INFLAMMATION ACROSS TISSUES, BUT THEIR ROLE IN OVARIAN AGING IS UNEXPLORED. WE WILL ASSESS THEIR PHENOTYPE, SPATIAL LOCALIZATION, AND IMPACT ON INFLAMMATORY SIGNALING AND FIBROTIC CHANGES FOLLOWING TARGETED DEPLETION. THIS RESEARCH WILL ALSO DETERMINE THE TEMPORAL HIERARCHY OF IMMUNE ACTIVATION IN OVARIAN AGING AND HOW INTERCELLULAR INFLAMMATORY SIGNALING EVOLVES ACROSS THE TISSUE. BY DELINEATING CELL-TYPE-SPECIFIC CONTRIBUTIONS TO CHRONIC INFLAMMATION, THIS WORK WILL ALSO PROVIDE A BLUEPRINT FOR UNDERSTANDING IMMUNE-MEDIATED AGING IN OTHER TISSUES. RATHER THAN FOCUSING ON REPRODUCTIVE ENDPOINTS, WE USE THE OVARY AS A MODEL OF IMMUNE-DRIVEN FIBROSIS AND STERILE INFLAMMATION—PROCESSES CENTRAL TO AGING IN MULTIPLE ORGANS. FINDINGS FROM THIS WORK WILL IDENTIFY NOVEL, IMMUNOLOGICAL TARGETS FOR INTERVENTION THAT COULD BE USED TO DELAY OVARIAN AGING AND BENEFIT OVERALL HEALTH.

EPSIN: A NOVEL REGULATOR IN PROSTATE CANCER PROGRESSION AND METASTASIS

EXPLORING THE THYMIC ORIGIN OF GROUP 2 INNATE LYMPHOID CELLS - ABSTRACT INNATE LYMPHOID CELLS (ILCS) PLAY DIVERSE ROLES IN SHAPING INNATE AND ADAPTIVE IMMUNITY. THESE CELLS EXIST AS HETEROGENEOUS POPULATIONS AND THEIR IDENTITIES ARE INFLUENCED BY THEIR TISSUE ENVIRONMENTS. LIKEWISE, THE ONTOGENY OF ILCS IS ALSO COMPLEX. ALTHOUGH ILCS ARE THOUGHT TO ARISE FROM BONE MARROW PROGENITORS OR TISSUE RESIDENT PROGENITORS DISTRIBUTED DURING EMBRYOGENESIS, WORK FROM OUR LABORATORY STRONGLY SUGGEST THAT ILCS AT LEAST ILC2S CAN ALSO BE GENERATED IN THE THYMUS, NOT ONLY FROM MULTIPOTENT PROGENITORS BUT ALSO FROM COMMITTED T CELL PRECURSORS. OUR RECENT DATA USING SINGLE CELL RNA SEQUENCING SUGGEST THAT A SUBSTANTIAL FRACTION OF THE ILC-ENRICHED POPULATION IN THE BLOOD OF WILD TYPE MICE (WT) COME FROM THE THYMUS, AND THEIR EQUIVALENTS CAN ALSO BE FOUND IN PERIPHERAL TISSUES. WE THUS HYPOTHESIZE THAT THE THYMUS EXPORTS A SUBSTANTIAL AMOUNT OF ILC PRECURSORS TO THE CIRCULATION, WHICH MAY REPLENISH ILC2S AND/OR OTHER ILCS IN PERIPHERAL TISSUES IN STEADY-STATE OR UPON IMMUNE CHALLENGES AND THE THYMUS-DERIVED ILCS MAY HAVE DISTINCT FUNCTIONS. IN THIS RENEWAL PROPOSAL, WE INTEND TO FOLLOW UP ON THESE NEW EXCITING FINDINGS AND DETERMINE THE FUNCTION OF THESE THYMUS-DERIVED ILC PRECURSORS AND THEIR BIOLOGICAL SIGNIFICANT. WE WILL FURTHER CHARACTERIZE THYMUS-DERIVED ILC-PRECURSORS IN THE LUNG AND SMALL INTESTINE AND DETERMINE THEIR FREQUENCIES DURING MOUSE DEVELOPMENT. WE WILL ALSO IDENTIFY THYMUS-DERIVED ILC PRECURSORS IN HUMAN BLOOD, THUS GAINING APPRECIATION OF THE CLINICAL RELEVANCE OF THESE CELLS. WE WILL THEN FOCUS ON LEARNING THE FUNCTION OF THESE THYMUS-DERIVED ILC PRECURSORS USING IN VITRO AND IN VIVO APPROACHES, AS WELL AS ADOPTIVE TRANSFER OF PURIFIED ILC PRECURSORS INTO RAG2-/-IL2G-/- MICE WHICH ARE DEVOID OF ILCS. THE BEHAVIORS OF THESE CELLS IN TYPE 2 IMMUNE REACTIONS WILL BE MONITORED. FINALLY, WE WILL ASSESS THE CELL-INTRINSIC FUNCTIONAL DIFFERENCES AMONG WT ILC2S GENERATED FROM BONE MARROW COMMON LYMPHOID PROGENITORS (CLP) AND THYMIC DN1 AND DN3 T CELL PRECURSORS. TAKEN TOGETHER, STUDIES OUTLINED IN THIS PROPOSAL WILL FURTHER OUR UNDERSTANDING OF THYMUS-DERIVED ILCS, WHICH WILL HELP ESTABLISH A NEW PARADIGM REGARDING TO THE PRODUCTION AND MAINTENANCE OF ILC POOLS. BECAUSE THE PRESENCE OF AN ACTIVE THYMUS REPRESENTS ONE OF THE MAJOR DIFFERENCES BETWEEN CHILDREN AND ADULTS, KNOWLEDGE ABOUT THYMUS-DERIVED ILCS MAY SHED LIGHT ON THEIR DIFFERENT IMMUNE RESPONSES SUCH AS THOSE DURING COVID-19 INFECTION.

CELL TYPE-SPECIFIC NEUROTENSIN SIGNALING IN METHAMPHETAMINE USE DISORDER - METHAMPHETAMINE (METH) USE DISORDER IS CHRONIC AND PROGRESSIVE, AND CURRENTLY NO THERAPEUTIC IS APPROVED BY THE FDA FOR ITS TREATMENT. DOPAMINE (DA) NEURONS IN THE SUBSTANTIA NIGRA PARS COMPACTA (SNC) AND VENTRAL TEGMENTAL AREA (VTA) ARE NECESSARY FOR LEARNING ASSOCIATED WITH DRUG REINFORCERS. OUR PUBLISHED AND PRELIMINARY WORK HIGHLIGHTS A ROLE FOR THE PEPTIDE TRANSMITTER NEUROTENSIN IN MODULATION OF METH SELF-ADMINISTRATION BEHAVIOR AND PLASTICITY OF INHIBITORY NEUROTRANSMISSION IN THE SNC/VTA. NEUROTENSIN RECEPTORS IN THE SNC/VTA ARE LOCALIZED NOT ONLY ON NEURONS BUT ALSO ON ASTROCYTES, WHICH ARE GLIAL CELLS THAT CAN ACT THROUGH LOCAL CIRCUITS TO AFFECT DA NEURON EXCITABILITY AND REWARD-RELATED BEHAVIOR. WE HAVE CREATED FLOXED NEUROTENSIN RECEPTOR MOUSE LINES THAT WILL ENABLE, FOR THE FIRST TIME, THE DETERMINATION OF THE ROLE OF SPECIFIC NEUROTENSIN RECEPTORS ON INDIVIDUAL CELL TYPES THAT CONTRIBUTE TO METH SELF-ADMINISTRATION AND RESPONDING TO RELATED CUES. FILLING THIS KNOWLEDGE GAP IS NECESSARY TO ASSESS THE FEASIBILITY OF TARGETING CELL TYPE-SPECIFIC NEUROTENSIN SIGNALING AND OTHER FORMS OF ASTROCYTE GLIAL MODULATION AS A VIABLE TREATMENT FOR METH USE DISORDER. THE OBJECTIVE OF THIS APPLICATION IS TO IDENTIFY THE ROLE OF SPECIFIC NEUROTENSIN RECEPTORS (NTSR1 AND NTSR2), CELL TYPES, AND CIRCUITS RESPONSIBLE FOR MODULATING INHIBITORY INPUT TO DA NEURONS AND METH SELF-ADMINISTRATION BEHAVIOR IN MICE. OUR CENTRAL HYPOTHESIS IS THAT REPEATED METH SELF-ADMINISTRATION AFFECTS VTA CIRCUITS THROUGH NTSR1 ON DA NEURONS AND NTSR2 ON ASTROCYTES, WHICH ENHANCES METH INTAKE AND RESPONDING FOR METH-RELATED CUES. THE EXPERIMENTS WILL COMBINE INTRAVENOUS METH SELF-ADMINISTRATION IN GENETICALLY-MODIFIED MICE, BRAIN SLICE ELECTROPHYSIOLOGY, AND VIRUS-INDUCED EXPRESSION OF CRE RECOMBINASE AND DESIGNER LIGAND-SENSITIVE PROTEINS TO DETERMINE THE CIRCUITS RESPONSIBLE FOR NEUROTENSIN EFFECTS IN THE SNC AND VTA. THE STUDIES IN AIM 1 WILL ELUCIDATE THE RECEPTOR SUBTYPES AND LOCALIZATION THAT ARE ESSENTIAL FOR INHIBITORY SYNAPTIC PLASTICITY USING PATCH CLAMP ELECTROPHYSIOLOGY IN MOUSE BRAIN SLICES. THE STUDIES IN AIM 2 WILL DETERMINE THE CONSEQUENCES OF METH SELF-ADMINISTRATION, AS WELL AS FORCED ABSTINENCE, ON NEUROTENSIN MODULATION OF DA NEURONS. EXPERIMENTS WILL COMBINE INTRAVENOUS SELF-ADMINISTRATION OF METH IN MICE WITH PATCH CLAMP ELECTROPHYSIOLOGY, RNASCOPE, PATCH-SEQ OF PREVIOUSLY-RECORDED DOPAMINE NEURONS, AND TRANSCRIPTOMICS IN MIDBRAIN ASTROCYTES. THE EXPERIMENTS IN AIM 3 WILL COMBINE CHEMOGENETICS AND BEHAVIOR TO PROBE THE EFFECTS OF DISTINCT RECEPTORS AND NEUROTENSIN INPUT ON SELF-ADMINISTRATION OF METH AND RESPONDING FOR METH-RELATED CUES AFTER A FORCED ABSTINENCE. THE KNOWLEDGE GLEANED FROM THESE STUDIES WILL CLARIFY THE ROLE OF SPECIFIC NEUROTENSIN RECEPTORS BY IDENTIFYING PLASTICITY MECHANISMS FOR INHIBITORY INPUT TO DA NEURONS, AND BY DETERMINING HOW NEUROTENSIN CONTRIBUTES TO MULTIPLE STAGES OF METH SELF-ADMINISTRATION (ACQUISITION, MAINTENANCE, RESPONDING FOR CUES AFTER ABSTINENCE). THE INCREASED UNDERSTANDING OF NEUROTENSIN AND INHIBITORY CIRCUITS IN THE SNC/VTA COULD OPEN THE DOOR TO NOVEL TARGETS FOR THE TREATMENT OF STIMULANT USE DISORDERS.

