The Rock Inc

DEVELOPING, DEMONSTRATING, AND DISSEMINATING INNOVATIVE PROGRAMS TO ACHIEVE TRANSLATIONAL SUCCESS

INTEGRATING INNATE AND ADAPTIVE PATHWAYS IN VACCINE RESPONSES

NATIONAL CENTER FOR DYNAMIC INTERACTOME RESEARCH

ONCOHISTONES: ROLE OF HISTONE H3 MUTATIONS IN THE ONCOGENESIS OF PEDIATRIC CANCERS

THE BROADBAND INFRASTRUCTURE DEPLOYMENT PROJECT PROPOSES TO INSTALL FIBER AND FIXED WIRELESS INFRASTRUCTURE TO DIRECTLY CONNECT 770 UNSERVED NATIVE AMERICAN HOUSEHOLDS WITH FIBER-TO-THE-HOME WITH 1 GBPS/1 GBPS AND/OR FIXED WIRELESS TO THE HOME WITH 100 MBPS/20 MBPS SERVICE

INTEGRIN ALPHAIIBBETA3 STRUCTURE, ACTIVATION, AND LIGAND BINDING

CHIPPEWA CREE TRIBE

A MINIMALLY INVASIVE SYNTHETIC BIO-DRIVEN APPROACH FOR NATURAL PRODUCTS DISCOVERY

THE CHIPPEWA CREE TRIBE OF THE ROCKY BOY RESERVATION

REGULATION OF EPIDERMAL DEVELOPMENT AND DIFFERENTIATION

CHIPPEWA CREE - ADULT DETENTION - 638 TRIBAL BASE CR1 DIST.

BROAD NEUTRALIZATION OF PANDEMIC THREAT CORONAVIRUSES - ABSTRACT-OVERALL THE RECURRENT EMERGENCE OF CORONAVIRUSES FROM ANIMAL RESERVOIRS, AND THE RESULTING COVID19 PANDEMIC, NECESSITATES THE DEVELOPMENT OF INTERVENTIONS THAT CAN TARGET DIVERSE PANDEMIC-THREAT CORONAVIRUSES. VACCINES ARE AMONG THE MOST POWERFUL MEANS FOR MITIGATING VIRAL EPIDEMICS BUT REQUIRE SIGNIFICANT BREADTH TO MAXIMIZE THE PROBABILITY OF EFFECTIVENESS AGAINST UNKNOWN VIRAL THREATS. CURRENTLY, FIRST GENERATION VACCINES ARE BEING DEPLOYED TO COMBAT SARS-COV-2, BUT THEIR EFFECTIVENESS AGAINST EMERGENT SARS-COV-2 VARIANTS AND, IMPORTANTLY, AGAINST OTHER POTENTIAL ZOONOTIC CORONAVIRUSES IS UNKNOWN. THIS PROGRAM WILL FOCUS ON NEUTRALIZING ANTIBODIES AS A DEMONSTRATED AND KEY COMPONENT OF PROTECTIVE IMMUNE RESPONSES. THE PROGRAM WILL IMPROVE PREPAREDNESS AGAINST CORONAVIRUSES, EMPLOYING A PROGRESSIVE MULTISTEP APPROACH TO INCREASE THE BREADTH OF VACCINE PROTECTION. A KEY COMPONENT OF THE RESEARCH WILL BE TO COMPREHEND HOW NEUTRALIZING ANTIBODY RESPONSES, ELICITED IN HUMANS FOLLOWING NATURAL INFECTION OR VACCINATION, TARGET THE SARS-COV-2 ENVELOPE SPIKE AND HOW ANTIBODY EVOLUTION LEADS TO INCREASED POTENCY AND BREADTH. THE IDENTIFICATION AND CHARACTERIZATION OF EPITOPES TARGETED BY SARS-COV-2 NEUTRALIZING ANTIBODIES, USING MULTIPLE APPROACHES, WILL GUIDE THE DESIGN OF IMMUNOGENS THAT AIM TO ELICIT NEUTRALIZING ANTIBODIES TARGETING AS DIVERSE A SPECTRUM OF CORONAVIRUSES AS POSSIBLE. SEVERAL IMMUNOGENS AND DELIVERY STRATEGIES WILL BE TESTED IN MICE AND HAMSTERS THAT WILL BE CHALLENGED WITH AUTHENTIC SARS-COV-2 OR A PANOPLY OF NEWLY DEVELOPED CHALLENGE MODELS INCORPORATING DIVERGENT CORONAVIRUS SPIKE PROTEINS. ANTIBODIES ELICITED IN THESE ANIMALS WILL BE ANALYZED AND COMPARED WITH THOSE FOUND IN SARS-COV-2 IMMUNIZED HUMANS AND IMMUNOGENS PROGRESSIVELY REFINED AND DOWN-SELECTED WITH THE GOAL OF PERFORMING VACCINE-CHALLENGE EXPERIMENTS IN NONHUMAN PRIMATES WITH THE MOST PROMISING CANDIDATES. THE EXPERTISE OF EACH PARTICIPATING TEAM IS HIGHLY COMPLEMENTARY AND THE PROGRAM WILL CAPITALIZE AND BUILD ON THE ALREADY EXISTING SCIENTIFIC SYNERGY TO ENSURE THE EFFICIENT AND TIMELY COMPLETION OF THE GOALS.

MOLECULAR MECHANISMS OF ANTIBODY-MEDIATED IMMUNOTHERAPIES

CHIPPEWA CREE - LAW ENFORCEMENT CR1 BASE FUNDING DIST.

CHIPPEWA CREE - FY 2013 TPA BASE CR 1

CHIPPEWA CREE - TPA BASE CR1 DISTRIBUTION

CELL ADHESION AND CYTOSKELETAL DYNAMICS IN SKIN

GENOME INSTABILITY IN CANCER: TELOMERES AND DNA REPAIR

CELLULAR DISSECTION OF HERPES SIMPLEX ENCEPHALITIS WITH IPS CELLS

OVERCOMING HOST RESTRICTION FACTORS TO DEVELOP BETTER ANIMAL MODELS FOR HIV/AIDS.

AN ENCYCLOPEDIA OF THE ADIPOSE TISSUE SECRETOME TO IDENTIFY MEDIATORS OF HEALTH AND DISEASE - WHITE AND BROWN ADIPOCYTES NOT ONLY PLAY A CENTRAL ROLE IN ENERGY STORAGE AND COMBUSTION, BUT ARE ALSO DYNAMIC SECRETORY CELLS THAT PRODUCE SIGNALING MOLECULES THAT LINK LEVELS OF ENERGY STORES TO OTHER VITAL PHYSIOLOGICAL SYSTEMS. DISRUPTION OF THE SIGNALING PROPERTIES OF ADIPOCYTES, AS OCCURS IN OBESITY, CONTRIBUTES TO INSULIN RESISTANCE, TYPE 2 DIABETES, AND OTHER METABOLIC DISORDERS. FAT CELLS HAVE BEEN ESTIMATED TO SECRETE MORE THAN 1,000 POLYPEPTIDES AND MICROPROTEINS AND EVEN LARGE NUMBER OF SMALL MOLECULE METABOLITES. THE GREAT MAJORITY OF THE ADIPOCYTE SECRETOME HAS NOT BEEN DEFINED OR CHARACTERIZED AND ADDRESSING THIS GAP IN KNOWLEDGE IS THE MAIN GOAL OF THIS COLLABORATIVE, INTERDISCIPLINARY TEAM PROJECT. A MAJOR OBSTACLE HAS BEEN THE LACK OF SUITABLE TECHNOLOGIES TO QUANTITATIVELY IDENTIFY CIRCULATING PROTEINS AND METABOLITES, DETERMINE THEIR CELLULAR ORIGIN, AND ELUCIDATE THEIR FUNCTION. BUILDING ON COMPELLING PRELIMINARY DATA AND KEY INNOVATIONS FROM MEMBERS OF THIS TEAM, WE WILL GENERATE THE FIRST ENCYCLOPEDIA OF THE WHITE AND BROWN ADIPOCYTE SECRETOME IN MOUSE MODELS AND HUMANS. SPECIFICALLY, WE WILL (1) GENERATE AN ENCYCLOPEDIA OF THE SECRETOME OF MURINE ADIPOCYTES, (2) CHARACTERIZE THE ADIPOCYTE SECRETOME IN RESPONSE TO PHYSIOLOGICAL STRESS AND IN PATHOLOGICAL STATES, (3) CHARACTERIZE THE ADIPOSE SECRETOME IN HUMANS, AND (4) CHARACTERIZE THE FUNCTION OF THE ADIPOCYTE SECRETOME. THESE STUDIES, WHICH SPAN FROM BASIC BIOLOGY TO HUMAN SUBJECT INVESTIGATION WILL ONLY BE POSSIBLE BY OPTIMIZING TOOLS WITHIN DIVERSE DISCIPLINES AND AT THEIR INTERSECTION. THIS PROJECT HAS THE POTENTIAL TO ADDRESS QUESTIONS CENTRAL TO THE MISSION OF THE NIDDK SUCH AS THE MOLECULAR BASIS FOR THE LINKS BETWEEN OBESITY AND TYPE 2 DIABETES AND UNDERSTANDING WHETHER THE ANTI-DIABETIC BENEFITS OF BROWN FAT ARE CONVEYED BY SECRETED MEDIATORS. OUR STUDIES HAVE THE POTENTIAL TO IDENTIFY NEW SECRETED MEDIATORS WITH ROLES IN OBESITY, TYPE 2 DIABETES AND METABOLIC DISEASES, CATALYZE THE DEVELOPMENT OF NEW TECHNOLOGIES, PROVIDE A CRUCIAL NEW RESOURCE FOR RESEARCHERS AND CLINICIANS, AND LEAD TO NEW BIOMARKERS AND THERAPIES.

COMBINING NEW MOLECULAR AND INFORMATIC STRATEGIES TO FIND HIDDEN WAYS TO TREAT BRAIN DISEASE

GLIAL CONTROL OF NEURON DEVELOPMENT AND FUNCTION

STRUCTURE, FUNCTION, AND REGULATION OF THE BACTERIAL TRANSCRIPTION CYCLE

FY 2013 TPA BASE DISTRIBUTION UNDER C.R. #1 (THROUGH MARCH 27, 2013)

MECHANISM OF THE TELOMERIC PROLIFERATION LIMIT

MENDELIAN GENETIC PREDISPOSITION TO HERPES SIMPLEX ENCEPHALITIS IN CHILDHOOD

INSTITUTIONAL CAREER DEVELOPMENT CORE

CENTER FOR SYSTEMS-LEVEL STUDY OF METASTASIS - PROJECT SUMMARY: METASTATIC DISEASE IS A COMPLEX, DYNAMIC AND EMERGENT PROCESS THAT REQUIRES COLLECTIVE AND COORDINATED INTERACTIONS BETWEEN MANY CELL TYPES, METABOLITES AND THE HOST. THERE IS SUBSTANTIAL CLINICO- PATHOLOGIC AND EXPERIMENTAL EVIDENCE FOR CRITICAL ROLES OF NEURAL INNERVATION, LYMPHATIC INTERACTIONS, METABOLITES AND ENDOTHELIAL CELLS IN REGULATING METASTATIC PROGRESSION BY ALTERING CANCER AND IMMUNE CELL FUNCTIONS. AS SUCH, THESE CELLULAR INTERACTIONS LIKELY SHAPE METASTATIC PROGRESSION, RESPONSES TO THERAPY AND METASTATIC DISSEMINATION. HOWEVER, WE HAVE A LIMITED UNDERSTANDING OF HOW THESE COMPONENTS COORDINATELY REGULATE METASTATIC PROGRESSION. THIS APPLICATION DESCRIBES A SERIES OF HIGHLY INNOVATIVE MULTIDISCIPLINARY MOLECULAR, CELL- BIOLOGICAL, METABOLIC, MASSIVELY-PARALLEL SINGLE-CELL SEQUENCING AND ORGANISMAL METHODS APPLIED TOWARDS DEFINING THE DYNAMIC AND EMERGENT MECHANISMS BY WHICH NEURAL CELLS, LYMPHATICS, IMMUNE CELLS AND METABOLITES INTERACT TO COORDINATELY REGULATE METASTATIC PROGRESSION—CONTRIBUTING TO A SYSTEMS-LEVEL UNDERSTANDING OF METASTASIS. WE AIM TO (I) DEFINE THE ROLE OF NEURAL INNERVATION ON METASTATIC PROGRESSION BY CHARACTERIZING NEURO- TUMOR AND NEURO-IMMUNE INTERACTIONS AND IDENTIFYING NEURAL SIGNALS AND THEIR PRO-METASTATIC MECHANISMS OF ACTION, (II) DETERMINE HOW ENDOTHELIAL CELLS REGULATE INNERVATION OF METASTATIC TUMORS, (III) DEFINE THE ROLE OF REGIONALIZED LYMPHATIC INTERACTIONS IN DRIVING METASTATIC PROGRESSION AND ANTI-METASTATIC IMMUNITY, (IV) ASSESS THE ROLE OF NEURO-IMMUNE AND NEURO-EPITHELIAL INTERACTIONS ON EARLY METASTATIC DISSEMINATION AND COLONIZATION, (V) IDENTIFY METABOLITE AND PROTEIN SIGNALS THAT DRIVE METASTATIC COLONIZATION, (VI) DISCOVER TUMORAL TRANSCRIPTION FACTORS AND RNA-BINDING PROTEINS THAT ACT DOWNSTREAM OF NEURAL AND METABOLIC SIGNALS TO DRIVE EMERGENT PRO- METASTATIC GENE EXPRESSION PROGRAMS, AND (VII) DETERMINE THE IMPACT OF STANDARD CHEMOTHERAPY ON THESE DIVERSE CELLULAR INTERACTIONS AND METABOLIC DETERMINANTS OF METASTATIC PROGRESSION. OUR PROPOSED METNET CENTER WILL ENHANCE OUR UNDERSTANDING OF HOW INTERACTIONS AND CROSSTALK BETWEEN CANCER CELLS WITH NERVOUS SYSTEM CELLS, LYMPHATICS, VASCULATURE AND IMMUNE CELLS ENABLES EMERGENCE OF METASTATIC DISEASE. WE WILL ALSO ASSESS HOW THERAPY IMPACTS SPECIFIC CELL-CELL AND METABOLIC INTERACTIONS OF METASTATIC CELLS AND PROVIDE INSIGHTS INTO THE IMPACT OF SPECIFIC CELLULAR INTERACTIONS IN THE PRIMARY MICROENVIRONMENT ON METASTATIC DISSEMINATION, INCLUDING EARLY DISSEMINATION. THESE FINDINGS WILL GENERATE AN INTEGRATED, SYSTEMS-LEVEL UNDERSTANDING OF METASTASIS, ENABLING DEVELOPMENT OF A NEW GENERATION OF ANTI-CANCER THERAPIES THAT PREVENT CRITICAL EMERGENT COORDINATED PRO-METASTATIC INTERACTIONS.

IMMUNOLOGIC CONTROL OF HIV-1 THROUGH COMBINATION BNABS AND BIOLOGICS.