FUNCTIONAL INTERACTION BETWEEN T-CELL AND B-CELL IMMUNE PATHWAYS IN NEURO- INFLAMMATORY DISORDERS

ROLE OF GATA-3 DURING CD4/CD8 LINEAGE COMMITMENT

SEGREGATION OF ERROR-PRONE CHROMOSOMES IN MEIOSIS

THE ROLE OF FBXL22 IN THE REGULATION OF SKELETAL MUSCLE MASS - PROJECT SUMMARY/ABSTRACT SKELETAL MUSCLE ATROPHY OCCURS AS A CONSEQUENCE OF MANY CHRONIC DISEASES AND CONDITIONS SUCH AS DISUSE. CENTRAL TO THE PROCESS OF MUSCLE ATROPHY IS PROTEIN DEGRADATION, FOR WHICH THE E3 UBIQUITIN LIGASES ARE CRITICAL IN TARGETING SPECIFIC PROTEINS FOR DEGRADATION. THE ROLES OF MANY E3 UBIQUITIN LIGASES IN MUSCLE ARE UNKNOWN AND SPECIFIC SUBSTRATES HAVE YET TO BE IDENTIFIED. THE OVERALL OBJECTIVE OF THIS PROPOSAL IS TO DETERMINE THE ROLE OF THE E3 LIGASE, F-BOX AND LEUCINE-RICH REPEAT PROTEIN 22 (FBXL22), IN SKELETAL MUSCLE ATROPHY. MY CENTRAL HYPOTHESIS IS THAT FBXL22 IS NECESSARY FOR THE PROCESS OF MUSCLE ATROPHY. ROBUST PRELIMINARY DATA SUPPORTS THIS HYPOTHESIS: 1) FBXL22 MRNA EXPRESSION IS INDUCED AFTER 3 DAYS IN A MODEL OF NEUROGENIC MUSCLE ATROPHY, 2) OVEREXPRESSION OF FBXL22 IN HINDLIMB MUSCLES RESULTS IN SIGNIFICANT ELEVATIONS OF TOTAL UBIQUITINATED PROTEINS. I PROPOSE TWO SPECIFIC AIMS: SPECIFIC AIM 1: DETERMINE THE FUNCTIONAL ROLE OF FBXL22 ISOFORMS IN SKELETAL MUSCLE ATROPHY, INJURY AND REGROWTH. I WILL ADDRESS THIS AIM USING KNOCKDOWN AND OVEREXPRESSION OF FBXL22 GENE ISOFORMS IN A MODEL OF NEUROGENIC MUSCLE ATROPHY AND ASSESS FBXL22 ISOFORM EXPRESSION IN AN ACUTE MUSCLE INJURY MODEL. SPECIFIC AIM 2: TO IDENTIFY SUBSTRATE TARGETS FOR FBXL22-DEPENDENT UBIQUITINATION. I WILL ADDRESS THIS AIM USING A COMBINED APPROACH OF FBXL22 GENE MANIPULATION WITH PROTEOMICS ANALYSIS AND POST-TRANSLATIONAL MODIFICATION IDENTIFICATION OF UBIQUITIN MOTIFS ON FBXL22-DEPENDENT SUBSTRATES. WITH THE COMPLETION OF THIS PROPOSAL, I EXPECT TO HAVE IDENTIFIED NOVEL MECHANISMS FOR UNDERSTANDING THE MUSCLE ATROPHY PROCESS. BY IDENTIFYING THE MECHANISMS OF SKELETAL MUSCLE ATROPHY, WE CAN EXPLORE NEW THERAPEUTIC STRATEGIES FOR IMPROVING AN INDIVIDUAL’S QUALITY OF LIFE WHERE MUSCLE WASTING IS PRESENT. THE UNIVERSITY OF IOWA HAS A LONGSTANDING HISTORY OF SUPPORT TO ITS TRAINEES AND JUNIOR FACULTY. MY MENTOR, PROF. BODINE IS A WORLD LEADER IN SKELETAL MUSCLE PHYSIOLOGY, ATROPHY AND HYPERTROPHY. THIS K01 AWARD WILL EXPAND MY RESEARCH SKILLS IN UBIQUITIN PROTEOMICS AND SKELETAL MUSCLE ATROPHY. THESE TOOLS MAY ENABLE THE IDENTIFICATION OF NEW THERAPEUTIC STRATEGIES FOR ALLEVIATING THE DETRIMENTAL IMPACT OF SKELETAL MUSCLE WASTING.