STRUCTURE-FUNCTION MAPPING OF THE NUCLEAR PORE COMPLEX

EPITOPE-FOCUSED VACCINE STRATEGIES AGAINST ZIKA VIRUS

FIFTY STATE SURVEYS RELATED TO FISHING, HUNTING, AND WILDLIFE-ASSOCIATED RECREATION

DEFINING THE MECHANISMS OF HIV RESISTANCE TO BNABS IN HUMANS - PROJECT SUMMARY COMBINATION ANTIRETROVIRAL THERAPY (ART) REVOLUTIONIZED THE TREATMENT AND PREVENTION OF HIV-1 INFECTION. HOWEVER, ART DOES NOT ERADICATE ESTABLISHED INFECTION AND WORLDWIDE HIV-1 INCIDENCE RATES REMAIN HIGH AND HAVE BEEN DECLINING SLOWLY. THUS, THE SEARCH FOR NOVEL PREVENTIVE AND THERAPEUTIC INTERVENTIONS REMAINS A HIGH PRIORITY. IN RECENT YEARS, BROADLY NEUTRALIZING ANTIBODIES (BNABS) EMERGED AS A LONG-ACTING ALTERNATIVE TO DAILY ART AND AS A PROMISING STRATEGY TO ACHIEVE LONG-TERM TREATMENT-FREE HIV-1 CONTROL. BNABS DIFFER FROM ART IN THAT THEY ENGAGE THE HOST IMMUNE SYSTEM BY VIRTUE OF THEIR FC EFFECTOR DOMAINS AND THEREFORE HAVE THE POTENTIAL TO MEDIATE KILLING OF INFECTED CELLS AND MODULATE OR ENHANCE HIV-SPECIFIC IMMUNE RESPONSES. HOWEVER, BNABS ARE VULNERABLE TO ESCAPE BY HIV-1 VARIANTS. DURING HIV-1 INFECTION, ANTIBODY RESPONSES CO-EVOLVE WITH A LARGE POPULATION OF RAPIDLY MUTATING VIRUSES, SUCH THAT VARIANTS THAT ARE RESISTANT TO INDIVIDUAL ANTIBODIES ARE FREQUENTLY ENCOUNTERED. CONSISTENT WITH THIS HIGH LEVEL OF DIVERSITY, SEVERAL CLINICAL STUDIES HAVE DEMONSTRATED THAT BNAB MONOTHERAPY LEADS TO TRANSIENT DECLINES IN VIREMIA WITH RAPID SELECTION OF BNAB-RESISTANT VIRAL STRAINS. IN CONTRAST, A COMBINATION OF TWO BNABS TARGETING NON-OVERLAPPING ENV EPITOPES MAINTAINED VIRAL SUPPRESSION IN PARTICIPANTS HARBORING ANTIBODY SENSITIVE VIRUSES WHO HAD ACHIEVED VIRAL SUPPRESSION WITH ART AND SUBSEQUENTLY RECEIVED REPEATED DOSES OF BNABS DURING ART INTERRUPTION. THESE EARLY STUDIES DEMONSTRATE THE POTENTIAL THERAPEUTIC APPLICATION OF BNABS BUT ALSO HIGHLIGHT THE NEED TO BETTER UNDERSTAND VIRAL ESCAPE PATHWAYS LEADING TO BNAB RESISTANCE. ALTHOUGH RESISTANCE TO SOME BNABS (I.E. ANTI-V3 LOOP) IS PREDICATED ON KNOWN FEATURES OF ENV, THE DETERMINANTS OF RESISTANCE ARE POORLY DEFINED FOR OTHER BNABS AND FOR COMBINATIONS OF BNABS. THE OVERARCHING GOALS OF THIS PROPOSAL ARE TO UNDERSTAND THE DIVERSITY OF PATHWAYS LEADING TO BNAB ESCAPE AND USE THIS INFORMATION TO GUIDE THE DESIGN OF MORE EFFECTIVE OPTIMIZED BNAB COMBINATIONS THAT PREVENT EMERGENCE OF RESISTANT VARIANTS. THIS PROPOSAL HAS FOUR INTERRELATED AIMS DIRECTED AT ACCOMPLISHING THESE GOALS: (1) DETERMINE THE SEQUENCE ELEMENTS THAT LEAD TO VIRAL RESISTANCE TO BNAB ADMINISTRATION IN HUMANS USING NEWLY DEVELOPED NEXT GENERATION DEEP SEQUENCING METHODS; (2) SYSTEMATICALLY MAP ALL POSSIBLE VIABLE BNAB RESISTANCE MUTATIONS TO IDENTIFY MECHANISMS OF ESCAPE ACROSS VIRAL STRAINS AND SUBTYPES BY PRODUCING AND TESTING COMPLETE LIBRARIES OF ENV MUTANTS; (3) DETERMINE THE NATURE OF CLINICALLY RELEVANT BNAB-RESISTANT HIV-1 VARIANTS THAT CAN BE SELECTED IN CELL CULTURE IN THE PRESENCE OR ABSENCE OF AUTOLOGOUS SERUM; (4) DEVELOP COMPUTATIONAL MODELS THAT DEFINE MECHANISMS OF HIV-1 BNAB RESISTANCE BY INTEGRATING THE SEQUENCE INFORMATION OBTAINED FROM AIMS 1-3.

EVALUATION OF THE FCGR MECHANISMS IN THE ANTIBODY-DEPENDENT ENHANCEMENT OF SARS-COV-2 INFECTION

THE YELLOW FEVER VACCINE SAGA: DEFINING THE VIRAL AND HOST DETERMINANTS RESPONSIBLE FOR SUCCESS VERSUS FAILURE

POTASSIUM CHANNEL GATING

DEFINITION OF SERUM RIBONUCLEOPROTEIN COMPOSITION AND ITS REGULATION AND FUNCTION

SKIN STEM CELLS: PURIFICATION AND CHARACTERIZATION

PANDEMIC PREPAREDNESS RESEARCH AND BIOCONTAINMENT INFRASTRUCTURE AT THE ROCKEFELLER UNIVERSITY - PROJECT SUMMARY/ABSTRACT ROCKEFELLER UNIVERSITY IS A UNIQUE INSTITUTION: WITH ONLY 70 HEADS OF LABORATORIES, THE FACULTY HAVE RECEIVED 3 NOBEL PRIZES IN MEDICINE IN THE LAST 10 YEARS AND 5 (PLUS 1 IN CHEMISTRY) IN THE LAST 21 YEARS, ALONG WITH APPOINTMENT OF 18 FACULTY AS HHMI INVESTIGATORS AND 29 TO MEMBERSHIP IN THE NATIONAL ACADEMY OF SCIENCES. ROCKEFELLER FACULTY HAVE PLAYED AN OUTSIZED ROLE IN CONTRIBUTIONS TO FOUNDATIONAL DISCOVERIES IN VIROLOGY AND INFECTIOUS DISEASE, BOTH HISTORICALLY AND PRESENTLY. RECOGNIZING THE IMPORTANCE OF THE CURRENT PANDEMIC, OVER THE PAST TWO YEARS, A DOZEN RESEARCH GROUPS AT ROCKEFELLER HAVE JOINED THE RESEARCH EFFORTS FOCUSED ON PANDEMIC THREAT VIRUSES, WITH NUMEROUS GRANTS SUPPORTING THE WORK AND OVER 50 PEER-REVIEWED PUBLICATIONS TO DATE. ROCKEFELLER LABS HAVE MADE SEMINAL CONTRIBUTIONS IDENTIFYING THE SPECTRUM OF NEUTRALIZING ANTIBODIES TO THE SARS-COV-2 SPIKE PROTEIN, THE IMPACT OF VARIANTS ON NEUTRALIZATION, AND CHARACTERIZING THE ATOMIC- RESOLUTION BINDING OF ANTIBODIES TO SPIKE PROTEIN. COLLECTIVELY, ROCKEFELLER LABORATORIES ARE WORKING ON ALPHAVIRUSES (E.G., SINDBIS, CHIKUNGUNYA VIRUSES), FLAVIVIRUSES (E.G., YELLOW FEVER, ZIKA, WEST NILE, POWASSAN VIRUSES), HEPACIVIRUSES (HEPATITIS C VIRUS, NORWAY RAT HEPACIVIRUS), HEPADNAVIRUSES (HEPATITIS B VIRUS), ORTHOMYXOVIRUSES (INFLUENZA), CORONAVIRUSES (SARS-COV-2 AND OTHER HUMAN CORONAVIRUSES) AND RETROVIRUSES INCLUDING HIV-1. MANY OF THE VIRUSES UNDER STUDY, INCLUDING CHIKUNGUNYA, YELLOW FEVER, WEST NILE, POWASSAN, AND SARS-COV-2, ARE CLASSIFIED AS RISK GROUP 3 (RG3) PATHOGENS, REQUIRING BSL3 CONTAINMENT FACILITIES AND PRACTICES. MOREOVER, SOME RESEARCH AT ROCKEFELLER INCLUDES THE GENERATION OF REPLICONS AND CHIMERIC (VSV- BASED) VIRUSES WHOSE STABILITY AND SAFETY SHOULD BE ESTABLISHED UNDER BSL3 CONDITIONS PRIOR TO USE AT LOWER BIOSAFETY CONTAINMENT LEVELS. THIS PROJECT SEEKS TO ENHANCE AND EXPAND SPACE AND SUPPORT FOR BOTH IN VIVO AND IN VITRO RESEARCH. SPECIFIC AIM 1 IS TO INCREASE THE ROCKEFELLER UNIVERSITY’S BSL3 CAPACITIES TO MEET THE SCIENTIFIC NEEDS BY ESTABLISHING A NEW BSL3 FACILITY IN AN EXISTING LABORATORY BUILDING. SPECIFIC AIM 2 IS TO EQUIP AND ENHANCE THE NEW FACILITY AND THE BSL3/ABSL3 FACILITIES THAT ALREADY EXIST AT THE UNIVERSITY. TO ACHIEVE THESE AIMS, AN EXTREMELY EXPERIENCED TEAM OF PERSONNEL AT ROCKEFELLER PROPOSE TO: ESTABLISH A NEW MULTIPLE- INVESTIGATOR BSL3 FACILITY; ADD EQUIPMENT TO THE EXISTING SINGLE-PI BSL3 FACILITY; REPLACE AN EXISTING AUTOCLAVE AND ADD AN INCREMENTAL AUTOCLAVE IN THE ABSL3; AND MODERNIZE THE SUPPORTING INFRASTRUCTURE IN THE ABSL3.

INTESTINAL REGULATION OF THPOK EXPRESSION AND CD4 HELPER T CELL FUNCTION

REGULATION OF MITOTIC CHROMOSOMES - REVISION - 2

ENZYMOLOGY AND FUNCTION(S) OF HISTONE PHOSPHORYLATION

MOLECULAR DEFINITION OF BRAIN CIRCUITS CONTROLLING ADDICTION

DISASTER GRANTS - PUBLIC ASSISTANCE (PRESIDENTIALLY DECLARED DISASTERS)

CHEMICAL BIOLOGY OF CELL DIVISION

PURCHASE LAND AND PROVIDE TECHNICAL ASSISTANCE TO FARMERS TO INCREASE CAPITAL AND MARKET ACCESS.

GENERATION OF IMMUNOLOGICAL MEMORY BY CRISPR-CAS SYSTEMS

QUANTIFYING CELL-CELL INTERACTIONS IN THE IMMUNE SYSTEM BY TRANS-SYNAPTIC LABELING

GENETICS AND CELL BIOLOGY

DYNAMICS OF ANTIGEN-DRIVEN SELECTION IN GERMINAL CENTERS

DETERMINANTS OF INFECTIOUS HIV-1 PARTICLE PRODUCTION

TYPE I INTERFERON-STIMULATED GENES AND THE ANTIVIRAL IMMUNE RESPONSE

CENTER FOR THERAPEUTIC TARGETING OF THE FUSION ONCOPROTEIN OF FIBROLAMELLAR HEPATOCELLULAR CARCINOMA

CLASS SWITCH RECOMBINATION IN B LYMPHOCYTES

NEUROENGINEERING A ROBUST VOCAL LEARNING PHENOTYPE IN MICE AS A MODEL FOR TREATING COMMUNICATION DISORDERS

ROLE OF FIBRINOGEN IN ALZHEIMER'S DISEASE

MOTOR PROTEIN DYNAMICS AND MITOTIC MECHANISMS

INDIAN HOUSING BLOCK GRANTS

OPTIMIZATION, APPLICATION, AND DISSEMINATION OF IMAGING MODULES FOR HIGH-SPEED MESOSCOPIC VOLUMETRIC RECORDING OF NEUROACTIVITY IN SCATTERING BRAINS - PROJECT SUMMARY / ABSTRACT A NUMBER OF RECENT OBSERVATIONS SUGGEST THAT COMPLEX BRAIN FUNCTIONS IN THE MAMMALIAN BRAIN EMERGE FROM HIGHLY PARALLEL COMPUTATION IN WHICH INFORMATION ABOUT SENSORY INPUTS, INTERNAL STATES, AND BEHAVIORAL PARAMETERS ARE MAPPED ONTO HIGHLY DISTRIBUTED BRAIN-WIDE NEURONAL POPULATIONS. THIS CALLS FOR NEUROTECHNOLOGIES THAT ALLOW FOR LARGE-SCALE RECORDING OF NEURO-ACTIVITY ACROSS TISSUE DEPTHS AND BRAIN REGIONS AT PHYSIOLOGICAL TIMESCALES AND CELLULAR RESOLUTION IN AWAKE AND BEHAVING ANIMALS. WHILE RECENT ADVANCEMENTS IN OPTICAL TOOL DEVELOPMENT BASED ON THE COMBINATION OF TWO-PHOTON SCANNING FLUORESCENCE MICROSCOPY (2P M) AND GENETICALLY-ENCODED CALCIUM INDICATORS (GECIS) AS REPORTERS OF NEURO-ACTIVITY HAVE BEEN AIMED AT ADDRESSING THESE NEEDS BY DEVELOPING FASTER, LARGER-SCALE, AND VOLUMETRIC CALCIUM (CA2+) IMAGING TECHNOLOGIES, A FUNDAMENTAL UNSOLVED CHALLENGE IN THIS CONTEXT IS NAVIGATING THE INHERENT TRADEOFFS BETWEEN SPEED, RESOLUTION, AND THE SIZE OF THE RECORDING VOLUME IN A PRINCIPLED AND SCALABLE MANNER. OUR LAB HAS RECENTLY ESTABLISHED CRITERIA FOR SUCH OPTIMAL RECORDING SCHEMES WHICH HAS LED TO THE REALIZATION OF A NEW HIGH-SPEED VOLUMETRIC CA2+ IMAGING APPROACH TERMED LIGHT BEADS MICROSCOPY (LBM). THROUGH LBM, WE HAVE DEMONSTRATED FLUORESCENCE LIFETIME LIMITED VOLUMETRIC RECORDING OF NEURO-ACTIVITY AT A SINGLE-CELL RESOLUTION OF UP TO 1 MILLION NEURONS WITHIN BOTH CORTICAL HEMISPHERES OF AWAKE, BEHAVING MICE. IN THIS PROJECT, WE WILL PURSUE A MULTIPRONGED STRATEGY TOWARDS THE OPTIMIZATION, BIOLOGICAL APPLICATIONS, AND EFFECTIVE DISSEMINATION OF OUR LBM TECHNOLOGY WHILE EXTENDING ITS PERFORMANCE. THIS WILL RESULT IN A MORE ROBUST, LESS COMPLEX, AND MORE USER-FRIENDLY VERSION OF OUR LBM TECHNOLOGY. TO ENABLE ITS BROAD AND EFFECTIVE DISSEMINATION, IN THE SECOND PART OF THE PROJECT, WE WILL UTILIZE FEEDBACK FROM OUR A-TESTERS TO DESIGN, BUILD, AND DISSEMINATE SS-PROTOTYPES OF OUR SYSTEM THAT WILL BE DISTRIBUTED TO SEVERAL END-USER LABORATORIES WHO WILL BE TESTING AND APPLYING OUR LBM TECHNOLOGY IN THE CONTEXT OF THEIR BIOLOGICAL QUESTIONS. THIS SS-PROTOTYPE WILL ALSO FORM THE BASIS FOR COMMERCIAL DISSEMINATION OF OUR TECHNOLOGY AS WELL AS A PARALLEL EFFORT FOR ITS OPEN-SOURCE DISSEMINATION.

INDIAN HOUSING BLOCK GRANTS

DISCOVERY AND MECHANISM OF ANTIRETROVIRAL FACTORS

GOVERNMENT TO GOVERNMENT AGREEMENT - CHIPPEWA CRESS

USING EVOLUTIONARY VARIATION TO PROBE THE NEURAL BASIS FOR BEHAVIOR

GENOME-WIDE SEARCH FOR INBORN ERRORS OF IL-17 IMMUNITY UNDERLYING CHRONIC MUCOCUTANEOUS CANDIDIASIS

B CELL CLONAL SELECTION IN GUT-ASSOCIATED GERMINAL CENTERS

OPTIMIZATION, APPLICATION AND DISSEMINATION OF HIGH-SPEED HYBRID MULTIPHOTON VOLUMETRIC IMAGING TECHNOLOGIES

CELL ADHESION AND CYTOSKELETAL DYNAMICS IN SKIN

INTESTINAL SURVEILLANCE BY INTRAEPITHELIAL LYMPHOCYTES

GENOME-WIDE DISSECTION OF MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE

MOLECULAR MECHANISMS UNDERLYING INSECT ODORANT RECEPTOR FUNCTION AND MODULATION

ROLE OF THE CONTACT SYSTEM IN ALZHEIMER'S DISEASE

FUNCTIONAL NETWORKS IN MICROGLIA AND ASTROCYTES REGULATING NEUROPATHOLOGY OF ALZHEIMER'S DISEASE

OPTIMIZATION OF FC EFFECTOR ACTIVITY OF ANTI-HIV ANTIBODIES TO TARGET HIV RESERVOIR

GRANT TO LOCAL GOVERNMENT FOR REPAIR OR REPLACEMENT OF DISASTER DAMAGED FACILITIES

INTERDISCIPLINARY STUDIES OF SLEEP AND CIRCADIAN RHYTHMS

MECHANISMS OF ANTIBODY-DEPENDENT ENHANCEMENT OF DENGUE DISEASE

DEVELOPMENT OF NOVEL GENOMIC APPROACHES FOR PROFILING CELLULAR TEMPORAL-SPATIAL DYNAMICS OF NEUROGENESIS IN AGING AND ALZHEIMER'S DISEASE - PROJECT SUMMARY ADULT NEUROGENESIS IS EMERGING AS AN IMPORTANT PLAYER IN MAINTAINING BRAIN HOMEOSTASIS AND NORMAL FUNCTIONS. THE DYSFUNCTIONS OF NEUROGENESIS HAVE BEEN ASSOCIATED WITH AGING AND NEUROLOGICAL DISORDERS, INCLUDING ALZHEIMER’S DISEASE (AD). THE ABILITY TO SYSTEMATICALLY MAP THE MOLECULAR DYNAMICS OF NEUROGENESIS AT SINGLE- CELL RESOLUTION COULD SERVE AS A FOUNDATION FOR A SYSTEMATIC EFFORT TO BETTER UNDERSTAND THE MOLECULAR EVENTS THAT GIVE RISE TO ABNORMAL CELL STATES IN AGING AND DISEASES. WHILE THE RAPID ADVANCES IN SINGLE-CELL GENOMICS ARE CREATING UNPRECEDENTED OPPORTUNITIES TO EXPLORE MOLECULAR HETEROGENEITY IN MAMMALIAN BRAINS, NEARLY ALL SUCH METHODS ARE RESTRICTED TO LOW THROUGHPUT AND FAIL TO RECOVER THE HETEROGENEITY AND DYNAMICS OF THE PROFOUNDLY RARE CELL STATES IN ADULT NEUROGENESIS (E.G., LESS THAN 0.1% OF THE CELL POPULATION IN THE BRAIN). HEREIN, WE PROPOSE TO DEVELOP NOVEL METHODOLOGIES THAT ENABLE A COMPREHENSIVE VIEW OF TEMPORAL-SPATIAL DYNAMICS OF NEUROGENESIS DURING AGING AND ALZHEIMER'S DISEASE (AD) IN BOTH HUMAN AND MOUSE BRAINS. SPECIFICALLY, WE WILL FIRST DEVELOP A NOVEL HIGH-THROUGHPUT, LOW-COST SINGLE-CELL GENOMICS APPROACH, SCINEXT1000, TO PROFILE THE MOLECULAR HETEROGENEITY OF FOUR MILLION CELLS FROM POST-MORTEM HUMAN HIPPOCAMPAL SAMPLES. THIS APPROACH WILL BE POWERFUL BECAUSE WE CAN NOT ONLY QUANTITATIVELY CHARACTERIZE THE FREQUENCY OF HUMAN ADULT HIPPOCAMPAL NEUROGENESIS AT SINGLE-CELL RESOLUTION, BUT ALSO IDENTIFY THE TRANSCRIPTOME FEATURES ASSOCIATED WITH IMPAIRED NEUROGENESIS IN AGING AND AD AT ISOFORM RESOLUTION. IN ADDITION, WE WILL DEVELOP ANOTHER NOVEL SINGLE-CELL GENOMIC TECHNIQUE, SCI-DIV- SEQ, TO ENHANCE THE DETECTION OF NEWBORN NEURONS, AND IDENTIFY THE CELLULAR DIFFERENTIATION TRAJECTORIES AND ASSOCIATED TRANSCRIPTOMIC FEATURES OF ADULT NEUROGENESIS IN YOUNG AND AGED MOUSE BRAINS. THE RESULTING DATASET WILL ADVANCE OUR UNDERSTANDING OF GENE REGULATION IN NEUROGENESIS ACROSS DIFFERENT NEURAL LINEAGES AND CONSTITUTE A SIGNIFICANT STEP TOWARDS A COMPREHENSIVE CHARACTERIZATION OF THE MOLECULAR MECHANISM UNDERLYING NEUROGENESIS IMPAIRMENT IN AGING. IN ADDITION TO THE INTERNAL MOLECULAR PROGRAMS, THE NEUROGENESIS PROCESS IS CONTROLLED BY ASPECTS OF ENVIRONMENTAL SIGNALS FROM THE NEURAL STEM NICHE. WE WILL APPLY A HIGH-THROUGHPUT SPATIAL TRANSCRIPTOMIC STRATEGY TO IDENTIFY THE CELLULAR INTERACTIONS AND LOCAL MICROENVIRONMENT INVOLVED IN ADULT NEUROGENESIS IN BOTH HUMAN AND MOUSE BRAINS. THESE MULTI-PRONGED APPROACHES WILL OPEN A NEW PARADIGM FOR UNDERSTANDING THE GLOBAL MOLECULAR PROGRAMS AND ENVIRONMENTAL REGULATION OF ADULT NEUROGENESIS, THEREBY INFORMING POTENTIAL THERAPEUTIC TARGETS TO RESTORE CELL POPULATION HOMEOSTASIS IN AGING AND BRAIN DISORDERS.

HAZARD MITIGATION GRANT

DISCOVERY OF ANTIBIOTICS FROM SOIL MICROBIOMES USING METAGENOMICS

3BNC117 AND 10-1074 TO SUPPRESS HIV-1 REPLICATION AND REDUCE THE RESERVOIR

NEURO-IMMUNE INTERACTIONS AT THE INTESTINAL SURFACE

LAUNCHING HBV WITH RNA TO ASSESS ANTIVIRAL RESISTANCE AND EXPLORE FUNDAMENTAL ASPECTS OF VIRUS-HOST BIOLOGY

INBORN ERRORS OF IMMUNITY IN PATIENTS WITH LIFE-THREATENING COVID-19 - PROJECT SUMMARY THERE IS IMMENSE INTERINDIVIDUAL CLINICAL VARIABILITY IN HUMANS INFECTED WITH SARS-COV-2, RANGING FROM SILENT INFECTION TO LETHAL COVID-19. THE FIRST BREAKTHROUGH TO CRACK THIS ENIGMA CAME FROM THE FIELD OF INBORN ERRORS OF IMMUNITY (IEI). IN AN INTERNATIONAL COHORT OF 659 PATIENTS, WE REPORTED 23 PATIENTS WITH IEIS AT EIGHT INFLUENZA SUSCEPTIBILITY LOCI THAT GOVERN TLR3- AND IRF7-DEPENDENT TYPE I INTERFERON (IFN) IMMUNITY (3.5%), INCLUDING FOUR UNRELATED PATIENTS WITH AUTOSOMAL RECESSIVE IRF7 OR IFNAR1 DEFICIENCY. WE ALSO REPORTED AN ADDITIONAL 101 PATIENTS WITH NEUTRALIZING AUTOANTIBODIES (AUTO-ABS) AGAINST TYPE I IFN (10.2% OF 987), WHO WERE AUTO-IMMUNE PHENOCOPIES OF THE PATIENTS WITH IEI. INTERESTINGLY, 94% OF THE PATIENTS WITH AUTO-AB AGAINST TYPE I IFN WERE MEN, AND ONE OF THE SIX SICK WOMEN HAD X-LINKED DOMINANT INCONTINENTIA PIGMENTI (IP), SUGGESTING X-LINKED INHERITANCE IN AT LEAST SOME OF THE PATIENTS. COLLECTIVELY, THESE PATIENTS ACCOUNT FOR ABOUT 13.5% OF LIFE-THREATENING COVID- 19 CASES STUDIED. WE NOW HYPOTHESIZE THAT OTHER IEI THAT RESULT IN ABNORMAL (I) PRODUCTION OR AMPLIFICATION OF TYPE I IFN, (II) ACTIVITY OF SOLUBLE TYPE I IFNS (VIA NEUTRALIZING AUTO-ABS), OR (III) RESPONSE TO TYPE I IFN (IN TERMS OF INTERFERON STIMULATED GENE (ISG) ACTIVITY), CAN UNDERLIE LIFE-THREATENING COVID-19 IN OTHER PATIENTS. TO TACKLE THESE THREE SPECIFIC AIMS, WE BENEFIT FROM AN INTERNATIONAL RECRUITMENT FROM THE COVID HUMAN GENETIC EFFORT (HTTPS://WWW.COVIDHGE.COM). OUR PRELIMINARY DATA ARE VERY STRONG. FIRST, WE HAVE FOUND 215 PATIENTS WITH PREDICTED LOSS-OF-FUNCTION (PLOF) VARIANTS AT 157 LOCI ASSOCIATED WITH PRODUCTION OR AMPLIFICATION OF TYPE I IFN, INCLUDING ONE PATIENT HOMOZYGOUS FOR A PLOF VARIANTS IN NLRC3, TWO PATIENTS HETEROZYGOUS FOR PLOF VARIANTS IN DDX58/RIG-I, AND SIX PATIENTS HETEROZYGOUS FOR PLOF VARIANTS IN SUBTYPES OF TYPE I OR III IFNS. SECOND, AMONG PATIENTS WITH AUTO-AB AGAINST TYPE I IFN, WE IDENTIFIED A PATIENT HEMIZYGOUS FOR A PLOF IN X-LINKED SASH3. IN ADDITION, WE FOUND THAT 25% OF PATIENTS WITH IP, WHICH IS ASSOCIATED WITH SEVERELY SKEWED X-INACTIVATION, HAVE AUTO-AB AGAINST TYPE I IFN, FURTHER SUGGESTING AN X-LINKED BASIS OF AUTO-AB TO TYPE I IFN PRODUCTION. THIRD, WE FOUND 24 PATIENTS WITH PLOF VARIANTS IN 18 ISGS. WE HAVE SHOWN THAT THE INTERNATIONAL PATH-BREAKING PROGRAM WE ESTABLISHED IN ONLY 6 MONTHS IS HIGHLY EFFICIENT, AS IT RESULTED IN A PARADIGM-SHIFTING DISCOVERY. OUR NEW PROGRAM WILL BENEFIT FROM THIS MOMENTUM. OUR FUTURE DISCOVERIES OF NEW INBORN ERRORS OF TYPE I IFN IMMUNITY UNDERLYING LIFE-THREATENING COVID-19 PNEUMONIA WILL PAVE THE WAY FOR NEW DIAGNOSTIC AND THERAPEUTIC STRATEGIES TO BETTER MANAGE PATIENTS INFECTED WITH SARS-COV-2 AT RISK OF SEVERE DISEASE. SELECTED PATIENTS MAY BENEFIT FROM SUBCUTANEOUS OR NEBULIZED IFN-A OR IFN-B (DEFECT IN TYPE I IFN PRODUCTION OR AMPLIFICATION), PLASMAPHERESIS AND/OR B CELL DEPLETION (NEUTRALIZING AUTO-ABS AGAINST TYPE I IFNS), OR OTHER THERAPIES, INCLUDING MABS AGAINST SARS-COV-2 (DEFECTS OF ISGS).

BIOLOGY AND GENETICS OF METASTATIC DISEASE - BIOLOGY AND GENETICS OF METASTATIC DISEASE MY LABORATORY STUDIES THE MOLECULAR ALTERATIONS THAT CONTRIBUTE TO METASTASIS FORMATION, A POORLY UNDERSTOOD PROCESS AND PRIMARY CAUSE OF SOLID CANCER DEATHS. IT HAS LONG BEEN THOUGHT THAT METASTASIS IS CAUSED BY SOMATIC METASTASIS DRIVER MUTATIONS—POSTULATED ALTERATIONS THAT HAVE YET TO BE IDENTIFIED. BY SHOWING THAT LEVELS OF SPECIFIC MICRORNAS BECOME ALTERED IN METASTATIC TUMORS AND IDENTIFYING THEIR TARGET GENES, MY LAB IDENTIFIED AND CHARACTERIZED CRITICAL PATHWAYS AND PROCESSES UNDERLYING METASTASIS FORMATION. BY STUDYING GERMLINE VARIANTS OF ONE SUCH TARGET METASTASIS GENE, WE DISCOVERED THAT METASTATIC POTENTIAL CAN ALSO PRE- DATE TUMOR FORMATION AND BE GENETICALLY INHERITED—REVEALING AN UNANTICIPATED GENETIC UNDERPINNING FOR METASTASIS AND OPENING UP A NEW DIRECTION FOR THE FIELD. SPECIFICALLY, WE DETERMINED THAT TWO COMMON HUMAN GERMLINE VARIANTS OF THE SECRETED GLYCOPROTEIN APOE PROMOTE OR SUPPRESS METASTASIS IN MELANOMA, WITH RECENT DATA SUGGESTING THIS PRINCIPLE MAY APPLY TO ADDITIONAL CANCERS. APOE SIGNALING WAS FOUND TO GOVERN VASCULAR AND IMMUNE INTERACTIONS AS WELL AS CELLULAR INVASIVENESS THAT COLLECTIVELY CONTRIBUTE TO METASTASIS FORMATION. THESE INSIGHTS HAVE SIGNIFICANT TRANSLATIONAL POTENTIAL AND FORMED THE BASIS OF CLINICAL TRIALS THAT ARE PROVIDING PROOF-OF-CONCEPT FOR ‘METASTASIS TARGETING THERAPY’, WHERE MULTIPLE METASTASIS REGRESSION RESPONSES WERE OBSERVED IN ADVANCED STAGE PATIENTS FOR WHOM STANDARD OF CARE AND IMMUNOTHERAPY TREATMENTS HAD FAILED. GOING FORWARD, WE WILL USE ALLELIC VARIANTS OF APOE AS POWERFUL GENETIC ENTRY POINTS TO UNDERSTAND THE MOLECULAR EVENTS UNDERLYING METASTASIS FORMATION, WHERE WE WILL DEFINE HOW APOE SIGNALS ARE RECEIVED BY CELLS AND HOW APOE MEDIATES INTRACELLULAR EVENTS. WE WILL ALSO EXTEND THE CONCEPT OF HEREDITARY METASTASIS GENETICS TO ADDITIONAL CANCERS AND GENES, APPLYING OUR REVERSE GENETIC AND MOUSE MODELING APPROACHES TO BREAST AND COLORECTAL CANCER METASTASIS. TO ACHIEVE THIS UNDERSTANDING, WE WILL EMPLOY INNOVATIVE OPTICAL, PHYSIOLOGICAL, GENETIC MODELING AND SCREENING METHODS TO INTERROGATE MOUSE AND HUMAN METASTATIC TRANSITIONS. THIS AWARD WILL ENABLE OUR GROUP TO ESTABLISH THE FIRST GENETICALLY GUIDED FRAMEWORK FOR UNDERSTANDING THE MOLECULAR MECHANISMS GOVERNING METASTASIS FORMATION—ENABLING NEW AVENUES FOR ITS THERAPEUTIC TREATMENT AND PREVENTION.

FUNCTIONS AND MECHANISMS OF TRANSCRIPTIONAL COACTIVATOR OCA-B IN B CELL DEVELOPMENT AND LYMPHOMAGENESIS

FUNCTION AND TARGETING OF A STABLE TRANSCRIPTION FACTOR COMPLEX IN LEUKEMIA

EXPANDING AND ENHANCING SCIENTIFIC INSTRUMENTATION PROTOTYPING AND FABRICATION AT THE ROCKEFELLER UNIVERSITY - ABSTRACT THE PROPOSED PROJECT INVOLVES FOCUSED CONSTRUCTION TO EXPAND THE ADVANCED FABRICATION FACILITY AND CAPABILITIES AT THE ROCKEFELLER UNIVERSITY IN MANHATTAN, NEW YORK CITY. THE PRECISION INSTRUMENTATION TECHNOLOGIES (PIT) RESOURCE CENTER PROVIDES A CENTRALIZED AND EXPERTLY STAFFED PROTOTYPING AND FABRICATION FACILITY THAT SUPPORTS MORE THAN 50 PRINCIPAL INVESTIGATORS AT THE ROCKEFELLER UNIVERSITY (RU). THIS PROJECT SEEKS TO EXPAND AND ENHANCE OUR DESIGN AND FABRICATION CAPABILITIES. THE PROJECT GOALS ARE TO: (1) RENOVATE AND ANNEX ADJACENT SPACE TO THE CURRENT PIT TO ACCOMMODATE ADDITIONAL INSTRUMENTATION, ACTIVITIES, AND PERSONNEL; (2) UNDERTAKE ELEVATOR REMOVAL AND RIGGING REQUIRED TO ADD INSTITUTIONALLY FUNDED FABRICATION EQUIPMENT (CNC MILL, CNC LATHE, WATER JET CUTTER); AND (3) CREATE A NEW MICROFABRICATION FACILITY.