PRE-CLINICAL 7T MRI FOR OKLAHOMA

BRIGHT FUNCTION IN THE IMMUNE SYSTEM

IDENTIFYING THE NOVEL SLE SUSCEPTIBILITY GENE ON 5P15.3

MECHANISM OF T CELL LYMPHOMA IN E PROTEIN DEFICIENCY

FINE MAPPING AND REPLICATION OF A GENOME-WIDE ASSOCIATION SCAN FOR SLE IN HISPANI

IMPROVING RECRUITMENT OF MINORITY PATIENTS TO LUPUS TRIALS THROUGH CONTINUING EDUCATION AND PROVIDER SUPPORT

INTRAVITAL MULTIPHOTON MICROSCOPE

HEALTH CARE AND OTHER FACILITIES

DEVELOPMENT OF A LACO/LACI BASED FLOURESCENCE REPORTER-OPERATOR SYSTEM TO STUDY CHROMOSOME DYNAMICS IN MICE - SUMMARY MEIOTIC CHROMOSOMES UNDERGO A RANGE OF MOTIONS PROMOTING HIGHLY REGULATED CHROMOSOME INTERACTIONS. THESE INTERACTIONS CULMINATE IN PAIRWISE ASSOCIATIONS OF MATERNAL AND PATERNAL HOMOLOGOUS CHROMOSOMES, WHICH ARE LATER STABILIZED VIA THE PROTEINACEOUS STRUCTURE CALLED SYNAPTONEMAL COMPLEX (SC) THAT FORMS BETWEEN THE HOMOLOGOUS CHROMOSOMES (SYNAPSIS). CHROMOSOME PAIRING AND SC FORMATION ARE INFLUENCED BY RAPID PROPHASE MOVEMENTS (RPMS). MUTATIONS THAT AFFECT CHROMOSOME MOTIONS OR SC DYNAMICS CAN LEAD TO COSTLY PHENOTYPES RANGING FROM PROBLEMS IN REPRODUCTIVE BIOLOGY AND FERTILITY TO SEVERE ANEUPLOID-BASED BIRTH DEFECTS. FUNDAMENTAL QUESTIONS REGARDING THE PROCESSES UNDERLYING RPMS, AND HOW RPMS LEAD TO STABLY HOMOLOGOUS CHROMOSOME PAIRING ARE POORLY UNDERSTOOD. EQUALLY IMPORTANT, DOUBLE-STRAND BREAKS ARE ESSENTIAL FOR MEIOTIC RECOMBINATION TO OCCUR ALLOWING HOMOLOGOUS INTERACTIONS. HOWEVER, HOW TIMING AND GENOME DISTRIBUTION OF DOUBLE STRAND ARE CONTROLLED IS POORLY UNDERSTOOD. A PROMINENT CANDIDATE FOR THIS CONTROL IS THE RECENTLY IDENTIFIED SPO11 PARTNER, THE ANKRD31 PROTEIN, WHOSE PRECISE MECHANISM OF ACTION AND TARGETS IN GERM CELLS ARE UNKNOWN. MOLECULAR STUDIES IN MOUSE HAVE BEEN TRADITIONALLY LIMITED BY THE LACK OF GENOME ENGINEERING TOOLS. THIS PROPOSAL BUILDS ON THE DEVELOPMENT OF AN INNOVATIVE APPROACH THAT DIRECTS PROTEINS TO CHROMOSOMAL LOCI IN MOUSE MEIOCYTES IN VIVO BY UTILIZING A FLUORESCENCE REPORTER-OPERATOR SYSTEM (FROS) USING LACO-LACR TECHNOLOGY. OUR PRELIMINARY DATA SHOWS THAT THIS SYSTEM HAS THE POTENTIAL TO ANSWER QUESTIONS THAT HAVE PREVIOUSLY BEEN INTRACTABLE FOR MOUSE MEIOSIS RELEVANT TO DIFFERENTIATION AND MATURATION OF MALE GERM CELLS. THE PLANNED EXPERIMENTS WILL ANSWER TWO SPECIFIC QUESTIONS: 1) HOW DO CHROMOSOME MOTIONS PROMOTE STABLE HOMOLOG PAIRING AND 2) WHAT IS THE ACTION MECHANISM OF ANKRD31 IN MEDIATING PROPER TIMING AND LOCATION OF MEIOTIC DOUBLE STRAND BREAKS? THE FIRST AIM WILL ASSESS THE MECHANISM AND REGULATION OF RPMS ON HOMOLOGOUS CHROMOSOME PAIRING. WE WILL USE LACO/LACR-GFP TO VISUALIZE AND QUANTIFY CHROMOSOME MOTIONS USING 3D TIME-LAPSE MOVIES IN MAMMALIAN LIVE GERM CELLS. WE WILL TEST HOW PREVIOUSLY UNRECOGNIZED CHROMOSOME CHARACTERISTICS (E.G. CHROMOSOME LOCATION WITHIN THE NUCLEUS AND CHROMOSOMAL LEVEL OF EXPRESSION) THAT MODULATE RPMS. ADDITIONALLY, OUR NEWLY DEVELOPED LONG-TERM SEMINIFEROUS TUBULE CULTURE SYSTEM WILL ALLOW US TO DIRECTLY TEST TWO COMPETING MODELS EXPLAINING HOW HOMOLOGOUS CHROMOSOMES INTERACT AND PAIR. THIS AIM WILL LEND NEW INSIGHTS INTO THE DYNAMIC FORCES THAT GOVERN HOMOLOG PAIRING IN SPACE AND TIME. AIM 2 WILL DETERMINE THE REQUIREMENTS OF THE ANKRD31 PROTEIN IN MEIOTIC DOUBLE STRAND FORMATION. TO THIS END WE PLAN TO GENERATE TRANSGENIC MICE CARRYING LACO REPEATS AND ANKRD31- GFP-LACR. TARGETING ANKRD31 FUSION TO SPECIFIC GENOMIC LOCI WILL ALLOW DIRECT EVALUATION OF ANKRD31 EFFECT ON LOCAL ACCUMULATION OF SPO11 AUXILIARY PROTEINS (REC114, MEI4, AND IHO1), DOWNSTREAM RECOMBINATION HOTSPOT INTERMEDIATES, AND FREQUENCY OF DOUBLE STRAND BREAK FORMATION.

SUSCEPTIBILITY GENES IN PRIMARY SJOGREN'S SYDROME

INTEGRATIVE SINGLE CELL AND SPATIAL TRANSCRIPTOMICS OF SALIVARY GLANDS IN SJOGREN'S SYNDROME

SALIVARY GLAND RESPONSE TO INNATE IMMUNE MEDIATORS DICTATES SJOGREN'S SYNDROME DEVELOPMENT - PROJECT SUMMARY SJÖGREN'S SYNDROME (SS) IS A SYSTEMIC AUTOIMMUNE DISORDER AFFECTING MULTIPLE ORGAN SYSTEMS. A DYSREGULATED IMMUNE RESPONSE TARGETING THE EXOCRINE SALIVARY AND LACRIMAL GLANDS REDUCES FLUID SECRETION, WHICH MANIFESTS INTO THE DRY MOUTH AND DRY EYE SYMPTOMS OF SS. RECENT EVIDENCE IN THE LITERATURE SUGGESTS THAT INNATE IMMUNE ACTIVATION IS A PROMINENT ETIOLOGIC FACTOR. NEVERTHELESS, HOW A SYSTEMIC OR LOCALIZED INNATE IMMUNE RESPONSE TRANSITIONS INTO AN ADAPTIVE AUTOIMMUNE RESPONSE AND TARGETS THE EXOCRINE GLANDS REMAINS UNCLEAR. THIS ISSUE IS ALSO HIGHLY RELEVANT IN THE CONTEXT OF THE ONGOING COVID-19 PANDEMIC. IN GENETICALLY SUSCEPTIBLE INDIVIDUALS, THE SYSTEMIC CYTOKINE STORM ELICITED BY THE SARS-COV-2 INFECTION AND THE SALIVARY GLAND TROPISM OF THIS VIRUS MAY HEIGHTEN THE RISK FOR DEVELOPING SS OR WORSENING ITS SEVERITY. THE PRESENCE OF LYMPHOCYTIC INFILTRATES IN EXOCRINE GLANDS IS A SIGNIFICANT FEATURE OF SS. THESE INFILTRATES ARE PREDOMINANTLY PERI-DUCTAL, SUGGESTING THAT DUCTAL CELLS ARE INVOLVED IN INITIATING INFLAMMATORY CELL INFILTRATION INTO THE SALIVARY GLANDS. BY USING THE INNOVATIVE COLLABORATIVE CROSS MICE AND POLY(I:C) AS A SURROGATE FOR VIRAL INFECTION, THIS PROPOSAL WILL INVESTIGATE HOW THE GENETIC REGULATION OF INNATE IMMUNITY IN SALIVARY GLAND EPITHELIAL CELLS (SGEC) INFLUENCES SS DEVELOPMENT. WE WILL TEST THE OVERALL HYPOTHESIS THAT IN GENETICALLY SUSCEPTIBLE INDIVIDUALS, THE SGEC RESPONSE TO INNATE IMMUNE STIMULI DICTATES LYMPHOCYTIC INFILTRATION INTO THE SALIVARY GLANDS AND SS DEVELOPMENT. TO TEST THIS HYPOTHESIS, IN AIM 1, WE WILL INVESTIGATE WHETHER THE HIERARCHY OF SYSTEMIC IFN RESPONSES IN COLLABORATIVE CROSS MICE INFLUENCES LYMPHOCYTIC INFILTRATION WITHIN THE SALIVARY GLANDS. IN AIM 2, WE WILL INVESTIGATE WHETHER THE GENETIC MAKEUP OF SGECS DICTATES THE MAGNITUDE OF THEIR RESPONSE TO INNATE IMMUNE MEDIATORS. THE SUCCESSFUL COMPLETION OF THIS PROPOSAL WILL HELP DECIPHER THE INFLUENCE OF A DIFFERENTIAL GRADIENT OF INNATE IMMUNE RESPONSIVENESS IN SS PATHOGENESIS. FURTHER, SS DEVELOPMENT IN ANY OF THE COLLABORATIVE CROSS MICE USED IN THIS PROPOSAL WILL PROVIDE THE SS RESEARCH COMMUNITY A MORE PATIENT-RELEVANT MODEL SYSTEM TO INVESTIGATE GENE-ENVIRONMENT INTERACTION(S) IN THE DISEASE.

CYTOSOLIC DNA SENSING PATHWAY IN THE PATHOGENESIS OF SJOGREN'S SYNDROME

ROLE OF TWO INTERFERON REGULATORY GENES IN LUPUS

INVESTIGATING THE ROLE OF SIRT3 IN METABOLIC FLEXIBILITY AND PROTEOSTASIS IN THE AGING HEART - CARDIOVASCULAR DISEASE IS THE LEADING CAUSE OF DEATH IN THE UNITED STATES AND ITS OCCURRENCE DRAMATICALLY INCREASES WITH AGE. PREVENTING OR DELAYING CARDIAC AGING CAN THEREFORE HAVE A SIGNIFICANT EFFECT ON LONGEVITY AND HEALTHSPAN. A CENTRAL GOAL OF THIS PROPOSAL IS TO EXPLORE A NOVEL MECHANISM BY WHICH MITOCHONDRIA DECLINE WITH AGE AND CONTRIBUTE TO LOSS OF CARDIAC FUNCTION. WE ARE FOCUSING ON THE POST-TRANSLATIONAL MODIFICATION, ACETYLATION, BECAUSE IT INCREASES WITH AGE AND THE ENZYME THAT REVERSES THIS MODIFICATION, SIRTUIN-3 (SIRT3), IS A KNOWN LONGEVITY FACTOR THAT DECREASES WITH AGE. HOWEVER, IF MITOCHONDRIAL ACETYLATION ACCELERATES CARDIAC AGING REMAINS UNRESOLVED AND CONTROVERSIAL. OUR OVERARCHING HYPOTHESIS IS THAT AN INCREASE IN ACETYLATION HAS TWO EFFECTS IN THE AGED HEART. FIRST, IT CAUSES METABOLIC INFLEXIBILITY BY DIRECTLY AFFECTING THE ACTIVITY OF KEY REGULATORY ENZYMES. SECOND, THE INCREASE IN ACETYLATION AFFECTS PROTEOSTASIS (PROTEIN SYNTHESIS AND DEGRADATION HOMEOSTASIS). DYSFUNCTIONAL PROTEOSTASIS IN TURN CONTRIBUTES TO METABOLIC INFLEXIBILITY BY CAUSING IMPROPER SYNTHESIS AND DEGRADATION OF MODIFIED MITOCHONDRIAL METABOLIC ENZYMES. FOR THIS PROPOSAL, WE GENERATED A CARDIOMYOCYTE (CM) SPECIFIC SIRT3 KNOCKOUT MICE (SIRT3CM-/-). OUR PRELIMINARY DATA SHOW THAT SIRT3CM-/- MICE EXHIBIT DRAMATIC HYPERTROPHY, LOSS OF CONTRACTILE FUNCTION, FIBROSIS, METABOLIC ABNORMALITIES, AND DYSFUNCTIONAL PROTEOSTASIS BY 10- MONTHS. THIS ACCELERATED MODEL OF AGING AND HYPERACETYLATION WILL BE USED TO TEST OUR HYPOTHESIS THROUGH THE FOLLOWING AIMS. AIM 1 WILL DETERMINE IF THE INCREASE IN ACETYLATION CAUSED BY THE LOSS OF SIRT3 AFFECTS METABOLIC FLEXIBILITY WITH AGE. WE HYPOTHESIZE THAT THE LOSS OF SIRT3 CAUSES METABOLIC INFLEXIBILITY BY AFFECTING THE ACTIVITY OF DISCRETE MITOCHONDRIAL REGULATORY ENZYMES THAT RESULT IN THE INCREASED RELIANCE ON GLUCOSE. THIS AIM WILL DETERMINE IF THE LOSS OF SIRT3 AFFECTS METABOLIC FLEXIBILITY BY MEASURING CARDIAC AND MITOCHONDRIAL FUNCTIONS, ENZYMATIC ACTIVITIES, AND METABOLIC FLEXIBILITY LONGITUDINALLY IN SIRT3CM-/- MICE AND ISOLATED ADULT CARDIOMYOCYTES. GLOBAL ACETYLATION AND THE ACETYLATION OF SPECIFIC METABOLIC ENZYMES WILL BE MEASURED BY MASS SPECTROMETRY. THE RESULTS OF THESE STUDIES WILL DETERMINE IF THERE IS A DIRECT CONSEQUENCE OF HYPERACETYLATION ON CARDIAC PATHOLOGY IN THE ABSENCE OF SIRT3. AIM 2 WILL DETERMINE HOW THE LOSS OF SIRT3 AFFECTS MITOCHONDRIAL PROTEOSTASIS. ACETYLATION OF PROTEINS CAN AFFECT STRUCTURE AND FUNCTION, YET LITTLE IS KNOWN REGARDING ITS ROLE IN GLOBAL CHANGES IN MITOCHONDRIAL PROTEIN QUALITY AND TURNOVER. THIS AIM WILL USE DEUTERIUM OXIDE (D2O) LABELING AND PROTEOMICS TO DETERMINE IF THE LOSS OF SIRT3 AFFECTS MITOCHONDRIAL PROTEIN SYNTHESIS AND THE RELATIVE TURNOVER RATES OF SPECIFIC METABOLIC REGULATORY ENZYMES. RESCUE EXPERIMENTS WILL BE PERFORMED BY AAV DELIVERY OF SIRT3. MECHANISTIC STUDIES IN CELL CULTURE WILL BE PERFORMED TO DEMONSTRATE HOW ACETYLATION AFFECTS PROTEOSTASIS. THESE WILL BE THE FIRST STUDIES TO EMPLOY IN VIVO LABELING TO DETERMINE HOW ACETYLATION AFFECTS PROTEIN SYNTHESIS AND TURNOVER. THE RESULTS WILL PROVIDE AN IMPETUS FOR FUTURE PROJECTS FURTHER DEFINING HOW ACETYLATION AND OTHER CARBON STRESSES AFFECT CARDIAC AGING.

VENTRAL TEGMENTAL AREA INVOLVEMENT IN ALZHEIMER'S PATHOLOGY - ALZHEIMER'S DISEASE (AD) IS MOST COMMONLY DIAGNOSED SUBSEQUENT TO MEMORY DEFICITS, BUT CO-MORBIDITIES INCLUDE A SLEW OF COGNITIVE AND NON-COGNITIVE IMPAIRMENTS INCLUDING DEPRESSION, APATHY, AND MOVEMENT DISORDERS. WHILE MOST WORK ON AD HAS THUS FAR FOCUSED ON EFFECTS IN THE CORTEX AND HIPPOCAMPUS, THE PATHOPHYSIOLOGY ASSOCIATED WITH AD IS FOUND THROUGHOUT THE BRAIN, AND MANY OF THE SECONDARY SYMPTOMS SUGGEST INVOLVEMENT OF THE MIDBRAIN DOPAMINERGIC SYSTEM. A RECENT MAJOR STUDY AS WELL AS PRELIMINARY DATA SUGGEST THAT DOPAMINE NEURONS OF THE VENTRAL TEGMENTAL AREA (VTA) MAY BE AFFECTED IN MOUSE MODELS OF AD PRIOR TO THE FORMATION OF AMYLOID PLAQUES AND NEUROFIBRILLARY TANGLES, SUGGESTING A POSSIBLE ROLE FOR THE VTA IN THE PRODROMAL PHASE OF THE DISEASE. THIS FIELD IS CURRENTLY LIMITED BY THE LACK OF INFORMATION ON THE STRUCTURAL AND FUNCTIONAL DEFICITS THAT DEVELOP IN SINGLE DOPAMINE NEURONS IN THE EARLY STAGES OF AD, AS WELL AS THEIR RELATIONSHIP WITH DECREMENTS IN HEDONIC AND REWARD LEARNING BEHAVIOR. THE EXPERIMENTS IN THIS PROPOSAL WILL FOCUS ON TWO ESTABLISHED MOUSE MODELS OF AD. APP/PS1 MICE EXPRESS A MUTATED HUMAN AMYLOID PRECURSOR PROTEIN AND A DELETION OF PRESENILIN 1, WHILE TRIPLE TRANSGENIC 3XTG-AD MICE ALSO EXPRESS A TRANSGENE FOR A HUMAN MUTANT TAU. THE GENERAL STRATEGY WILL BE TO MEASURE DEFICITS IN DOPAMINE-MEDIATED BEHAVIOR (SUCROSE PREFERENCE, AN OPERANT LEARNING TASK, AND LOCOMOTOR ASSAYS) IN MUTANT MICE AGED 3, 6, AND 10 MONTHS, AND NON-TRANSGENIC CONTROLS, FOLLOWED BY SACRIFICE FOR ELECTROPHYSIOLOGY AND RNA SEQUENCING OF SINGLE VTA DOPAMINE NEURONS. AIM 1 WILL FOCUS ON BOTH INTRINSIC AND SYNAPTIC CONDUCTANCES THAT AFFECT DOPAMINE NEURON FIRING AND HAVE BEEN IDENTIFIED IN PRELIMINARY STUDIES AS POSSIBLY BEING AFFECTED IN AD. AIM 2 WILL TEST IF A COMMONLY USED ANTI-AGING INTERVENTION, DIETARY RESTRICTION, CAN MITIGATE SOME OR ALL DECREMENTS IN ION CHANNEL PHYSIOLOGY, BEHAVIOR, AND NEURONAL MORPHOLOGY. AS THE PREVALENCE OF ADRD CONTINUES TO INCREASE, WE ARE IN DESPERATE NEED OF AD TREATMENTS THAT NOT MERELY ALLEVIATE SYMPTOMS BUT SLOW OR HALT THE PROGRESSION OF THE DISEASE. THESE TREATMENTS DO NOT CURRENTLY EXIST BECAUSE WE LACK A FUNDAMENTAL UNDERSTANDING OF THE DECREMENTS IN CELLULAR AND CIRCUIT FUNCTION THAT OCCUR DURING THE EARLY STAGES OF PATHOLOGY. THE WORK IN THIS PROPOSAL WILL ADVANCE THE FIELD TOWARD BETTER TREATMENTS BY ESTABLISHING THE INVOLVEMENT OF DOPAMINERGIC PROCESSES IN AD MOUSE MODELS. DELINEATING THE SEQUENCE OF PATHOLOGICAL EVENTS WILL ALLOW FOR THE IDENTIFICATION OF MOLECULAR TARGETS FOR INTERVENTION IN EARLY AD.