DEVELOPMENTAL AND DYNAMIC REGULATION OF THE CROSSTALK BETWEEN ADIPOCYTES AND THE SYMPATHETIC NERVOUS SYSTEM

DEVELOPMENT OF NEXT GENERATION MASS SPECTROMETRIC INSTRUMENTATION FOR PROTEOMICS

FUNCTIONAL AND MECHANISTIC STUDY OF HISTONE CROTONYLATION IN HEMATOLOGICAL MALIGNANCIES

REMOTE ELECTROMAGNETIC CONTROL OF NEURAL ACTIVITY FOR TREATMENT OF PARKINSON'S DISEASE

STRUCTURE/FUNCTION ANALYSES OF ESSENTIAL MYCOBACTERIAL TRANSCRIPTION REGULATORS

MOLECULAR CONTROL OF GERMINAL CENTER SELECTION AND AFFINITY MATURATION

FIRST-IN-HUMAN STUDY OF A POTENT ANTI-HBSAG NEUTRALIZING ANTIBODY - PROJECT SUMMARY HEPATITIS B VIRUS (HBV) REMAINS A MAJOR GLOBAL HEALTH PROBLEM AND CHRONIC HBV (CHB) IS A MAJOR CAUSE OF LIVER CIRRHOSIS AND HEPATOCELLULAR CARCINOMA. WHILE ANTIVIRAL THERAPIES ACHIEVE LONG-TERM VIRAL SUPPRESSION, THEY CAN RARELY CLEAR THE INFECTION OR ACHIEVE A STATE OF FUNCTIONAL CURE WHERE LONG-TERM VIRAL SUPPRESSION IS MAINTAINED IN THE ABSENCE OF TREATMENT. ALONG WITH PERSISTENCE OF VIRAL ANTIGENS, IMPAIRED HBV-SPECIFIC IMMUNITY CONTRIBUTES TO THE CHRONICITY OF INFECTION. CHRONIC EXPOSURE TO HIGH LEVELS OF HBSAG MAY RENDER HBV- SPECIFIC IMMUNE CELLS OVERLY ACTIVATED AND FUNCTIONALLY TOLERIZED THUS, DECREASING SERUM HBSAG COULD BE A VALUABLE THERAPEUTIC STRATEGY, DUE TO ITS POTENTIAL TO ALLEVIATE FUNCTIONAL EXHAUSTION AND CONFER IMMUNE CONTROL. PASSIVE TRANSFER OF ANTIBODIES IS A POTENTIAL STRATEGY IN CHB FOR THEIR DUAL FUNCTIONALITY. ANTIBODIES DIFFER FROM DIRECT-ACTING ANTIVIRALS IN THAT THEY CAN RECRUIT IMMUNE EFFECTOR FUNCTIONS THROUGH THEIR FC DOMAINS TO ACCELERATE CLEARANCE OF VIRUSES AND INFECTED CELLS. IN ADDITION, IMMUNE COMPLEXES ARE POTENT IMMUNOGENS THAT CAN FOSTER DEVELOPMENT OF HOST IMMUNE RESPONSES. HEPB MONOCLONAL ANTIBODY (MAB)19 IS A HUMAN MONOCLONAL ANTIBODY TO THE A-DETERMINANT OF THE EXTRACELLULAR LOOP OF HBSAG AND BINDS THE MAJOR HBV SEROTYPES. HEPB MAB19 SHOWED EXCEPTIONAL IN VITRO NEUTRALIZATION ACTIVITY WITH IC50 IN THE NANOGRAM RANGE AND IN VIVO ANTIVIRAL ACTIVITY IN AN ANIMAL MODEL OF INFECTION. THE OBJECT OF THIS PROPOSAL IS TO CONDUCT A FIRST-IN-HUMAN DOSE-ESCALATION STUDY OF A LONG-ACTING VARIANT OF HEPB MAB19 IN INDIVIDUALS WITH CHB ON ANTIVIRAL NUCLEOS(T)IDE ANALOGUE (NRTI) THERAPY. THE HYPOTHESIS TO BE TESTED IS THAT THE ADMINISTRATION OF HEPB MAB19-LS DURING SUPPRESSIVE NRTI THERAPY WILL BE SAFE AND WELL TOLERATED, WILL LEAD TO DECREASED LEVELS OF CIRCULATING HBSAG, AND ENHANCE HOST INNATE AND ADAPTIVE IMMUNE RESPONSES TO HBV.

GLIAL CONTROL OF SENSORY NEURON FUNCTION

IMMUNITY AND INFECTIOUS DISEASE

ANALYSIS OF IMMUNITY, VIRAL ADAPTATION AND PATHOGENESIS IN A NEW MOUSE MODEL OF HCV-RELATED RODENT HEPACIVIRUS INFECTION

DEFINING THE ROLE OF T CELL HELP IN GERMINAL CENTERS BY INTERCELLULAR ENZYMATIC LABELING - PROJECT SUMMARY GENERATION OF HIGH AFFINITY ANTIBODIES IN GERMINAL CENTERS (GCS) IS A CRITICAL STEP IN A WIDE VARIETY OF CLINICALLY RELEVANT PROCESSES, FROM PROTECTION AGAINST PATHOGENS BY PRIOR INFECTION OR VACCINATION TO THE DEVELOPMENT OF ALLERGIES AND AUTOIMMUNE DISEASES. ANTIBODY AFFINITY MATURATION FOLLOWS A PROTOTYPICAL DARWINIAN FRAMEWORK, IN WHICH GC B CELLS INTRODUCE RANDOM MUTATIONS INTO THE ANTIGEN-BINDING PORTIONS OF THEIR IMMUNOGLOBULIN (IG) GENES, GENERATING VARIATIONS IN AFFINITY WITHIN THEIR PROGENY. RARE B CELLS THAT ACQUIRE AFFINITY-INCREASING MUTATION ARE THEN SELECTIVELY EXPANDED WITHIN THE GC POPULATION, THUS INCREASING THE AVERAGE AFFINITY OF GC B CELLS AS A WHOLE, IN A PROCESS WE REFER TO AS POSITIVE SELECTION. DESPITE DECADES OF WORK, THE PRECISE CELLULAR MECHANISMS OF POSITIVE SELECTION—IN OTHER WORDS, HOW GCS “PICK OUT” B CELLS WITH THE HIGHEST AFFINITY—REMAINS A TOPIC OF DEBATE. MORE THAN 10 YEARS AGO, WE PROVIDED THE FIRST IN VIVO EVIDENCE IN MICE FOR A ROLE FOR T FOLLICULAR HELPER (TFH) CELLS AS ARBITERS OF THIS SELECTIVE PROCESS. IN OUR MODEL, TFH CELLS WOULD SENSE HOW MUCH PEPTIDE A B CELL COULD PRESENT ON ITS SURFACE (WHICH IN TURN DEPENDED ON THE B CELL’S AFFINITY), PROVIDING HELP SELECTIVELY TO THE HIGHEST-AFFINITY B CELLS. HOWEVER, DESPITE ACCUMULATING FUNCTIONAL EVIDENCE FOR THIS MODEL, SELECTIVE DELIVERY OF T CELL HELP TO B CELLS BASED ON THEIR AFFINITY HAS NEVER BEEN DIRECTLY DEMONSTRATED IN PHYSIOLOGICAL SETTINGS. TO ACHIEVE THIS, WE DEVELOPED LIPSTIC, A METHOD THAT ALLOWS US TO DIRECTLY RECORD T CELL HELP TO B CELLS WITH GREAT PRECISION IN VIVO. IN AIM 1 OF THIS PROJECT, WE PROPOSE TO USE LIPSTIC AS A MEANS TO DIRECTLY TEST THE T CELL HELP MODEL IN CLASSIC HAPTEN-CARRIER INDUCED GC SELECTION MODELS. IN AIM 2, WE WILL FOLLOW UP ON THIS BY TESTING OUR FINDINGS FROM MOUSE LIPSTIC IN HUMAN VACCINE-INDUCED GCS. IN AIM 3, WE USE THE ORIGINAL LIPSTIC IN CONJUNCTION WITH TWO NOVEL VERSIONS ON THIS STRATEGY TO INVESTIGATE THE DYNAMICS OF MULTI-ANTIGEN DRIVEN SELECTION IN INFLUENZA-INDUCED GCS.

TRACKING SARS-COV-2 ONE MOLECULE AT A TIME: SPATIOTEMPORAL INVESTIGATION OF CORONAVIRUS REPLICATION DYNAMICS AND HOST RESPONSE IN SINGLE CELLS IN VITRO AND IN VIVO - PROJECT SUMMARY THE CURRENT PANDEMIC HAS HIGHLIGHTED FUNDAMENTAL GAPS IN OUR KNOWLEDGE ABOUT THE REPLICATION STRATEGIES OF CORONAVIRUSES, AND HOW THESE ARE AFFECTED BY THE HOST AT BOTH THE ORGANISMAL AND CELLULAR LEVEL. THERE IS A PRESSING NEED TO UNDERSTAND HOW SARS-COV-2 INFECTION AND HOST-CELL RESPONSES TRIGGER SUCH A DIVERSE SET OF PATHOLOGIES, AND THE ROLES PLAYED BY VIRAL VARIATION, HOST GENETICS AND UNDERLYING PRECONDITIONS. AS STUDIES OF SARS-COV-2 FREQUENTLY UTILIZE POPULATION-BASED ASSAYS THAT LOOK HOURS TO DAYS POST INFECTION, INFORMATION ON CELLULAR AND SPATIAL VARIABILITY ARE LOST. FURTHERMORE, HOST RESPONSES ARE COMMUNICATIVE SPATIAL PROCESSES SUBJECT TO SIGNALING GRADIENTS THAT VARY BETWEEN CELLS. THUS, AVERAGES OVER POPULATIONS OBSCURE HETEROGENEITY AND SPATIAL SEPARATIONS, AND MISS THE EARLIEST VIRAL AND HOST BEHAVIORS DUE TO LACK OF SENSITIVITY. TO FILL THIS GAP, WE DEVELOPED EXPERIMENTAL AND COMPUTATIONAL APPROACHES TO QUANTIFY INDIVIDUAL VIRION ENTRANCE, ESTABLISHMENT OF THE FIRST REPLICATIVE EVENTS, AND PRODUCTION OF VIRAL RNAS AND HOST RESPONSES IN SINGLE CELLS, ALL WHILE MAINTAINING SAMPLE SPATIAL INTEGRITY. THIS PROJECT’S LONG-TERM OBJECTIVE IS TO APPLY THIS NOVEL APPROACH TO GAIN INSIGHTS INTO SARS-COV-2 BIOLOGY DISTINCT FROM THOSE GLEANED USING TRADITIONAL STRATEGIES. THIS KNOWLEDGE WILL PROVIDE NEW INSIGHT INTO THE SPECTRUM OF COVID-19 DISEASE OUTCOMES AND HELP GUIDE FUTURE THERAPEUTIC STRATEGIES. TO THIS END, SINGLE-MOLECULE IN SITU ANALYSES, INCLUDING SINGLE MOLECULE FLUORESCENCE IN SITU HYBRIDIZATION (SMFISH) AND MULTIPLEXED ERROR-ROBUST FISH (MERFISH) WILL BE APPLIED TO THE STUDY OF SARS-COV-2. AIM 1 WILL QUANTIFY SARS-COV-2 ENTRY, REPLICATION AND SPREAD, AND HOST TRANSCRIPTIONAL RESPONSES IN CELLS OF VARYING TISSUE ORIGIN. THESE DATA WILL BE USED TO DEVELOP A STOCHASTIC COMPUTATIONAL MODEL TO ADDRESS THE DETERMINANTS OF EARLY VIRAL REPLICATION AND THE RESULTING CELLULAR RESPONSE. AIM 2 WILL EXAMINE THE EFFECT OF HOST MUTATIONS OR PRE-EXISTING CONDITIONS THAT AFFECT THE TYPE I INTERFERON (IFN) RESPONSE AND HAVE BEEN ASSOCIATED WITH SEVERE COVID-19, AS WELL AS EMERGING VIRAL VARIANTS OF CONCERN. AIM 3 WILL MODEL PATIENT COMORBIDITIES IN VIVO USING MOUSE MODELS OF SARS-COV-2 INFECTION, IDENTIFYING THE FUNCTIONAL AND SPATIAL CONSEQUENCES OF HOST RESPONSES, INCLUDING IFN AND OTHER CYTOKINE EXPRESSION, IN THE RESPIRATORY TRACT AND LUNG. WE WILL FURTHER UTILIZE THESE IN VIVO MODELS TO UNDERSTAND WHY PATHOGENESIS AND DISEASE OUTCOME DIFFER DEPENDING ON THE INOCULUM DOSE AND THE AGE OF THE ANIMAL. TOGETHER, OUR MULTIDISCIPLINARY APPROACH UTILIZING TECHNIQUES AND INFORMATION FROM SYSTEMS-LEVEL VIROLOGY, SPATIAL TRANSCRIPTOMICS, HOST GENETICS, COMPUTATIONAL BIOLOGY, AND INNATE IMMUNITY PROVIDES A POWERFUL MEANS OF PROBING QUESTIONS CENTRAL TO UNDERSTANDING CLINICAL OUTCOME AND INFORMING LIFE-SAVING INTERVENTIONS.

CORRELATING MOLECULAR BEHAVIORAL PHENOTYPES IN A MARMOSET MODEL OF HUNTINGTONS DISEASE - ABSTRACT THE COMMON MARMOSET PROVIDES A VERY RELEVANT PRIMATE MODEL FOR UNDERSTANDING THE ORGANIZATION OF THE HUMAN NERVOUS SYSTEM AND THE DISEASES THAT AFFECT IT. LIKE HUMANS, MARMOSETS ALSO DEMONSTRATE COOPERATIVE SOCIAL BEHAVIOR AND HAVE ADVANCED COGNITIVE PROCESSES, MAKING THEM OF GREAT INTEREST IN THE FIELD FOR MODELING DEVELOPMENTAL AND PSYCHIATRIC DISEASES AND THEIR THERAPIES. THEY ARE ALSO IDEAL FOR MULTIGENERATIONAL GENETIC EXPERIMENTS AS THEY GIVE BIRTH TWICE A YEAR AND MATURE FASTER THAN MOST PRIMATES. HOWEVER, WHILE THE CRISPR/CAS9 SYSTEM HAS BEEN USED TO KNOCKOUT GENES AND CREATE KNOCK-INS OF SINGLE AMINO ACIDS IN A HERITABLE MANNER IN MARMOSETS, IT HAS BEEN A CHALLENGE IN THE FIELD TO CREATE GERMLINE TRANSMISSIBLE MODELS OF GENE REPORTERS AND TRINUCLEOTIDE REPEAT GENES ANALOGOUS TO THEIR MURINE COUNTERPARTS. THE VERY LOW EFFICIENCY OF HOMOLOGOUS RECOMBINATION (HR) IN PRIMATES HAS PRECLUDED KNOCKING-IN CODING SEQUENCES BY SIMPLY INJECTING CAS9 PROTEIN AND A GUIDE RNA INTO EMBRYOS DURING IN VITRO FERTILIZATION (IVF) AS IS DONE FOR CREATING KNOCKOUTS. THIS LIMITATION HAS PREVENTED MODELLING OF MORE GENETICALLY COMPLEX NEUROLOGICAL DISEASES SUCH AS HUNTINGTON’S DISEASE (HD) AND FOR CREATING CONDITIONAL REPORTERS IN MARMOSETS, BOTH OF WHICH ARE MAINSTAYS IN THE MOUSE NEUROGENETICS FIELD. IN ADDITION TO LOW HR FREQUENCY, OTHER BARRIERS TO CREATING GERMLINE TRANSMISSION OF KNOCK- INS INCLUDE THE ABSENCE OF A WELL ANNOTATED MARMOSET GENOME UNTIL RECENTLY, LACK OF PROTOCOLS FOR DERIVATION OF GROUND STATE MARMOSET PLURIPOTENT STEM CELLS (CJPSCS), THE LOW PERCENTAGE OF MARMOSET PREGNANCIES AFTER EMBRYO REIMPLANTATION, AND A GENERAL DEFICIENCY OF DEVELOPMENTAL BIOLOGY EXPERTISE IN THE MARMOSET FIELD. WE PROPOSE TO HARNESS OUR LABS’ EXPERTISE IN DEVELOPMENTAL BIOLOGY, IVF TECHNOLOGIES, AND TRANSGENIC STEM CELL BIOLOGY TO OVERCOME THIS BARRIER TO WIDESPREAD USE OF MARMOSETS. WE AIM TO CREATE TRANSGENIC KNOCK-IN CJPSCS, CONVERT THEM INTO GROUND-STATE PLURIPOTENT STEM CELLS AND THEN INJECT THEM INTO IVF MORULA TO CREATE A CHIMERIC FOUNDER MARMOSET THAT CARRIES THE MODIFIED GENOME. WE THEN AIM TO SCREEN THE TRANSGENIC GAMETES FROM THE FOUNDER MARMOSETS TO CREATE THE F1 PROGENY AND USE THEM TO CORRELATE THE MOLECULAR-BEHAVIORAL PHENOTYPE OF HD. AS PROOF-OF-PRINCIPLE, WE WILL FOCUS ON THREE KNOCK-IN REPORTER LINES TO BROADLY TARGET EXCITATORY, INHIBITORY, AND PERIPHERAL NEURONAL POPULATIONS. TOGETHER, IF SUCCESSFUL, OUR AIMS WILL RESULT IN CREATION OF THE FIRST PRIMATE MODEL WITH NEURON-SPECIFIC REPORTERS, ESTABLISH THE MARMOSET AS A VALID MODEL OF HD, ENABLE ACCESS TO SINGLE- CELL TRANSCRIPTOMIC CHANGES AT THE EARLY STAGES OF HD IN A PRIMATE DISEASE MODEL, AND FINALLY CORRELATE THESE MOLECULAR CHANGES WITH THE BEHAVIORAL PHENOTYPE. THESE AIMS WILL PROVIDE FUNDAMENTAL INSIGHTS INTO THE BIOLOGY OF HD AND THE ROLE OF HUNTINGTIN PROTEIN IN DIFFERENT CLASSES OF NEURONS. THE OUTCOME OF THIS PROJECT WILL ALSO INFLUENCE A BETTER UNDERSTANDING OF POLY-GLUTAMINE NEURODEGENERATIVE DISEASES THAT AFFECT HUMANS. IN ADDITION, THE TRANSGENIC MARMOSETS THAT WE GENERATE WILL BE BROADLY AVAILABLE TO THE RESEARCH COMMUNITY AND ENABLE NEW STUDIES INTO NEURAL CIRCUITS, DEVELOPMENT, BEHAVIOR, AND A WIDE RANGE OF OPTOGENETIC APPLICATIONS.