CONNECTING THE GAP BETWEEN GWAS AND FUNCTIONAL TARGETS FOR LUPUS SUSCEPTIBILITY - ABSTRACT SYSTEMATIC LUPUS ERYTHEMATOSUS (SLE OR LUPUS) IS A CURRENTLY INCURABLE AUTOIMMUNE DISEASE, CHARACTERIZED BY ABNORMAL IMMUNE CELL (E.G., B-LYMPHOCYTES) RESPONSE AND THE PRODUCTION OF NUMEROUS PATHOGENIC AUTOANTIBODIES, CULMINATING IN MULTI-ORGAN DAMAGE (E.G., KIDNEYS, SKIN). WHILE THE ETIOLOGY OF SLE IS INCOMPLETELY UNDERSTOOD, A SUBSTANTIAL GENETIC CONTRIBUTION IS WELL ESTABLISHED. SEVERAL GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED OVER 100 SLE PREDISPOSING LOCI (P<5X10-8), MOSTLY SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS). MOST OF THESE SNPS DO NOT DIRECTLY ALTER PROTEIN PRODUCTS, AND PREVIOUS WORK FROM US AND OTHERS HAVE SHOWN THAT MANY SUCH SNPS ARE ENRICHED WITHIN CIS REGULATORY ELEMENTS (CRE) (I.E., ENHANCERS AND SILENCERS) AND LIKELY TO MODULATE GENE EXPRESSION. HOWEVER, PINPOINTING THE CAUSAL, PREDISPOSING SNPS WITHIN CRES AND DECIPHERING THEIR PRECISE MECHANISMS REPRESENT MAJOR OBSTACLES TO PROGRESS IN THE FIELD. CONSEQUENTLY, THIS KNOWLEDGE GAP HAS SEVERELY HINDERED THE TRANSLATION OF GWAS FINDINGS INTO CLINICAL APPLICATIONS. HENCE, THERE IS A PROFOUND NEED FOR UNBIASED, COMPREHENSIVE, AND HIGH-THROUGHPUT APPROACHES TO ADDRESS THE MECHANISTIC LINK BETWEEN HUNDREDS OF POTENTIAL REGULATORY SNPS (RSNPS) AND SLE SUSCEPTIBILITY. WE HYPOTHESIZE THAT SLE- PREDISPOSING RSNPS ABERRANTLY INDUCE CRE ACTIVITIES THAT INFLUENCE THE EXPRESSION OF TARGET GENES IN UNSTIMULATED AND/OR STIMULATED B-CELLS. TO SYSTEMATICALLY DELINEATE RSNPS AND THEIR IMPACT ON TARGET GENES, WE PROPOSE TO ESTABLISH A HIGH-THROUGHPUT EXPERIMENTAL PIPELINE TO DETERMINE AND VALIDATE RSNPS UNDERLYING GWAS LOCI. IN AIM 1, WE WILL APPLY THE HIGH-THROUGHPUT TECHNIQUE “SELF-TRANSCRIBING ACTIVE REGULATORY REGION-SEQUENCING” (STARR-SEQ) TO FUNCTIONALLY QUANTIFY THE REGULATORY ACTIVITIES OF HUNDREDS OF SNP-CONTAINING REGIONS SIMULTANEOUSLY. USING RAJI CELLS (B-LYMPHOCYTE) IN BOTH UNSTIMULATED AND STIMULATED CONDITIONS, WE WILL APPLY STARR-SEQ TO EVALUATE 756 SELECTED RSNPS WITHIN 79 DISTINCT GWAS LOCI FOR SLE SUSCEPTIBILITY. IN AIM 2A, WE WILL APPLY NEXT-GENERATION (NG)-CAPTURE-C TO DETECT SNP-SPECIFIC EFFECTS ON CIS INTERACTIONS WITH ENDOGENOUS, COGNATE TARGET GENES IN RAJI CELLS, NEEDING NO STRONG A PRIORI HYPOTHESIS OF TARGET GENES AND FUNCTIONAL CONSEQUENCES. WE WILL EVALUATE THE SAME SET OF 756 SNPS IN AIM 1 AND AIM 2A. WE ANTICIPATE THIS TWO-PRONG TANDEM STRATEGY WILL BRIDGE THE GAP BETWEEN GWAS-DERIVED RSNPS AND MECHANISTIC LINKS TO THEIR TARGET GENES. TO VALIDATE THE EFFECTIVENESS AND ACCURACY OF THE PROPOSED METHODS, AIM 2B WILL USE CRISPR-BASED (EPI)GENETIC EDITING OF A SELECTED RSNP TO VALIDATE THE ALLELE-SPECIFIC FUNCTIONAL EFFECTS ON THE ENDOGENOUS TARGET GENE(S) IN ISOGENIC CELLS. COLLECTIVELY, THE PROPOSED UNBIASED APPROACHES FOR DISCOVERING AND VALIDATING SLE “CAUSAL” SNPS IS HIGH RISK/HIGH REWARD AND MAY LEAD TO BREAKTHROUGHS IN THE UNDERSTANDING OF SLE ETIOLOGY AND INTERVENTION STRATEGIES. DISCOVERY OF RSNPS, CRES, AND THEIR TARGET GENES WILL SIGNIFICANTLY ADVANCE OUR KNOWLEDGE OF SLE GENETICS, AND YIELD DIRECTIONS FOR FUTURE IN-DEPTH MECHANISTIC RESEARCH ON THE CONTRIBUTION OF RSNPS AND TARGET GENES IN LUPUS SUSCEPTIBILITY, WHICH COULD DEFINE FUTURE THERAPEUTIC TARGETS DERIVED FROM GWAS.