STRUCTURE, FUNCTION, AND INHIBITION OF THE SARS-COV-2 REPLICATION-TRANSCRIPTION COMPLEX - PROJECT SUMMARY COVID-19, CAUSED BY THE CORONAVIRUS SARS-COV-2, CONTINUES TO DEVASTATE THE WORLD. IN LESS THAN A YEAR, THERE HAVE BEEN MORE THAN 20 MILLION CASES WITH OVER 700,000 DEATHS. THE VIRAL RNA-DEPENDENT RNA POLYMERASE (RDRP) IS THE CENTRAL ENZYME RESPONSIBLE FOR TRANSCRIPTION AND REPLICATION OF THE VIRAL RNA GENOME. THIS ENZYME IS ALSO A TARGET FOR THE CURRENT ANTIVIRAL, REMDESIVIR, USED TO AMELIORATE THE SEVERITY AND DURATION OF THIS DISEASE. THE VIRUS ALSO ENCODES SEVERAL NUCLEIC ACID PROCESSING ENZYMES, IN ADDITION TO THE RDRP, INCLUDING A HELICASE, AN ENDONUCLEASE, AN EXONUCLEASE, AND METHYLTRANSFERASES. HOWEVER, IT IS UNKNOWN HOW THESE ENZYMES COORDINATE TO TRANSCRIBE AND REPLICATE THE VIRAL GENOME. THIS PROPOSAL BUILDS UPON PRELIMINARY DATA OF THE STRUCTURE OF THE HELICASE, NSP13, IN COMPLEX WITH THE RDRP AND A PRIMED SUBSTRATE RNA (NSP13-REPLICATION/TRANSCRIPTION COMPLEX OR NSP13-RTC). THE AIMS HERE INCLUDE COMPLETING THE STRUCTURAL ANALYSIS OF THIS COMPLEX BY UTILIZING ADDITIONAL DATA COLLECTED. THE RESULT OF THIS AIM WILL PROVIDE HIGHER RESOLUTION (BETTER THAN 2.7 Å IN SOME PARTS THE RDRP), PROVIDING A RICH BASIS FOR THE DEVELOPMENT OF ANTIVIRAL INHIBITORS. ALSO, HAVING THIS STRUCTURE IN HAND ALLOWS FOR THE COLLABORATION WITH EXPERT DEVELOPERS OF ANTIMICROBIALS, ALSO PART OF THE AIMS, INCLUDING THE INVESTIGATION OF THE STRUCTURAL DETAILS OF THE PRE-INCORPORATION STATE OF REMDESIVIR AND ANTIVIRALS PRODUCED BY HUMAN MICROBIOME. THE MODELS RESULTING FROM THE STRUCTURE OF NSP13-RTC SERVE AS FOUNDATIONS TO TEST HOW THE HELICASE AND EXONUCLEASE FUNCTION TOGETHER WITH THE RDRP. SPECIFICALLY, REAL-TIME FLUORESCENCE ASSAYS, SINGLE-MOLECULE FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET), AND MULTI-COLOR FLUORESCENCE MICROSCOPY WILL BE USED TO PROBE THE ROLE OF THE HELICASE AND THE EXONUCLEASE IN UNWINDING SUBSTRATE RNA, BACKTRACKING, AND PROOFREADING. ANOTHER AIM APPLIES THE PIPELINE USED TO CHARACTERIZE THE NSP13-RTC ASSEMBLY, WHICH YIELDED A HIGH- RESOLUTION STRUCTURE OF THE COMPLEX, TO OTHER RTC ASSEMBLIES. SPECIFICALLY, NATIVE ELECTROPHORETIC MOBILITY ASSAYS WILL BE USED AS A STARTING POINT TO PROBE LARGER ASSEMBLIES OF THE RTC. NATIVE MASS-SPECTROMETRY WILL THEN BE USED TO DETERMINE THE COMPOSITION AND STOICHIOMETRY OF THE COMPLEXES. FINALLY, CRYO-EM WILL BE APPLIED TO SOLVE THE STRUCTURES OF THESE MACROMOLECULAR MACHINES. THE RESULTING STRUCTURES WILL PROVIDE A STARTING POINT TO ELUCIDATE THE COORDINATED FUNCTIONS OF THESE ENZYMES, PROVIDE INSIGHT INTO THEIR MECHANISMS, AND ESTABLISH NOVEL TARGETS FOR THERAPEUTICS. IN SUMMARY, THIS PROPOSAL AIMS TO UNDERSTAND AT THE MOLECULAR AND STRUCTURAL LEVEL HOW THE SARS-COV-2 NUCLEIC ACID PROCESSING ENZYMES COORDINATE TO REPLICATE AND TRANSCRIBE THE VIRAL GENOME, AND TO PROVIDE STRUCTURE-GUIDED TARGETS FOR DRUG DISCOVERY, WITH THE ULTIMATE GOAL OF PROVIDING RELIEF FOR THE COVID-19 PANDEMIC.

ROLE OF ASTN2 IN CEREBELLAR CIRCUIT FUNCTION AND ASD-RELATED BEHAVIORS - PROJECT SUMMARY THE PROPOSED RESEARCH ADDRESSES A CRITICALLY IMPORTANT QUESTION IN AUTISM SPECTRUM DISORDER (ASD) RESEARCH: HOW DEFECTS IN CEREBELLAR CIRCUITS CONTRIBUTE TO ASD. IN PARTICULAR, IT EXAMINES THE ROLE OF THE PREDOMINANTLY CEREBELLAR GENE ASTN2 IN CEREBELLAR CIRCUIT FUNCTION AND ASD-RELATED BEHAVIORS. COPY NUMBER VARIATIONS (CNVS) IN ASTN2 HAVE BEEN IDENTIFIED AS A SIGNIFICANT RISK FACTOR FOR ASD (LIONEL ET AL, 2014), SUGGESTING THAT ASTN2 MUTATIONS SUCH AS THOSE FOUND IN PATIENTS WITH ASTN2 CNVS, LEAD TO ALTERED CEREBELLAR SYNAPTIC FUNCTION. IN ADDITION, WE RECENTLY REPORTED A FAMILY WITH A PATERNALLY INHERITED INTRAGENIC ASTN2 DUPLICATION, WHICH CAUSED A HETEROZYGOUS LOSS OF FUNCTION OF ASTN2. THE FAMILY MANIFESTED A RANGE OF NEURODEVELOPMENTAL DISORDERS, INCLUDING ASD, LEARNING DIFFICULTIES AND SPEECH AND LANGUAGE DELAY (BEHESTI ET AL, 2018). OUR CELLULAR AND MOLECULAR STUDIES ON MOUSE CEREBELLUM SHOW THAT ASTN2 BINDS TO AND REGULATES THE TRAFFICKING OF MULTIPLE SYNAPTIC PROTEINS, INCLUDING NEUROLIGINS, WHICH HAVE BEEN GENETICALLY LINKED TO ASDS, AND MODULATES CEREBELLAR PURKINJE CELL (PC) SYNAPTIC ACTIVITY (BEHESTI ET AL, 2018). TO PROVIDE A GENETIC MODEL TO STUDY CEREBELLAR CIRCUIT FUNCTION, WE GENERATED BOTH A GLOBAL LOSS OF FUNCTION ASTN2 LINE AND A FLOXED ASTN2 LINE FOR CONDITIONAL KNOCKOUT EXPERIMENTS. NEW, PRELIMINARY EVIDENCE INDICATES THAT PCS IN MICE LACKING ASTN2 HAVE A DECREASE IN EVOKED EXCITATION RELATIVE TO INHIBITION IN PCS AND REDUCED PC DENDRITIC SPINE DENSITY, SUGGESTING SPECIFIC CEREBELLAR CIRCUIT DEFECTS. IN ADDITION, PRELIMINARY EVIDENCE SHOWS MILD MOTOR DEFICITS AND DEFECTS IN USVS AND AN OPEN FIELD ASSAY, ASD-RELATED BEHAVIORS. AS OTHER PRELIMINARY FINDINGS DO NOT INDICATE MAJOR DEFECTS IN CEREBELLAR DEVELOPMENT, WE HYPOTHESIZE THAT THE BEHAVIORAL DEFECTS WE OBSERVED RELATE TO DEFECTS IN THE CEREBELLAR CIRCUITRY WITH UNDERLYING CHANGES IN RECEPTOR TRAFFICKING. IN THE PROPOSED RESEARCH, WE WILL 1) TEST HOW LOSS OF ASTN2 ALTERS INTRINSIC EXCITABILITY IN PCS AND THE SYNAPTIC EFFICACY OF THEIR PRESYNAPTIC INPUTS FROM GCS AND MOLECULAR LAYER INTERNEURONS, 2) USE PROTEOMICS TO IDENTIFY CHANGES IN THE LEVELS OF SYNAPTIC PROTEINS AND LIVE IMAGING TO ASSESS WHETHER SUCH CHANGES RELATE TO CHANGES IN THE RATE OF ENDOCYTOSIS, 3) COMPARE CHANGES IN PC DENDRITIC BRANCHING AS WELL AS THE REGIONAL DISTRIBUTION OF PC SPINES IN WILD TYPE AND MUTANT ANIMALS TO PROVIDE INSIGHT ON WHETHER THERE ARE CHANGES IN THE ORGANIZATION OF PC INPUTS DURING THE ESTABLISHMENT OF THE CEREBELLAR CIRCUITRY, AND 4) ANALYZE CHANGES IN SOCIAL BEHAVIOR AND ULTRASONIC VOCALIZATION IN ASTN2 WILD TYPE, HETEROZYGOUS AND MUTANT ANIMALS. TAKEN TOGETHER, THE PROPOSED RESEARCH WILL PROVIDE A NEW MOUSE MODEL THAT ALLOWS US TO LINK AN ASD-RELATED GENE THAT IS PREDOMINANTLY EXPRESSED IN THE CEREBELLUM WITH SPECIFIC CEREBELLAR CIRCUIT FUNCTION AND MOLECULAR PATHWAYS.

A CLEAR VIEW OF ENCEPHALITIS: A SINGLE CELL APPROACH TO DETERMINE THE BASIS OF FLAVIVIRAL PATHOGENESIS IN THE CENTRAL NERVOUS SYSTEM - ENCEPHALITIC FLAVIVIRUS INFECTIONS AFFECT THOUSANDS OF PEOPLE GLOBALLY EVERY YEAR CAUSING ACUTE ENCEPHALOMYELITIS AND PLACING SIGNIFICANT BURDEN ON HEALTHCARE SYSTEMS. CURRENTLY, NO VIRUS-SPECIFIC TREATMENTS ARE AVAILABLE FOR THESE LIFE-THREATENING CONDITIONS. THE CENTRAL NERVOUS SYSTEM (CNS) ENCOMPASSES DOZENS OF CELL TYPES WITH DIVERSE PROPERTIES AND FUNCTIONS. LIMITED BY A LACK OF ADEQUATE TOOLS THAT COMBINE THROUGHPUT, DEPTH AND RESOLUTION, THE INTERACTIONS BETWEEN VIRUSES AND THIS COMPLEX ENVIRONMENT REMAIN LARGELY A MYSTERY. TO COMPLICATE MATTERS, THE CNS IS ALSO EXTENSIVELY CONNECTED TO THE PERIPHERY BY BOTH PHYSICAL NEURAL PROJECTIONS AND PERIPHERAL IMMUNE SIGNALING. OUR PRELIMINARY STUDIES DEMONSTRATE THAT TROPISM OF THE IMPORTANT ENCEPHALITIC VIRUS WEST NILE (WNV) WITHIN THE CNS AFTER DIRECT INTRACRANIAL INOCULATION OF MICE DIFFERS FROM THAT SEEN AFTER SPREAD TO THE CNS FOLLOWING PERIPHERAL INFECTION. WE HYPOTHESIZE THAT CNS TROPISM IS LARGELY DETERMINED BY RESIDENT NEURAL AND GLIAL INNATE IMMUNE PROFILES, WHICH CAN BE READILY MODIFIED BY IMMUNE SIGNALS GENERATED DURING PERIPHERAL INFECTION. TO ADDRESS THIS HYPOTHESIS, WE WILL UTILIZE WNV TO STUDY IMMUNE INTERACTIONS BETWEEN CELL TYPES IN AN IN VIVO MOUSE MODEL AND IN VITRO USING HUMAN INDUCED PLURIPOTENT STEM CELL (HPSC) MODELS. TISSUE CLEARING TECHNIQUES AND WHOLE MOUNT IMAGING WILL BE USED TO VISUALIZE VIRAL ANTIGENS ACROSS THE ENTIRE BRAIN AND SPINAL CORD USING LIGHT SHEET MICROSCOPY, CREATING A COMPLETE TIME-RESOLVED 3-DIMENSIONAL MAP OF INFECTION. RESPONSES OF SINGLE CELLS WILL BE EXAMINED USING A COMBINATION OF CUTTING-EDGE NUCLEAR RNA SEQUENCING AND MICROSCOPY-BASED SPATIAL TRANSCRIPTOMICS. CO-CULTURES OF HPSC-DERIVED NEURONS AND GLIA WILL BE INTERROGATED USING HIGH-THROUGHPUT MICROSCOPY AND SEQUENCING TO IDENTIFY RESISTANCE FACTORS AND RESPONSES IN HUMAN CELL TYPES. HITS WILL BE MECHANISTICALLY STUDIED USING BLOCKING ANTIBODIES AND CRISPR-MEDIATED KNOCKOUTS. LASTLY, BY USING SYSTEMIC AND CELL-TYPE SPECIFIC KNOCK OUT ANIMALS OR CYTOKINE NEUTRALIZING ANTIBODIES, WE WILL INVESTIGATE THE ROLE OF TYPE I INTERFERON IN MODULATING CNS TROPISM AND DISEASE. THIS PROJECT WILL PROVIDE NEW DATA ON FLAVIVIRAL ENCEPHALITIS AT UNPARALLELED RESOLUTION TO HELP BRIDGE CURRENT INFORMATION GAPS AND IMPROVE FUNDAMENTAL KNOWLEDGE BY DEFINING CELLULAR TROPISM AND CNS INFLAMMATORY RESPONSES AT THE SINGLE CELL LEVEL AND EVALUATING HOW CHANGES IN PERIPHERAL SIGNALING INFLUENCE INFECTION OF THE BRAIN. IDENTIFIED PERIPHERAL FACTORS RESTRICTING CNS INFECTION ARE POSSIBLE TARGETS FOR IMMUNOMODULATORY THERAPY, THUS PROMOTING RESEARCH THAT MAY IMPROVE TREATMENT FOR OTHER FORMS OF VIRAL ENCEPHALITIS. FINALLY, THE RESULTING EXPERIMENTAL PIPELINE WILL BE BROADLY APPLICABLE TO THE STUDY OF CNS STRESS AND INFLAMMATION, WITH RELEVANCE TO OTHER DISEASES LIKE LYME NEUROBORRELIOSIS OR CHRONIC DEBILITATING CONDITIONS LIKE TRAUMATIC BRAIN INJURY.

MOLECULAR MECHANISMS OF ESTROGEN RECEPTOR-DEPENDENT TRANSCRIPTION REGULATION

BRIDGING THE GAP FROM GENES TO CIRCUITS TO BEHAVIOR IN UNDERSTANDING COGNITIVE DYSFUNCTION

NOVEL TRANSGENIC MOUSE MODELS ADDRESSING OUTSTANDING TRANSLATIONAL BARRIERS IN ANTIBODY-BASED THERAPEUTICS

THE GENETIC AND EPIGENETIC MECHANISMS OF PHENOTYPIC INNOVATIONHTTPS://APPS.ERA.NIH.GOV/GM/REPORTCHECKLIST.DO?APPLICATIONID=9798249

A SYSTEMATIC ANALYSIS OF SMALL RNA INTERACTIONS DURING RNA VIRUS INFECTIONS

DEFINING MOLECULAR CONTRIBUTIONS OF LINE-1 RETROTRANSPOSONS TO AD / ADRD - PROJECT SUMMARY: ALZHEIMER'S DISEASE (AD) AND AD-RELATED DEMENTIAS (ADRDS; E.G. FRONTOTEMPORAL DEMENTIA, LEWY BODY DEMENTIA, ETC.) ARE CRIPPLING NEURODEGENERATIVE DISORDERS. THE ONSET OF THESE DISEASES IS STRONGLY CORRELATED WITH AGING. CURES FOR AD AND ADRDS REMAIN ELUSIVE; ALZHEIMER'S HAS BECOME THE 6TH MOST FREQUENT CAUSE OF DEATH IN THE USA. AN EMERGING CANDIDATE CONTRIBUTOR TO ALZHEIMER'S AND ADRD IS THE L1 RETROTRANSPOSON, WHICH BECOMES DYSREGULATED DURING AGING AND CORRELATES WITH ALZHEIMER'S / ADRD ONSET. ONE HYPOTHETICAL MECHANISM BY WHICH L1 MAY CONTRIBUTE TO ALZHEIMER'S / ADRD IS THROUGH ITS EXACERBATION OF CELLULAR SENESCENCE. SENESCENCE IS A PHENOMENON BY WHICH NORMAL CELLS STOP DIVIDING; THESE CELLS ACCUMULATE WITH ADVANCING AGE AND ARE FOUND AT THE LOCATIONS OF DYSFUNCTION IN AGE-RELATED DISEASES. IN MICE, SENESCENT CELLS HAVE BEEN SHOWN TO SHORTEN LIFE AND ACTIVELY DRIVE AGE-RELATED NEURODEGENERATION; PREVENTING SENESCENT CELL ACCUMULATION DECREASES TAU-DEPENDENT DEGENERATION AND COGNITIVE DECLINE. AD PATIENTS EXHIBIT INCREASED INDICATORS OF CELLULAR SENESCENCE. IT IS INCREASINGLY CLEAR THAT SENESCENT CELLS ARE NOT INERT, BUT INSTEAD DRIVE TISSUE DETERIORATION VIA THE SENESCENCE-ASSOCIATED SECRETORY PHENOTYPE - SECRETING A VARIETY OF GROWTH FACTORS AND PRO- INFLAMMATORY CYTOKINES. L1 RETROTRANSPOSONS HAVE RECENTLY BEEN SHOWN TO DRIVE PROGRESSION OF THE SENESCENCE- ASSOCIATED SECRETORY PHENOTYPE, AND THUS, L1 IS AN IMPORTANT AGENT OF CELLULAR SENESCENCE. L1 ACTIVATION IS ALSO ASSOCIATED WITH AD-RELATED TAU PATHOLOGIES. THE L1 ENCODED ORF2P ENZYME (ENDONUCLEASE AND REVERSE TRANSCRIPTASE) IS OFTEN FLAGGED AS A SOURCE OF PATHOLOGICAL CELLULAR INSULTS, E.G. VIA NEW, MUTAGENIC L1 INSERTIONS AND CONTRIBUTIONS TO CHROMOSOMAL INSTABILITY. HOWEVER, THE EFFECTS OF L1 EXPRESSION EXTEND BEYOND DNA DAMAGE. NUMEROUS MECHANISMS MAY BE AT PLAY, INCLUDING THE TITRATION OF NORMALLY HOMEOSTATIC HOST FACTORS AWAY FROM THEIR PHYSIOLOGIC FUNCTIONS AND INTO L1 RIBONUCLEOPROTEIN GRANULES, AS WELL AS THE PRODUCTION OF IMMUNITY-AND-INFLAMMATION-TRIGGERING CYTOPLASMIC L1 DNA:RNA HYBRIDS; INDEED, THE LATTER IS NOW UNDERSTOOD TO BE A KEY COMPONENT OF L1’S ROLE IN CELLULAR SENESCENCE. MOREOVER, L1 ALSO MOBILIZES ALU AND OTHER NON- AUTONOMOUS RETROTRANSPOSONS. OBJECTIVES: IN AIM 1, WE WILL USE TARGETED MASS SPECTROMETRY TO PROFILE L1 ORF1 PROTEIN EXPRESSION IN THE CEREBROSPINAL FLUID (CSF) AND POST-MORTEM BRAIN TISSUES OF PATIENTS EXHIBITING AD/ADRD AND COGNITIVE DECLINE, AS WELL AS IN SENESCENT CELLS AND NEURONAL IPSCS; IN AIM 2 (IN VITRO CELL CULTURE) AND AIM 3 (CLINICAL SAMPLES) WE WILL PROFILE L1-ASSOCIATED PROTEIN-PROTEIN AND PROTEIN-RNA INTERACTIONS IN THE SAME BIOLOGICAL SAMPLES AS AIM 1; AND IN AIM 4 WE WILL TAKE A CANDIDATE-BASED APPROACH USING MOLECULAR GENETICS TO DISSECT THE MECHANISMS OF ACTION OF L1 IN SENESCENT CELLS.

EMOTIONS AS COMPUTATIONS

IN SEARCH OF AN HBV CURE: NOVEL MODEL SYSTEMS AND TARGETS

HETEROGENEITY OF PARKINSON'S DISEASE PATIENTS IDENTIFICATION AND CHARACTERIZATION...

DEFINING THE NEURAL CIRCUITS OF ATTENTION CONTROL: A NEW HYPOTHESIS

LENSLESS, HIGH-SPEED AND MULTI-REGION VOLUMETRIC CA2+ IMAGING OF UP TO 1CM2 BRAIN SURFACE ACROSS MODEL ANIMALS

TRANSCRIPTIONAL REGULATORY MECHANISMS IN B CELL DEVELOPMENT AND LEUKEMOGENESIS

ELUCIDATION OF COMMENSAL BACTERIA MECHANISMS REQUIRED FOR HOST PROTECTION

TRIBAL CYBERSECURITY GRANT PROGRAM

CORTEX-WIDE VOLUMETRIC IMAGING OF NEURONAL ACTIVITY.

ENGAGING IMMUNOSUPPRESSIVE MYELOID CELLS IN THE TME FOR THE TREATMENT OF PANCREATIC CANCER - ABSTRACT ALTHOUGH THE PAST DECADE WAS MARKED BY THE TREMENDOUS SUCCESS OF IMMUNOTHERAPEUTICS IN ONCOLOGY, FEW HAVE PROVIDED SIGNIFICANT BENEFIT TO PATIENTS WITH PANCREATIC CANCER. THE LONG-TERM GOAL OF THIS PROJECT IS TO DEVELOP THERAPEUTICALLY USEFUL IMMUNOTHERAPIES FOR THE CLINICAL TREATMENT OF PANCREATIC CANCER. THE OVERALL OBJECTIVES IN THIS APPLICATION ARE TO (I) CHARACTERIZE THE ROLE FOR MARCO IN THE TME, (II) TO EVALUATE THE THERAPEUTIC EFFICACY OF ANTIBODIES BLOCKING THIS RECEPTOR, AND (III) IMPROVE ANTIGEN PRESENTATION WITHIN THE PANCREATIC TME WITH A COMBINATION OF FC-ENHANCED ANTIBODIES TARGETING MARCO AND CD40. OUR CENTRAL HYPOTHESIS IS THAT PANCREATIC CANCER REPOLARIZES TUMOR ASSOCIATED MACROPHAGES TO AN M2 PHENOTYPE AN THIS CAN BE REVERSED THROUGH ENGAGEMENT OF MARCO WITH ANTIBODIES, THEREFORE PROMOTING ANTI-TUMOR RESPONSES. THE RATIONALE FOR THIS PROJECT IS THAT A DETERMINATION OF THE IMMUNOSUPPRESSIVE ROLE OF TAMS EXPRESSING MARCO IN PANCREATIC CANCER WILL LIKELY OFFER A STRONG SCIENTIFIC FRAMEWORK WHEREBY NEW IMMUNOTHERAPIES CAN BE DEVELOPED. THE CENTRAL HYPOTHESIS WILL BE TESTED BY PURSUING THREE SPECIFIC AIMS: 1) DEVELOP ANTI-HUMAN TAM ANTIBODIES FOR CANCER IMMUNOTHERAPY AND SET UP NOVEL ASSAYS FOR LARGE-SCALE EVALUATION OF ANTI-TAM ANTIBODIES, 2) DEFINE THE ROLE FOR MARCO IN THE POLARIZATION OF TAMS IN PANCREATIC CANCER, AND 3) AUGMENT TAM REPOLARIZATION IN THE PANCREATIC CANCER TME USING THE FC-ENHANCED CD40 AGONIST ANTIBODY 2141-V11, ALONE OR IN COMBINATION WITH ANTI- HMARCO ANTIBODIES.. UNDER THE FIRST AIM, NOVEL ANTIBODIES WILL BE GENERATED AGAINST HUMAN MARCO AND OPTIMIZED FOR FC-RECEPTOR BINDING. IN THE SECOND AIM, MICE GENETICALLY ENGINEERED TO EXPRESS HUMAN MARCO WILL BE STUDIED FOR THEIR ROLE IN THE TME OF PANCREATIC CANCER. FOLLOWING CHARACTERIZATION IMMUNE CELLS EXPRESSING MARCO THAT CAN BE TARGETED BY ANTIBODY THERAPY, A PANEL OF ANTIBODY VARIANTS WILL BE TESTED TO DETERMINE THE FC-REQUIREMENTS FOR OPTIMAL IN VIVO ACTIVITY. MARCO EXPRESSION AND CHARACTERIZATION OF THE IMMUNE MICROENVIRONMENT WILL ALSO BE EVALUATED IN HUMAN PANCREATIC CANCER SPECIMENS. FINALLY, IN THE THIRD AIM, WE WILL USE KNOWLEDGE GAINED IN AIMS 1 AND 2 TO TEST NOVEL COMBINATIONS TARGETING THE MYELOID AXIS IN PANCREATIC TUMORS. HERE, THE ANTI-TUMOR ACTIVITY OF THE FC-ENHANCED ANTI-CD40 ANTIBODY WILL BE TESTED ALONE OR IN COMBINATION WITH ANTI-MARCO ANTIBODIES IN PANCREATIC CANCER MODELS. THE RESEARCH PROPOSED IN THIS APPLICATION IS INNOVATIVE, IN THE INVESTIGATOR’S OPINION, BECAUSE IT FOCUSES ON DEFINING NOVEL PATHWAYS ON MYELOID CELLS CONTRIBUTING TO THE IMMUNE SUPPRESSIVE TME OF PANCREATIC CANCER, WHILE IDENTIFYING LEAD THERAPEUTIC CANDIDATES THROUGH FC- ENGINEERING. THE PROPOSED RESEARCH IS SIGNIFICANT BECAUSE IT IS EXPECTED TO PROVIDE STRONG SCIENTIFIC JUSTIFICATION FOR THE DEVELOPMENT OF MACROPHAGE TARGETING IMMUNOTHERAPIES. ULTIMATELY, SUCH KNOWLEDGE HAS THE POTENTIAL OF OFFERING NEW OPPORTUNITIES FOR THE TRANSLATION OF INNOVATIVE THERAPIES TO THE TREATMENT OF PANCREATIC CANCER.

EFFECTS OF INTERFERON ON PRIMATE LENTIVIRUSES - ABSTRACT THIS PROPOSAL IS A MULTI PI PROPOSAL THAT AIMS TO UNDERSTAND HOW ANTIVIRAL PROTEINS LIMIT HOST RANGE. PRIOR WORK HAS DEMONSTRATED THAT TYPE I INTERFERON (IFN) INDUCED PROTEINS LIMIT PRIMATE LENTIVIRUS REPLICATION IN NATURAL AND NON-NATURAL HOSTS. INDEED, OUR PREVIOUS STUDIES HAVE LED TO THE DISCOVERY OF ANTIVIRAL INTERFERON STIMULATED GENES (ISGS), ELUCIDATED THEIR MECHANISM OF ACTION AND UNCOVERED WAYS IN WHICH PRIMATE LENTIVIRUSES EVOLVE EVADE OR COUNTERACT ANTIVIRAL PROTEINS. ADDITIONALLY, WE HAVE EXPLOITED THIS KNOWLEDGE TO GENERATE NOVEL CHIMERIC VIRUSES THAT BETTER REPRESENT THE HIV-1 STRAINS CIRCULATING IN HUMANS FOR USE IN NON-HUMAN PRIMATE MODELS, INCLUDING A MINIMALLY MODIFIED HIV-1 STRAIN (STHIV) THAT CAN CAUSE AIDS IN PIGTAIL MACAQUES WHEN CD8+ CELLS ARE TRANSIENTLY DEPLETED DURING THE ACUTE INFECTION. IN THIS NEW PROPOSAL, WE WILL EXPLORE THE ROLE OF TYPE I IFN AND NOVEL ANTIVIRAL ISGS IN LIMITING PRIMATE LENTIVIRUS REPLICATION IN VITRO AND IN VIVO. SPECIFIC AIM 1 WILL EXPLORE THE MECHANISM OF ACTION OF ANTIVIRAL ISGS AFFECTING VIRAL ENTRY. USING A CRISPR SCREEN, WE HAVE FOUND NOVEL ISGS WHOSE EXPRESSION APPEARS TO INHIBIT HIV-1 INFECTION, SPECIFICALLY AT THE VIRUS ENTRY STEP. KNOCKOUT OF ONE OF THESE GENES ENHANCES HIV-1 INFECTION IN A MANNER THAT VARIES DRAMATICALLY ACCORDING TO THE PARTICULAR STRAIN FROM WHICH THE ENV PROTEIN IS DERIVED. IMPORTANTLY, THE MAGNITUDE OF EFFECT OF THE ISG ON HIV-1 INFECTION MEDIATED BY A PARTICULAR ENV VARIANT CORRELATES WITH THE SENSITIVITY OF A VIRUS CARRYING THAT ENV VARIANT TO INHIBITION BY TYPE I IFN. WE WILL DETERMINE THE MOLECULAR DETAILS OF THE HOW IFN/ISGS INHIBITS HIV-1 ENTRY BY ELUCIDATING VIRAL AND HOST DETERMINANTS OF INHIBITION, ACROSS CELL TYPES AND SPECIES, AND USE IMAGING AND OTHER APPROACHES TO DETERMINE HOW INHIBITION AFFECTS INCOMING VIRON FATE. WE WILL ALSO DETERMINE HOW NOVEL ISGS CONTRIBUTE TO THE DIFFERENTIAL IFN SENSITIVITY OF PRIMARY TRANSMITTED FOUNDER AND CHRONIC HIV-1 STRAINS AND ADAPTED/UNADAPTED SHIVS. IN SPECIFIC AIM 2, WE WILL USE NEWLY DEVELOPED, MORE EFFECTIVE METHODS TO PERTURB TYPE I IFN IN MACAQUES TO DETERMINE THE EFFECT OF IFN ON PRIMATE LENTIVIRUS REPLICATION THEREIN. IN PARTICULAR, WE WILL DETERMINE THE EFFECT OF TYPE I IFN BLOCKADE ON STHIV REPLICATION AND CLINICAL COURSE IN PIGTAIL MACAQUES. WE WILL ALSO USE SHIV TO DETERMINE WHETHER HIV-1 ENV VARIANTS THAT EXHIBIT DIFFERENTIAL TYPE I IFN SENSITIVITY IN VITRO, ALSO DO SO IN VIVO. FINALLY, WE WILL DETERMINE HOW TYPE I IFN AFFECTS SHIV DISSEMINATION AND COMPETITIVENESS USE BARCODED SHIVS, BEARING IFN/ISG-SENSITIVE AND RESISTANT HIV-1 ENV PROTEINS, DEFINED IN AIM 1, BY DETERMINING HOW EFFICIENTLY EACH SHIV DISSEMINATES IN MACAQUES IN THE PRESENCE AND ABSENCE OF TYPE I IFN BLOCKADE.