THE ROLE OF SHORT-FORM RON (SFRON) KINASE IN THE PATHOGENESIS OF HIGH-GRADE SEROUS OVARIAN CANCER

TREATMENT STRATEGIES FOR AUTOIMMUNE DEMYELINATING OPTIC NEURITIS

E PROTEIN ACTIVITY REGULATES EFFECTOR LINEAGE DIFFERENTIATION OF NKT AND ILCS

LOCALIZING FUNCTIONAL VARIANTS IN SLE SUSCEPTIBILITY GENES

DEVELOPMENT OF A MOUSE MODEL OF PEPTIDOGLYCAN-INDUCED PATHOLOGY

MECHANISTIC INVESTIGATIONS OF TRYPTOPHAN CATABOLITES FOR VISION IMPROVEMENT IN MULTIPLE SCLEROSIS - MULTIPLE SCLEROSIS (MS) IS AN AUTOIMMUNE DISEASE CHARACTERIZED BY INFLAMMATION, DEMYELINATION AND NEURODEGENERATION WITHIN THE CENTRAL NERVOUS SYSTEM. OPTIC NEURITIS IS THE MOST PREVALENT OCULAR COMPLICATION EXPERIENCED BY PEOPLE WITH MS (PWMS), AND REPEATED EPISODES CAN CULMINATE IN IRREVERSIBLE VISION LOSS. YET, EFFECTIVE TREATMENTS REMAIN AN IMPORTANT UNMET NEED. HEALTHY DIETS HAVE BEEN LINKED TO BETTER OUTCOMES FOR PWMS, BUT THERE IS CURRENTLY NO CONSENSUS AS TO WHICH DIETARY RECOMMENDATIONS CONFER THE MOST BENEFIT. USING A RODENT MODEL OF AUTOIMMUNE-MEDIATED DEMYELINATION CALLED EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE), WE RECENTLY DISCOVERED THAT A THERAPEUTIC KETOGENIC DIET (KD) RESTORED VISUAL FUNCTION AND OPTIC NERVE INTEGRITY WHEN FED TO SYMPTOMATIC MICE EXPERIENCING OPTIC NEURITIS AND VISION LOSS. THE GOAL OF THIS APPLICATION IS TO CHARACTERIZE METABOLITES AND PATHWAYS THAT CONFER TO THE KD THE CAPACITY TO RESTORE VISION AND OPTIC NERVE INTEGRITY. RNA-SEQ ANALYSIS OF OPTIC NERVES IDENTIFIED CHANGES IN GENE EXPRESSION COORDINATED BY A NUCLEAR RECEPTOR TRANSCRIPTION FACTOR CALLED THE ARYL HYDROCARBON RECEPTOR (AHR) THAT IS LINKED TO INFLAMMATION SUPPRESSION AND NEUROPROTECTION. UNTARGETED METABOLOMICS OF CECAL MATERIAL AND PLASMA REVEALED THAT THE KD ELEVATED MULTIPLE LIGANDS FOR THE AHR, INCLUDING SEVERAL INDOLE DERIVATIVES OF TRYPTOPHAN CATABOLISM PRODUCED EXCLUSIVELY BY THE GUT MICROBIOTA. THE PROPOSED EXPERIMENTS TEST THE HYPOTHESIS THAT INDOLE DERIVATIVES PRODUCED BY THE GUT MICROBIOTA ACTIVATE THE AHR ON OPTIC NERVES TO PROMOTE KD-MEDIATED RESTORATION OF VISION. STUDIES INCLUDE ADMINISTERING THE INDOLE DERIVATIVES IN THE DRINKING WATER AS STAND-ALONE TREATMENTS, EVALUATING KD EFFICACY IN THE PRESENCE OF AHR PHARMACOLOGICAL INHIBITORS AND IN AN AHR LIGAND-BINDING MUTANT MOUSE STRAIN, AND PERFORMING LONGITUDINAL MICROBIOME ANALYSES TO IDENTIFY THE BACTERIAL SPECIES ENRICHED BY THE KD THAT PRODUCE THE INDOLE DERIVATIVES OF INTEREST. READOUTS FOR THE ABOVE STUDIES INCLUDE: (A) MEASURING OCULAR FUNCTION BY OPTOKINETIC TRACKING, FLASH VISUAL EVOKED POTENTIALS, AND PHOTOPIC NEGATIVE RESPONSE ERGS, (B) MEASURING INDOLE DERIVATIVE LEVELS IN PLASMA BY GC-MS, (C) QUANTIFYING RETINAL GANGLION CELL SPARING, AND (D) QUANTIFYING OPTIC NERVE (DE)MYELINATION, LESION NUMBER AND SIZE, AND AHR TARGET GENE EXPRESSION. IMMUNE CELL PROFILING OF OPTIC NERVES AND SPINAL CORDS WILL REVEAL THE IMPACTS OF THE KD AND THE INDOLE DERIVATIVES ON THE BALANCE BETWEEN PRO- AND ANTI-INFLAMMATORY IMMUNE CELL POPULATIONS. TOGETHER, THESE STUDIES ARE ANTICIPATED TO OPTIMIZE AND ADVANCE DIETARY ADJUVANTS THAT IMPROVE VISUAL OUTCOMES FOR PWMS.

AGING, SEX BIAS, AND CD8 T CELL EXHAUSTION IN SALIVARY GLAND INFLAMMATION - PROJECT SUMMARY SJÖGREN’S DISEASE (SJD) IS A SYSTEMIC AUTOIMMUNE DISORDER MOST FREQUENTLY TARGETING EXOCRINE GLANDS AND CAUSING ORAL AND OCULAR DRYNESS. SJD IS CHARACTERIZED BY SERUM ANTIBODIES TO NUCLEAR AND CYTOPLASMIC ANTIGENS AND SALIVARY GLAND INFLAMMATION (SIALOADENITIS), WHICH IS ASSOCIATED WITH AN INCREASED RISK OF LYMPHOMA. FOR UNKNOWN REASONS, SJD DISPROPORTIONATELY AFFECTS FEMALES AND IS TYPICALLY DIAGNOSED IN THE 5TH- 6TH DECADE OF LIFE. THUS, FEMALE SEX AND AGING ARE INTRICATELY LINKED WITH IMMUNE DYSFUNCTION AND AUTOIMMUNITY IN SJD. THE LONG-TERM GOAL OF THIS PROJECT IS TO UNDERSTAND THE AGE- AND SEX-RELATED IMMUNE MECHANISMS INVOLVED IN THE SALIVARY GLAND PATHOLOGY OF SJD. THE PROPOSED STUDIES WILL UTILIZE MALE AND FEMALE MICE THAT SPONTANEOUSLY DEVELOP THE SALIENT CHARACTERISTICS OF SJD AND MIMIC THE AGE- AND SEX-BIAS OF THE DISEASE. WHILE BOTH MALES AND FEMALES DEVELOP SYSTEMIC AUTOIMMUNITY, ONLY THE FEMALES PROGRESS TO SEVERE SIALOADENITIS. WE WILL USE A CONCEPTUALLY INNOVATIVE APPROACH BY INVESTIGATING THE IMMUNOREGULATORY MECHANISMS THAT PROTECT MALES FROM SEVERE SIALOADENITIS. AIM 1 WILL INVESTIGATE WHETHER THE AGED MALE SALIVARY GLAND MICROENVIRONMENT HARBORS IMMUNOSUPPRESSIVE SIGNALS THAT INHIBIT PROGRESSIVE INFLAMMATION. WE WILL CHARACTERIZE GENE EXPRESSION CHANGES IN IMMUNE, EPITHELIAL, AND STROMAL COMPARTMENTS USING SINGLE- CELL TRANSCRIPTOMIC PROFILING OF MALE AND FEMALE SALIVARY GLANDS AT KEY AGING MILESTONES. RECENT LITERATURE SUGGESTS A PROMINENT ROLE FOR CD8 T CELLS IN THE GLANDULAR PATHOLOGY OF SJD. AIM 2 WILL EXAMINE THE DIFFERENTIATION TRAJECTORY OF CD8 T CELLS TARGETING SALIVARY GLAND ANTIGENS IN MALE AND FEMALE MICE. WE WILL LEVERAGE A NOVEL MOUSE MODEL EXPRESSING OVALBUMIN AS A SURROGATE SELF-ANTIGEN IN THE SALIVARY GLANDS TO TRACK ANTIGEN-SPECIFIC CD8 T CELL ACTIVATION. OUR FINDINGS WILL INFORM THE DEVELOPMENT OF INNOVATIVE THERAPEUTIC STRATEGIES AIMED AT LOCAL IMMUNE MODULATION TO ALLEVIATE SALIVARY GLAND PATHOLOGY IN SJD.