SOURCES AND CONSEQUENCES OF NOISE IN CELL CYCLE REGULATION

EMPOWERING THE PARTICIPANT VOICE: COLLABORATIVE INFRASTRUCTURE AND VALIDATED TOOLS FOR COLLECTING PARTICIPANT FEEDBACK TO IMPROVE THE CLINICAL RESEARCH ENTERPRISE

BIOCHEMICAL MECHANISM AND STRUCTURE OF THE EUKARYOTIC REPLICATION FORK

DYNAMIC READOUT OF TGFB SIGNALING AND MODELING OF CELL FATE SPECIFICATION IN HUMA

THE MOLECULAR UNDERPINNINGS OF COMPLEX SOCIAL BEHAVIOR

NEURAL MECHANISMS OF FACE RECOGNITION

EMPLOYING VIRUSES TO UNRAVEL THE FUNCTIONAL SIGNIFICANCE OF THE M5C EPITRANSCRIPTOME - 5-METHYLCYTOSINE (M5C) IS AN IMPORTANT RNA MODIFICATION STUDIED MOSTLY FOR ITS ROLE IN TRNA BIOLOGY. HOWEVER, ITS ROLES IN OTHER ASPECTS OF RNA BIOLOGY REMAIN UNDERSTUDIED. OUR PRELIMINARY RESULTS, USING BISULFITE TREATMENT OF RNA FOLLOWED BY HIGH-THROUGHPUT SEQUENCING, SHOW THAT THE GENOMES OF MANY RNA VIRUSES ARE M5C METHYLATED IN A SITE-SPECIFIC MANNER, INCLUDING SINDBIS VIRUS (SINV), CHIKUNGUNYA VIRUS (CHIKV) AND COXSACKIEVIRUS B3 (CVB3). THE PRESENCE OF M5C IN DIVERSE VIRUSES, WHOSE RNAS UNDERGO MANY PROCESSES INCLUDING TRANSLATION, REPLICATION, TRANSCRIPTION, AND VIRION PACKAGING, PROVIDES AN ATTRACTIVE STARTING POINT FOR UNDERSTANDING THE BROADER SIGNIFICANCE OF THIS MODIFICATION IN REGULATING RNA FUNCTION. A SINGLE DOMINANT M5C SITE IN SINV ALLOWED US TO GENERATE AN M5C-NULL MUTANT THAT EXHIBITED CELL-TYPE DEPENDENT EFFECTS ON VIRUS REPLICATION. THE HOST TRNA METHYLTRANSFERASE (MTASE), NSUN2, WHICH IS IMPORTANT FOR HOST NEURONAL DEVELOPMENT AND STEM CELL DIFFERENTIATION, APPEARS TO BE THE “WRITER” REQUIRED FOR M5C MODIFICATION OF SINV. NSUN5, AN MTASE OF RIBOSOMAL RNA IS REQUIRED FOR CVB3 METHYLATION. WE HYPOTHESIZE THAT M5C PLAYS A ROLE IN REGULATING VIRAL RNA FUNCTIONS IMPACTING VIRUS-HOST INTERACTIONS AND VIRAL LIFE CYCLES. IN THREE AIMS, USING VIROLOGIC, MOLECULAR, BIOCHEMICAL, HIGH-THROUGHPUT SEQUENCING, AND SMALL ANIMAL MODEL APPROACHES: I) SINV WILL BE EXPLOITED TO LEARN HOW M5C IS DEPOSITED AND HOW IT REGULATES RNA FUNCTIONS AND VIRAL INFECTION AND PATHOGENESIS; STUDIES OF THE RELATED ALPHAVIRUS CHIKV WILL ALLOW CONSERVED AND VIRUS-SPECIFIC FEATURES TO BE UNCOVERED, II) HOW M5C REGULATES CVB3 RNA AND WHAT EFFECTS THE MODIFICATION HAS ON VIRUS REPLICATION AND CVB3-ASSOCIATED MYOCARDITIS WILL BE DETERMINED, AND III) SINV AND CVB3 WILL BE LEVERAGED TO CHARACTERIZE UNKNOWN FUNCTIONS OF THE NSUN2 AND NSUN5 MTASES, RESPECTIVELY, AND AS PROBES TO DISCOVER NOVEL M5C BINDING PROTEINS THAT CAN EXERT THEIR EFFECT BY DIRECT BINDING (“READERS”) OR BY REMOVAL OF THE M5C MARK (“ERASERS”). THIS WORK WILL CONTRIBUTE TO OUR UNDERSTANDING OF HUMAN BIOLOGY BY REVEALING FUNDAMENTAL PRINCIPLES AND FUNCTIONS OF THIS WIDESPREAD MARK IN THE EPITRANSCRIPTOME WITH IMPLICATIONS FOR ITS ROLES IN MAINTAINING CELLULAR HOMEOSTASIS. SINCE M5C METHYLATION IS A CELLULAR PROCESS EXPLOITED BY NUMEROUS VIRUSES, THIS STUDY COULD YIELD NEW TARGETS FOR ANTIVIRAL INTERVENTION.

GENOME-WIDE DISSECTION OF MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE - PROJECT SUMMARY MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE (MSMD) IS A GENETIC AND SELECTIVE PREDISPOSITION TO CLINICAL DISEASE CAUSED BY WEAKLY VIRULENT MYCOBACTERIA, SUCH AS BACILLUS CALMETTE-GUERIN (BCG) VACCINES AND ENVIRONMENTAL MYCOBACTERIA (EM). PATIENTS WITH MSMD ARE OCCASIONALLY VULNERABLE TO OTHER INTRA-MACROPHAGIC PATHOGENS (E.G. SALMONELLA). THE PATHOGENESIS OF MSMD REMAINED UNCLEAR UNTIL 1996, WHEN ITS FIRST GENETIC ETIOLOGY WAS DECIPHERED IN CHILDREN WITH INTERFERON- RECEPTOR 1 (IFN-R1) DEFICIENCY. GENETIC STUDIES OVER THE LAST 25 YEARS HAVE IDENTIFIED 16 MSMD-CAUSING GENES, INCLUDING 14 AUTOSOMAL (IFNG, IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, IL12RB2, IL23R, IRF8, SPPL2A, RORC, ISG15, TYK2, JAK1) AND 2 X-LINKED GENES (NEMO, CYBB). THE HIGH LEVEL OF ALLELIC HETEROGENEITY AT THESE LOCI HAS DEFINED 31 DISTINCT DISORDERS. THERE IS HOWEVER PHYSIOLOGICAL HOMOGENEITY, AS ALL DISORDERS IMPAIR IFN- IMMUNITY. MUTATIONS IN 5 GENES (RORC, ISG15, TYK2, JAK1, STAT1) CAN UNDERLIE AN ATYPICAL, SYNDROMIC FORM OF MSMD, WITH AN ASSOCIATED PHENOTYPE. WITH HINDSIGHT, MSMD IS A MISNOMER, AS MOST GENETIC ETIOLOGIES SHOW INCOMPLETE PENETRANCE FOR MSMD. THIS SERENDIPITOUSLY LED TO THE DISCOVERY OF GENETIC ETIOLOGIES OF BONA FIDE TUBERCULOSIS. REMARKABLY, ONLY ABOUT HALF OF THE 900 INTERNATIONAL PATIENTS STUDIED IN OUR LAB CARRY MSMD-CAUSING LESIONS IN THE EXONS AND FLANKING INTRON REGIONS AT ANY OF THESE 16 LOCI. IN THIS RENEWAL APPLICATION, WE HYPOTHESIZE THAT UNEXPLAINED MSMD CASES CAN RESULT FROM NOVEL MONOGENIC INBORN ERRORS OF IMMUNITY, POSSIBLY BUT NOT NECESSARILY INVOLVING IFN- MEDIATED IMMUNITY. WE AIM TO IDENTIFY NEW MSMD-CAUSING GENES BY FOLLOWING A GENOME-WIDE (GW) APPROACH, BASED PRIMARILY BUT NOT EXCLUSIVELY ON WHOLE-EXOME SEQUENCING (WES). WE WILL ENROLL AT LEAST 50 MSMD PATIENTS EACH YEAR. WE WILL SEARCH FOR NOVEL GENETIC ETIOLOGIES BY TESTING A HYPOTHESIS OF GENETIC HOMOGENEITY, I.E. SEARCHING FOR GENES MUTATED IN TWO OR MORE FAMILIES. WE WILL ALSO TEST A HYPOTHESIS OF GENETIC HETEROGENEITY, I.E. SEARCHING FOR GENES MUTATED IN A SINGLE FAMILY. THIS SEARCH WILL BENEFIT FROM OUR 12-YEAR-LONG DEVELOPMENT OF COMPUTATIONAL TOOLS TO ANALYZE WES. CAUSAL RELATIONSHIPS BETWEEN CANDIDATE GENOTYPES AND MSMD WILL BE ESTABLISHED EXPERIMENTALLY IN GREAT MECHANISTIC DEPTH AT THE MOLECULAR, CELLULAR, AND IMMUNOLOGICAL LEVELS, TAKING ADVANTAGE OF CUTTING-EDGE TECHNOLOGIES AND OUR 25-YEAR-LONG STUDY OF MSMD. IN PATIENTS WITHOUT CANDIDATE GENOTYPES BY WES, WE WILL SEARCH FOR CANDIDATE REGULATORY VARIATIONS IN KNOWN AND UNKNOWN MSMD-CAUSING GENES BY WHOLE GENOME SEQUENCING (WGS). OUR PRELIMINARY RESULTS ARE EXCITING, AS WE HAVE IDENTIFIED MSMD-CAUSING MUTATIONS IN GENES KNOWN TO BE CRUCIAL FOR IFN- IMMUNITY (TBX21, IRF1) AND IN OTHER GENES THAT PROBABLY DISRUPT IFN- IMMUNITY BY NOVEL MECHANISMS (ZNFX1, MCTS1). FROM AN IMMUNOLOGICAL STANDPOINT, THIS RESEARCH WILL PROVIDE NOVEL INSIGHTS INTO THE MECHANISMS OF HUMAN IMMUNITY TO MYCOBACTERIA. FROM A MEDICAL STANDPOINT, THIS WORK WILL PROVIDE MOLECULAR DIAGNOSES FOR MSMD PATIENTS AND GENETIC COUNSELING FOR FAMILIES, WHILE OFFERING THE USE OF THERAPEUTIC IFN-, AT LEAST IN PATIENTS WHOSE GENETIC DISORDER DOES NOT ABOLISH CELLULAR RESPONSES TO IFN-.

SINGLE CELL DYNAMICS ON A WHOLE ORGANISM SCALE - PROJECT SUMMARY THE FUNCTIONS OF MAMMALIAN ORGANS ARE MAINTAINED BY THE BEHAVIORS AND DYNAMICS OF INDIVIDUAL CELLS. THE ABILITY TO SYSTEMATICALLY MAP EACH CELL TYPE'S TEMPORAL DYNAMICS IS CENTRAL TO THE UNDERSTANDING OF MANY ASPECTS OF BIOLOGICAL CHANGES THAT MAMMALS UNDERGO IN DEVELOPMENT. HOWEVER, CONVENTIONAL METHODS ARE RESTRICTED BY INADEQUATE THROUGHPUT AND THE LIMITED RANGE OF CELLULAR CONTENTS THAT CAN BE MEASURED. WHILE SINGLE-CELL GENOMIC TECHNIQUES HAVE BEEN DEVELOPED TO CHARACTERIZE CELL STATE HETEROGENEITY WITH HIGH RESOLUTION, NEARLY ALL SUCH METHODS CAPTURE ONLY A STATIC SNAPSHOT AT A SINGLE TIME POINT, WITH BOTH TEMPORAL AND SPATIAL INFORMATION LOST DURING CELL ISOLATION. HEREIN, THE PROPOSED PROJECTS AIM TO DEVELOP NOVEL METHODOLOGIES THAT ENABLE A COMPREHENSIVE VIEW OF SINGLE-CELL SPATIOTEMPORAL DYNAMICS ACROSS THE LIFESPAN OF AN ENTIRE MAMMALIAN ORGANISM. SPECIFICALLY, I WILL EXPAND ON THE HIGH-THROUGHPUT SINGLE-CELL RNA-SEQ PLATFORM (SCI-RNA- SEQ), TO DEVELOP A NOVEL METHOD FOR CONCURRENTLY PROFILING TRANSCRIPTOME, EPIGENOME, AND CELLULAR TEMPORAL DYNAMICS (E.G., PROLIFERATION, APOPTOSIS) IN EACH OF MILLIONS OF CELLS. THE TECHNIQUE WILL BE EMPLOYED TO INVESTIGATE HOW AGING REGULATES THE STATUS OF A WHOLE MAMMALIAN BODY BY SYSTEMATICALLY MONITORING SINGLE CELL STATE DYNAMICS ACROSS A BROAD RANGE OF TISSUES IN YOUNG AND AGED MICE. THIS APPROACH WILL BE POWERFUL BECAUSE WE CAN NOT ONLY VISUALIZE IN-VIVO PROLIFERATION AND APOPTOSIS BEHAVIORS OF EACH CELL TYPE BUT ALSO DISSECT ITS CONNECTION WITH INTERNAL TRANSCRIPTOME/EPIGENOME STATES. IN ADDITION TO THE INTERNAL MOLECULAR PROGRAMS, CELL STATE DYNAMICS ARE CONTROLLED BY ASPECTS OF TISSUE ARCHITECTURE SUCH AS CELL-CELL INTERACTIONS AND EXTRACELLULAR MATRIX ABUNDANCE. TO PROFILE SINGLE CELL MICROENVIRONMENT WITH HIGH THROUGHPUT AND ACCURACY, WE WILL DEVELOP A NOVEL TECHNIQUE CALLED "MICROTISSUE-SEQ", FOR CO-PROFILING SINGLE-CELL MOLECULAR CONTENTS, CELLULAR SPATIAL INTERACTIONS, AND EXTRACELLULAR MATRIX (ECM) PROTEINS ACROSS TENS OF THOUSANDS OF SPATIAL LOCATIONS IN A SINGLE EXPERIMENT. WE WILL EMPLOY THIS TECHNIQUE TO INTERROGATE HOW CELLULAR MICROENVIRONMENT REGULATES ORGANISMAL-SCALE CELL STATE DYNAMICS IN DIFFERENT AGE GROUPS OF MICE. OVERALL, THE PROPOSED PROJECTS WILL ESTABLISH A TECHNICAL FRAMEWORK FOR COMPREHENSIVE PROFILING SINGLE-CELL SPATIOTEMPORAL DYNAMICS AT AN UNPRECEDENTED SCALE OF A WHOLE MAMMALIAN ORGANISM. BY PROFILING CELL-STATE SPECIFIC DYNAMIC BEHAVIORS ACROSS THE LIFESPAN OF MICE, THESE TECHNOLOGIES AND EXPERIMENTS WOULD UNIQUELY ENABLE ACCURATE MODELING OF THE EXQUISITE PROGRAM UNDERLYING MAMMALIAN SYSTEM MAINTENANCE AND BREAKDOWN WITH AGE AT SINGLE CELL RESOLUTION. THESE MULTI-PRONGED APPROACHES ALSO OPEN A NEW PARADIGM FOR UNDERSTANDING THE GLOBAL MOLECULAR PROGRAMS REGULATING CELL STATES AND DYNAMICS DURING AGING, THEREBY INFORMING POTENTIAL PATHWAYS TO DELAY THE AGING PROCESS AS WELL AS THE RATIONAL DESIGN OF EFFECTIVE THERAPIES TO RESTORE TISSUE HOMEOSTASIS FOR PATIENTS WITH AGING-RELATED DISEASES.