FUNCTIONAL ANALYSIS OF GWAS LOCI ASSOCIATED WITH HEARING LOSS. - HEARING LOSS IS A HIGHLY PREVALENT AND DEBILITATING NEUROSENSORY DISORDER ASSOCIATED WITH SUBSTANTIALLY REDUCED QUALITY OF LIFE AND OVERALL HEALTH. IT CURRENTLY AFFECTS 430 MILLION PEOPLE WORLDWIDE; BY 2050 THIS IS EXPECTED TO INCREASE TO NEARLY 2.5 BILLION AND RESULT 1 IN 10 PEOPLE REQUIRING REHABILITATION. ABOUT 50% OF CASES ARE PREDICTED TO HAVE A GENETIC BASIS, HOWEVER HEARING LOSS CAN ALSO BE CAUSED BY OTHER FACTORS SUCH AS AGE, OTOTOXIC DRUGS, NOISE, INFECTION OR INJURY. LOW-COST NEXT GENERATION SEQUENCING TECHNOLOGIES HAVE FACILITATED MANY GENOME-WIDE ASSOCIATION STUDIES (GWAS) AND EXOME SEQUENCING PROJECTS THAT HAVE IDENTIFIED HUNDREDS OF VARIANTS AND GENES ASSOCIATED WITH HEARING LOSS. THERE ARE CURRENTLY MORE THAN 150 LOCI AND OVER 100 GENES ASSOCIATED WITH NON- SYNDROMIC HEARING LOSS, HOWEVER FEW CANDIDATE GENES HAVE BEEN IDENTIFIED FOR COMPLEX PHENOTYPES SUCH AS AGE-RELATED HEARING LOSS (ARHL) OR PRESBYCUSIS, WHICH IS BECOMING INCREASINGLY COMMON AS THE POPULATION AGES. A GWAS CONDUCTED TO IDENTIFY CANDIDATE GENES ASSOCIATED WITH ARHL IDENTIFIED 44 INDEPENDENT GENOMIC LOCI ASSOCIATED WITH HEARING LOSS. A NEAREST GENE WAS MAPPED FOR EACH SNP IDENTIFIED IN THIS STUDY, YET HOW THIS SNP INFLUENCES GENE FUNCTION IN HEARING LOSS HAS NOT BEEN DETERMINED. ESTABLISHING A LINKAGE BETWEEN THE TARGET GENES AND THE DISEASE PHENOTYPE IS A HUGE CHALLENGE THAT ULTIMATELY AFFECTS THE CORRECT DIAGNOSIS; GENERATING SIMILAR PHENOTYPES UPON GENE INACTIVATION IN ANIMAL MODELS CAN ESTABLISH A STRONG SUPPORT FOR A CANDIDATE GENE. WE IDENTIFIED 39 ORTHOLOGS OF 44 GWAS CANDIDATE GENES IN ZEBRAFISH, AND FURTHER SELECTED 29 NOVEL GENES THAT WILL BE TESTED FUNCTIONALLY FOR THEIR ROLE IN HEARING LOSS BY (1) GENERATING A LIBRARY OF ZEBRAFISH MUTANTS FOR 29 (AND PARALOGS) CANDIDATE GENES ASSOCIATED WITH ARHL (2) ANALYZING THESE MUTANTS VIA A HIGH-THROUGHPUT PHENOTYPING PIPELINE INCLUDING MORPHOLOGICAL, CELLULAR, AND BEHAVIORAL PHENOTYPES. IDENTIFYING THE FUNCTIONAL CONSEQUENCES OF THE CANDIDATE GENES IN ZEBRAFISH WILL YIELD MECHANISTIC INSIGHTS IN DISEASE PATHOGENESIS.

NADPH-OXIDASE AND SLE SUSCEPTIBILITY

A NOVEL MECHANISM FOR MITOCHONDRIAL REGULATION OF MAMMALIAN LONGEVITY

NOTCH REGULATION OF AIRWAY EPITHELIAL-IMMUNE CELL CROSS-TALK IN SARS-COV-2 INFECTION - ABSTRACT SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2) IS THE CAUSATIVE AGENT OF COVID-19. INFECTION WITH SARS-COV-2 BEGINS IN EPITHELIAL CELLS OF THE UPPER AIRWAYS, WHICH TRIGGERS MULTIPLE ANTIVIRAL RESPONSES THAT ACTIVATE MYELOID IMMUNE CELLS, COORDINATE THE EARLY INNATE IMMUNE RESPONSE, AND LIMIT VIRAL SPREAD. NOTABLY, OUR UNDERSTANDING OF THE CELL SIGNALING MECHANISMS THAT REGULATE EARLY EPITHELIAL-IMMUNE CELL INTERACTIONS AND INDUCTION OF TISSUE PATHOLOGY DURING SARS-COV-2 INFECTION OF THE HUMAN AIRWAY REMAINS LIMITED. TO INVESTIGATE THESE PROCESSES, WE HAVE DEVELOPED A NOVEL IN VITRO ALL HUMAN 3D MODEL OF THE UPPER AIRWAYS (HUA) COMPOSED OF LAYERS OF PRIMARY DIFFERENTIATED AIRWAY EPITHELIAL CELLS EXPRESSING ACE2, LUNG FIBROBLASTS IN A COLLAGEN MATRIX, AND PULMONARY ENDOTHELIAL CELLS, WITH MYELOID CELLS PRESENT IN EACH LAYER. SARS-COV-2 INFECTION OF THE HUA MODEL RESULTS IN VIRUS REPLICATION AND INDUCTION OF AN IMMUNE RESPONSE REMINISCENT OF IN VIVO INFECTION, AND THE PRESENCE OF MYELOID CELLS LIMITS VIRAL REPLICATION. THEREFORE, WE WILL USE THE HUA MODEL TO PERFORM KINETIC STUDIES OF THE MECHANISMS BY WHICH HUMAN AIRWAY CELLS INTERACT TO REGULATE VIRUS-HOST INTERACTIONS, INFLAMMATION, AND TISSUE PATHOLOGY ASSOCIATED WITH SARS-COV-2. WE WILL FOCUS ON THE NOTCH PATHWAY, WHICH ACTS VIA DIRECT CELL- TO-CELL SIGNALING TO REGULATE AIRWAY EPITHELIAL CELL FATE DECISIONS AS WELL AS MYELOID CELL MAINTENANCE, TOLL-LIKE RECEPTOR SIGNALING, PRO-INFLAMMATORY POLARIZATION, AND ANTIVIRAL FUNCTIONS. HOWEVER, THE MECHANISMS BY WHICH NOTCH SIGNALING REGULATES AIRWAY EPITHELIAL-MYELOID CELL INTERACTIONS DURING SARS-COV-2 INFECTION REMAIN UNKNOWN. OUR PRELIMINARY ANALYSES SHOWED THAT AIRWAY EPITHELIAL CELLS AND MYELOID CELLS EXPRESS MULTIPLE NOTCH LIGANDS AND RECEPTORS IN UNINFECTED HUA MODELS, SUGGESTING THAT THE NOTCH PATHWAY WILL MEDIATE BIDIRECTIONAL CROSSTALK DURING THE AIRWAY RESPONSE TO INFECTION. WE WILL TEST THE CENTRAL HYPOTHESIS THAT NOTCH RECEPTOR SIGNALING IN MYELOID CELLS REGULATES THEIR PRO-INFLAMMATORY PHENOTYPE, THEREBY PROMOTING THE HOST INNATE ANTIVIRAL IMMUNE RESPONSE AND CONTRIBUTING TO AIRWAY EPITHELIAL CELL DAMAGE AND REMODELING DURING SARS-COV-2 INFECTION. USING LENTIVIRUS VECTORS AND INDUCIBLE EXPRESSION SYSTEMS, WE WILL ATTENUATE EXPRESSION OF INDIVIDUAL NOTCH LIGANDS AND RECEPTORS IN A CELL-TYPE SPECIFIC AND TEMPORAL MANNER. FOLLOWING SARS-COV-2 INFECTION, WE WILL QUANTIFY THE IMPACT OF REDUCING NOTCH SIGNALING ACTIVITY ON VIRUS REPLICATION, MYELOID CELL PHENOTYPES, THE HOST IMMUNE RESPONSE, AND AIRWAY EPITHELIAL REMODELING. THE DATA COLLECTED IN THIS STUDY WILL ADVANCE OUR UNDERSTANDING OF THE MECHANISMS THAT REGULATE AIRWAY EPITHELIAL-IMMUNE CELL INTERACTIONS DURING SARS-COV-2 INFECTION AND MAY IDENTIFY CANDIDATE THERAPEUTIC TARGETS IN THE NOTCH PATHWAY TO ENHANCE ANTIVIRAL IMMUNITY AND REDUCE EPITHELIAL INJURY AND REMODELING IN THE UPPER AIRWAY.

THE ROLE OF TETRASPANIN-REGULATED ENDOLYSOSOMAL TRAFFICKING IN ALZHEIMER'S DISEASE - ALTHOUGH THE ACCUMULATION OF AΒ AND SUBSEQUENT CHRONIC INFLAMMATION AND NEURONAL DYSFUNCTION LIKELY CONTRIBUTE TO THE DEVELOPMENT OF ALZHEIMER’S DISEASE (AD) PATHOLOGY AND CLINICAL MANIFESTATION, THE MECHANISMS FOR AD INITIATION AND PROGRESSION IN SPORADIC, LATE-ONSET CASES REMAIN UNCLEAR. AN ABUNDANCE OF PATHOLOGICAL AND GENETIC EVIDENCE POINTS TOWARDS A ROLE OF THE ENDOLYSOMAL NETWORK AND ITS EFFECTS ON APP TRAFFICKING AND PROCESSING AS A POTENTIAL INITIATOR OF THE DISEASE. WE RECENTLY FOUND THAT TETRASPANIN CD151 RESTRAINS THE RELEASES OF I) APP, II) Α-SECRETASE, AND III) PRO-INFLAMMATORY MOLECULES. OUR RECENT STUDIES ALSO SHOW THAT CD151 SUSTAINS PROPER STRUCTURE AND FUNCTION OF ENDOLYSOSOMES, E.G., SUPPORTING PROPER TURNOVER OF PROTEINS IN ENDOLYSOSOMES. THESE OBSERVATIONS UNDERLINE A PUTATIVE MECHANISTIC CONNECTION BETWEEN DYSREGULATED ENDOLYSOSOME FUNCTION, RESULTING FROM THE DIMINISHED CD151 LEVEL IN INFLAMMATION AND AGING, AND ABNORMAL PROCESSING OF APP, LEADING TO CHRONIC INFLAMMATION AND AD. THIS NOTION FORMS THE BASIS OF OUR HYPOTHESIS FOR THIS RESEARCH PROJECT, I.E., CD151-DEPENDENT ENDOLYSOSOME TRAFFICKING IS REQUIRED FOR PROPER DEGRADATION AND LIMITED RELEASE OF APP. THE GOAL OF THIS PROJECT IS TO UNDERSTAND THE MECHANISTIC ROLES OF CD151-REGULATED ENDOLYSOSOMAL PROTEOLYSIS IN THE PATHOGENESIS OF AD. SPECIFICALLY, WE WILL ASSESS BOTH IN VITRO AND IN VIVO EFFECTS OF CD151 REMOVAL ON THE I) ENDOLYSOSOMAL TRAFFICKING AND/OR PROCESSING OF APP AND/OR ITS RELEVANT SECRETASES, II) SECRETION OF PRO-INFLAMMATORY MOLECULES, AND III) AD-RELATED PATHOLOGICAL AND BEHAVIOR CHANGES. FROM THESE IN-DEPTH MECHANISTIC STUDIES, WE WILL UNDERSTAND IF AND HOW CD151-DEPENDENT REGULATION OF ENDOLYSOSOMES IS IMPORTANT FOR THE PROGRESSION OF AD, LIKELY ESTABLISH A NOVEL PARADIGM BETWEEN MOLECULAR IDENTITY OF ENDOLYSOSOMES AND PROTEOLYTIC DETERMINATION OF APP, AND POSSIBLY DELINEATE THE MOLECULAR SWITCH THAT GOVERNS THE AΒ DEPOSITION IN BRAIN. AT THE END OF THIS PROJECT, WE WILL DEVELOP AN INTEGRATED UNDERSTANDING OF THE UNIQUE FEATURES OF ENDOLYSOSOME AND TETRASPANIN CD151 IN THE PATHOGENESIS OF AD, WHICH WILL ULTIMATELY LEAD TO THE DEVELOPMENT OF THERAPEUTIC STRATEGIES AGAINST AD.

REGULATION OF DENDRITIC CELLS BY ESTROGEN RECEPTORS DURING INFLUENZA INFECTION

ASB2 IN CD4+ T CELL LINEAGE DIFFERENTIATION AND ITS PLASTICITY

REGULATION OF NKT CELL DEVELOPMENT AND FUNCTION BY C-MYB

IDENTIFICATION OF LUPUS PREDISPOSING VARIANTS BY COMPARING MULTIPLE POPULATIONS

TARGETING SFRON-S6K1 SIGNALING AND MITOTIC KINESIN EG5 IN OVARIAN CANCER: A NOVEL SYNERGISTIC TREATMENT STRATEGY - PROJECT SUMMARY OVARIAN CANCER REMAINS THE DEADLIEST OF ALL GYNECOLOGIC MALIGNANCIES NATIONWIDE. UNFORTUNATELY, CURRENT OVARIAN CANCER TREATMENT LEADS TO CHEMORESISTANCE AND A RAPID TUMOR RECURRENCE IN MORE THAN 85% OF PATIENTS. THE PROJECT OUTLINED HERE REPRESENTS A MUCH NEEDED NEW OPPORTUNITY FOR THERAPEUTIC INTERVENTION. OUR PREVIOUS PUBLISHED AND ONGOING WORK, HAS REVEALED THAT SHORT-FORM RON (SFRON), A TRUNCATED ISOFORM OF THE FULL-LENGTH RON (FLRON) RECEPTOR TYROSINE KINASE, CAN DRIVE OVARIAN CANCER DEVELOPMENT AND PROGRESSION. PREVIOUSLY, WE SHOWED THAT SFRON PREFERENTIALLY SIGNALS THROUGH THE PI3K PATHWAY, AND THAT THE KEY SFRON EFFECTOR S6K1 IS CRUCIAL FOR TUMOR PROMOTING EFFECTS. OUR PRELIMINARY FINDINGS REVEALED A NOVEL FUNCTION OF SFRON-S6K1 SIGNALING IN INDUCING MITOTIC PROGRESSION OF THE CELL CYCLE. THESE DATA LED TO DRUG SCREENING STUDIES SHOWING THAT THE INHIBITION OF SFRON OR S6K1 (BY BMS777608 OR AD80, RESPECTIVELY) RENDERS OVARIAN CANCER CELLS PARTICULARLY VULNERABLE TO MITOTIC KINESIN EG5 INHIBITOR ISPINESIB. WE HYPOTHESIZE THAT A SYNERGISTIC COMBINATION THERAPY SIMULTANEOUSLY BLOCKING SFRON OR S6K1 AND MITOTIC KINESINS COULD PROVE AN EFFECTIVE PRECISION TREATMENT FOR ADVANCED OVARIAN CANCER. TO TEST THIS HYPOTHESIS, WE PROPOSE TWO SPECIFIC AIMS: (1) WE WILL DETERMINE THE MECHANISM BY WHICH SFRON PROMOTES THE MITOTIC PROGRESSION OF CELL CYCLE. WE WILL TEST IF DEPLETION OF SFRON AND/OR S6K1 SIGNALING BY GENETIC AND PHARMACOLOGICAL APPROACHES AFFECTS CELL PROLIFERATION, CELL CYCLE DISTRIBUTION, AND MITOTIC PROGRESSION OR PROPER FUNCTION OF CRITICAL MITOTIC REGULATORS SUCH AS AURORA B AND/OR PLK1. FURTHER, WE HYPOTHESIZE THAT THE DEPLETION OF S6K1 CONTRIBUTES TO MITOTIC SLIPPAGE OF OVARIAN CANCER CELLS SENSITIZING THEM TO ISPINESIB. TO TEST THAT, WE WILL COMPARE THE EFFECTS OF SFRON, S6K1, AND MITOTIC KINESIN INHIBITORS AS MONOTHERAPY OR IN COMBINATION AND DETERMINE THE DRUG-INDUCED CELL FATE INCLUDING MITOTIC ARREST, MITOTIC SLIPPAGE, AND CELL DEATH. (2) WE WILL DETERMINE IF SYNERGISTIC COMBINATION THERAPY BLOCKING SFRON-S6K1 SIGNALING AND MITOTIC KINESINS LEADS TO A POTENT AND SUSTAINED ANTITUMOR EFFICACY USING CLINICALLY RELEVANT PATIENT-DERIVED XENOGRAFTS (PDXS). WE WILL USE CHEMOTHERAPY NAÏVE PDXS AND DETERMINE IF THE INHIBITION OF SFRON SYNERGISTIC PATHWAYS RESULTS IN A MORE SUSTAINED ANTI-TUMOR RESPONSE THAN STANDARD CHEMOTHERAPY. IN RECURRENT DISEASE SETTING, WE WILL USE OUR CHEMORESISTANT PDX MODELS AND INVESTIGATE IF THE COMBINATION THERAPY IS AN EFFECTIVE TREATMENT REGIMEN FOR RECURRENT OVARIAN TUMORS THAT CURRENTLY HAVE VERY LIMITED TREATMENT OPTIONS. THIS PROPOSAL WILL EXPAND OUR UNDERSTANDING OF THE MECHANISM BY WHICH SFRON AND/OR S6K1 REGULATES THE MITOTIC PROGRESSION OF CELL CYCLE. IT WILL ALSO PROVIDE NEW INSIGHT INTO WHY SFRON-S6K1 INHIBITION RENDERS OVARIAN CANCER CELLS PARTICULARLY VULNERABLE TO MITOTIC KINESIN INHIBITORS. IN THE LONG-TERM, THIS PROJECT COULD OFFER A NEW PRECISION THERAPY DESIGNED TO ACHIEVE A CLINICALLY MEANINGFUL AND SUSTAINED ANTI-TUMOR RESPONSE TO EVEN CHEMORESISTANT OVARIAN TUMORS.