TRAPPING AND RECONSTITUTING EARLY STAGES OF EUKARYOTIC RIBOSOME ASSEMBLY

DISSECTING TUMOR METABOLIC HETEROGENEITY IN VIVO

PROBING SYMMETRY BREAKING IN EPIGENETIC INHERITANCE: FROM SINGLE MOLECULES TO SYSTEMS BIOLOGY

IDENTIFICATION OF METABOLIC REGULATORS OF GLYCEROLIPID SYNTHESIS AND STORAGE

CROSS-REGULATION BETWEEN LOOP EXTRUSION, CHROMATIN FIBER STRUCTURE AND CHROMATIN-ASSOCIATED RNAS - THE COHESIN COMPLEX IS A MAJOR FACTOR DRIVING THE 3-D ORGANIZATION OF MAMMALIAN GENOMES AT THE SCALE OF TENS TO KILOBASES TO MEGABASES. RECENT SINGLE-MOLECULE EXPERIMENTS HAVE SHOWN THAT IT CAN EXTRUDE LOOPS OF DNA, WHICH ARE AN ORGANIZING PRINCIPLE OF GENOME ARCHITECTURE. TOGETHER WITH CTCF, A DNA-BINDING PROTEIN THAT STALLS COHESIN’S TRANSLOCATION AND DEFINES LOOP BOUNDARIES, AND SEVERAL REGULATORS OF COHESIN THAT PROMOTE LOADING, SUCH AS NIPBL, OR RELEASE FROM CHROMATIN, SUCH AS WAPL, COHESIN DEFINES INTERACTION DOMAINS IN CHROMOSOMES THAT AFFECT PATTERNS OF GENE EXPRESSION DURING DEVELOPMENT AND CAN LEAD TO DEVELOPMENTAL DISEASES OR CANCER WHEN DISRUPTED. ALTHOUGH LOOP EXTRUSION ON NAKED DNA HAS BEEN STUDIED, COHESIN IN CELLS MUST NAVIGATE NUCLEOSOME-PACKED CHROMATIN FIBERS THAT RESTRICT ACCESS TO BINDING SITES ON DNA, SELF-ORGANIZE INTO COMPARTMENTS OF SIMILAR EPIGENETIC STATE THAT ARE INDEPENDENT OF AND COMPETE WITH LOOP DOMAINS, POTENTIALLY REGULATE DNA SUPERCOILING, AND ARE DECORATED WITH CHROMATIN-ASSOCIATED RNAS. HOW COHESIN AND CTCF INTERACT WITH CHROMATIN IN CELLS IS THE NEXT FRONTIER IN UNDERSTANDING 3-D GENOME ORGANIZATION. PROGRESS IN THIS ARENA WILL REQUIRE A MULTI-SCALE APPROACH, WITH METHODS THAT PROBE BOTH NUCLEOSOME-SCALE AND MEGABASE-SCALE FEATURES. I PROPOSE EXPERIMENTS TO PROBE (1) HOW LOOP EXTRUSION BY COHESIN PERTURBS THE LOCAL STRUCTURE OF THE CHROMATIN FIBER; (2) HOW THE LOCAL STRUCTURE OF THE CHROMATIN FIBER, MODULATED BY DEPLETION OF LINKER HISTONES AND DESTABILIZATION OF NUCLEOSOMES, REGULATES COHESIN’S ABILITY TO LOAD AND EXTRUDE LOOPS; (3) HOW CHANGES IN THE BALANCE OF SUPERCOILING DUE TO EXCESS COHESIN LOOPING AFFECT LOCAL NUCLEOSOME-NUCLEOSOME INTERACTIONS; AND (4) THE CHROMATIN-ASSOCIATED RNA INTERACTOME OF CTCF AT RNA-DEPENDENT AND RNA-INDEPENDENT LOOP BOUNDARIES. TO DISSECT THE SPECIFIC EFFECTS OF COHESIN, ITS REGULATORS AND THE CHROMATIN FIBER, WE WILL USE A COMBINATION OF STABLE PROTEIN DEPLETION, ACUTE DEGRADATION, AND PHARMACOLOGICAL INHIBITION IN HUMAN AND MOUSE CELL LINES. WE WILL READ OUT CHANGES IN CHROMATIN FIBER STRUCTURE AND CHROMATIN-ASSOCIATED RNAS USING RICC-SEQ, A METHOD I RECENTLY DEVELOPED FOR MEASURING DNA-DNA CONTACTS AT SUB-NUCLEOSOME RESOLUTION IN INTACT CELLS, AND USING NOVEL TECHNOLOGY DEVELOPMENT TO PROBE THE CHROMATIN-ASSOCIATED RNA INTERACTOME OF SPECIFIC PROTEINS. THESE METHODS WILL BE COMBINED WITH MORE ESTABLISHED EPIGENOME AND TRANSCRIPTION PROFILING TOOLS AND WITH COARSE- GRAINED SIMULATIONS TO DEVELOP AND TEST MULTI-SCALE MODELS FOR THE INTERACTION OF LOOP EXTRUSION MACHINERY WITH THE CHROMATIN FIBER. I ANTICIPATE THAT THE RESULTS OF THESE EXPERIMENTS WILL SHED NEW LIGHT ON HOW LOOP EXTRUSION AND CHROMATIN’S SELF-ASSOCIATION INTERACT IN SPECIFIC CONTEXTS, WHICH MODELS FOR COHESIN’S ENGAGEMENT WITH DNA ARE RELEVANT TO LOOP EXTRUSION, HOW SUPERCOILING IS DISSEMINATED ACROSS CHROMOSOMES, AND HOW THE LOCAL MOLECULAR CONTEXT DEFINES LOOP BOUNDARIES. THIS KNOWLEDGE MAY REVEAL NEW STRATEGIES FOR COMPENSATING TRANSCRIPTIONAL DYSREGULATION DUE TO MUTATIONS IN COHESIN OR ITS REGULATORS USING TARGETABLE FACTORS THAT REGULATE THE CHROMATIN FIBER, COHESIN’S NATIVE SUBSTRATE.

APPLYING RNA LOGIC IN SPACE AND TIME TO NEUROLOGIC DISEASE - 2024 R35 PROJECT SUMMARY THIS PROPOSAL PRESENTS A SERIES OF INTERRELATED NEW TECHNIQUES AND CONCEPTS AIMED AT DISCOVERING OTHERWISE HIDDEN THERAPEUTIC TARGETS FOR NEUROLOGIC DISEASES. THIS TREATMENT-ORIENTED APPROACH COMBINES THE URGENCY OF A PRACTICING NEUROLOGIST WITH THE KNOWLEDGE AND TECHNOLOGY THAT MODERN MOLECULAR BIOLOGY BRINGS TO NEUROSCIENCE. FROM THE BASIC SCIENCE PERSPECTIVE, UNDERSTANDING THE FUNDAMENTAL ROOT MECHANISMS OF DISEASE IS AN UNCOMPROMISING GOAL. FROM THE NEUROLOGIST’S PERSPECTIVE, THE PERFECT SHOULD NOT BE THE ENEMY OF THE GOOD. THIS LEADS TO SEVERAL FUNDAMENTAL POINTS: • GENETIC (DNA) ETIOLOGIES OF BRAIN DISEASE SET THE STAGE FOR OUR FOCUS—THE DOWNSTREAM MANIFESTATIONS OF SUCH DEFECTS. • DIFFERENT CELL TYPES CONTRIBUTE TO DIFFERENT BRAIN DISORDERS, BUT THE DIFFERENCE BETWEEN INDIVIDUAL CELLS IS UNKNOWN. THESE DIFFERENCES ARE MANIFEST AT THE LEVEL OF RNA, MEDIATED BY THE STOICHIOMETRY, VARIATION AND LOCALIZATION OF CELL-SPECIFIC REGULATORY SYSTEMS, AND THEIR CONSEQUENT EFFECTS ON PROTEINS WITHIN AFFECTED CELLS. • NEURONS ARE SPATIALLY COMPLEX AND TEMPORALLY DYNAMIC, REQUIRING COMMENSURATE FOCUS AND TOOL DEVELOPMENT THAT ADDRESS THESE ASPECTS OF RNA REGULATION. • DEVELOPING AND VALIDATING NEW MODEL SYSTEMS WILL LEAD TO UNEXPECTED DISCOVERIES. • UNDERSTANDING HUMAN NEUROLOGIC DISEASE IS COMPLICATED; THUS, THE BEST MODEL SYSTEM FOR UNDERSTANDING NEUROLOGIC DISORDERS IS THE HUMAN. THEREFORE MODEL SYSTEMS MUST BE COMPLIMENTED AND VALIDATED BY STUDYING HUMAN NEURONS. • BUILDING THE BRIDGE BETWEEN MODEL SYSTEMS AND HUMAN NEUROBIOLOGY ALONGSIDE COMPUTATIONAL BIOLOGIC ANALYSIS OF LARGE DATASETS IS NECESSARY TO DEVELOP NEW INSIGHTS AND PREDICTIONS FULLY. • PROVIDING NEW STRATEGIES FOR FUNDAMENTAL DISCOVERY AND FOCUSING ON ACTIONABLE SUBSETS FOR NEUROLOGIC DISEASE IS OUR PRIMARY STRATEGY. A SIGNIFICANT INNOVATION OUR LAB HAS DEVELOPED SUPPLEMENTS THE STANDARD ANALYSIS OF RNA QUANTITY (RNASEQ) TO UNDERSTAND THE REGULATION OF RNA QUALITY THROUGH THE DEVELOPMENT OF CLIP, A RAPIDLY EXPANDING TECHNOLOGY. RECENT WORK HAS ESTABLISHED THE POTENTIAL OF THIS TECHNOLOGY, AND WE PROPOSE TO FURTHER MAP THE ABILITY TO STUDY RNA REGULATION IN SINGLE NEURONAL CELL TYPES IN THE BRAIN, SUBCELLULAR DOMAINS OF NEURONS, AND DURING THE RAPID CHANGES THAT ACCOMPANY NEURONAL DEPOLARIZATION. IMPORTANTLY, WE PROPOSE TO INTEGRATE DATA FROM MODEL SYSTEMS WITH A NOVEL CONCEPT TO STUDY MOLECULAR VARIATION IN NEURONS OBTAINED FROM HUMAN AUTOPSY. WE WILL ANALYZE COMPLEX MOLECULAR DATASETS WITH COMPUTATIONAL BIOLOGY. SUCCESS IN THE TREATMENT OF BRAIN DISEASES TO DATE EMPHASIZES THE IMPORTANCE OF FOCUSING ON ACCESSIBLE MOLECULES WHICH WILL GUIDE OUR APPROACH TO BIG DATA ANALYSIS.

AN INTEGRATED PIPELINE FOR NEXT-GENERATION PROTEIN INTERACTOMICS

AMERICAN INDIAN VOCATIONAL REHABILITATION SERVICES

HOW BRAINS BUILD NAVIGATIONAL VARIABLES AND USE THEM TO GUIDE BEHAVIOR - PROJECT SUMMARY / ABSTRACT OUR BRAIN PROVIDES US WITH A SENSE OF WHERE WE ARE IN SPACE. THE IMPORTANCE OF THIS SENSE IS CLEAR WHEN WE BECOME SPATIALLY DISORIENTED, LIKE WHEN ONE IS CONFUSED ABOUT ONE’S ORIENTATION AFTER EXITING A SUBWAY STATION. CENTRAL TO THE UNDERSTANDING OF HOW BRAINS GIVE RISE TO SPATIAL COGNITION HAS BEEN THE DISCOVERY OF PLACE CELLS IN THE 1970’S (I.E., NEURONS THAT ARE ACTIVE WHEN ANIMALS ARE IN ONE LOCATION IN SPACE), HEAD-DIRECTION CELLS IN THE 1980’S (I.E., NEURONS THAT ARE ACTIVE WHEN ANIMALS FACE ONE COMPASS DIRECTION), AND GRID CELLS IN THE EARLY 2000’S (I.E., NEURONS THAT ARE ACTIVE WHEN ANIMALS ARE IN A GRID OF LOCATIONS IN SPACE). A FUNDAMENTAL NEXT STEP IN OUR UNDERSTANDING OF SPATIAL COGNITION WOULD BE TO DESCRIBE THE CIRCUIT-LEVEL INTERACTIONS THAT GIVE RISE TO SUCH PHYSIOLOGICAL ACTIVITY PATTERNS AND TO UNDERSTAND HOW SUCH SIGNALS ULTIMATELY INFLUENCE NAVIGATIONAL BEHAVIOR. WE WISH TO LEVERAGE THE ADVANCED GENETIC, BEHAVIORAL, ANATOMICAL AND PHYSIOLOGICAL TOOLS IN DROSOPHILA, TO ACHIEVE THREE BROAD GOALS. FIRST, WE WISH TO RIGOROUSLY CHARACTERIZE NEURAL CIRCUITS THAT EXPLAIN HOW NAVIGATIONAL SIGNALS ARE BUILT. SECOND, WE WISH TO IMPROVE THE TASKS THAT FLIES PERFORM WHILE WE RECORD FROM THEIR BRAIN, WHICH WILL ALLOW US TO ISOLATE CELLS AND CIRCUITS REQUIRED FOR THE FORMATION OF SPATIAL WORKING MEMORIES. THIRD, WE AIM TO REVEAL MOLECULAR, CELLULAR AND CIRCUIT MECHANISMS BY WHICH SUCH MEMORIES ARE FORMED AND GUIDE BEHAVIOR. THIS WORK SHOULD ALLOW US TO MORE RIGOROUSLY LINK MOLECULAR FACTORS, THROUGH THEIR EFFECTS ON CELLS AND CIRCUITS, TO THEIR FUNCTION IN SPATIAL-COGNITION. OUR DISCOVERIES SHOULD ULTIMATELY HELP TO INFORM HOW HUMANS PERFORM NAVIGATIONAL TASKS LIKE DRIVING HOME FROM WORK OR FINDING A CAR IN A PARKING LOT, ALONGSIDE HOW TO APPROACH NEUROLOGICAL CONDITIONS IN WHICH SUCH ABILITIES ARE IMPAIRED, LIKE IN ALZHEIMER’S DISEASE.

GRADUATE RESEARCH FELLOWSHIP PROGRAM (GRFP)

NEURAL CIRCUITS FOR SPATIAL NAVIGATION

UNDERSTANDING THE ROLE OF QUANTITATIVE INTERNAL SIGNALS IN BEHAVIORAL FLEXIBILITY - PROJECT SUMMARY / ABSTRACT THIS GRANT FOCUSES ON HOW VERY RECENT EXPERIENCES––OVER THE PAST FEW SECONDS TO MINUTES––ALLOW BRAINS TO UPDATE EXPECTATIONS ABOUT THE WORLD AND THEN USE THESE EXPECTATIONS TO GUIDE BEHAVIOR. THE ABILITY TO FLEXIBLY ADJUST ONE'S COURSE OF ACTION IN THIS MANNER IS A HALLMARK OF ADAPTIVE HUMAN BEHAVIOR. AT THE NEURAL LEVEL, RELEVANT CELLULAR-ACTIVITY CORRELATES HAVE BEEN DESCRIBED IN NON-HUMAN PRIMATES AND OTHER VERTEBRATE MODEL SYSTEMS. FOR EXAMPLE, RAMPING NEURAL ACTIVITY HAS BEEN OBSERVED IN THE FEW HUNDRED MILLISECONDS, OR SECONDS, LEADING UP TO BEHAVIORAL DECISIONS AND THE RATE OF RISE OF THESE RAMPS TRACKS THE GRADUAL ACCUMULATION OF INFORMATION RELEVANT FOR THE DECISION BEING MADE. RAMPING ACTIVITY IS THUS A CORRELATE OF AN INCREASING EXPECTATION THAT AN IMPORTANT DECISION NEEDS TO BE MADE AND THE MOMENT AT WHICH THE RAMP REACHES A THRESHOLD LEVEL TYPICALLY SIGNIFIES WHEN A FINAL DECISION IS TAKEN. ANOTHER SALIENT CORRELATE OF INTERNAL EXPECTATIONS ARE REWARD-PREDICTION ERROR SIGNALS: BURSTS OF DOPAMINE-NEURON ACTIVITY WHEN AN ANIMAL RECEIVES AN UNEXPECTED REWARD OR A REWARD IS SURPRISINGLY OMITTED. REWARD-PREDICTION ERROR SIGNALS SEEM WELL POISED TO ADJUST ANIMAL AND HUMAN BEHAVIOR BASED ON LEARNED EXPECTATIONS. A CLEARER PICTURE OF HOW QUANTITATIVE INTERNAL SIGNALS––LIKE RAMPING AND REWARD-PREDICTION ERROR ACTIVITY––CONTRIBUTE TO BEHAVIORAL FLEXIBILITY WOULD BE AN IMPORTANT STEP FORWARD FOR COGNITIVE NEUROSCIENCE. HERE, WE PROPOSE TO DEVELOP TWO NEW BEHAVIORAL TASKS IN TETHERED DROSOPHILA, WHERE WE CAN PERFORM SIMULTANEOUS NEUROPHYSIOLOGY. OUR FIRST AIM IS TO USE ONE OF THESE TASKS TO TEST THE HYPOTHESIS THAT RAMPING NEURAL SIGNALS ARE FUNDAMENTAL IN FORMING BEHAVIORAL DECISIONS OVER TENS-OF-SECONDS TO MINUTES TIMESCALES IN ETHOLOGICALLY RELEVANT CONTEXTS, RATHER THAN JUST ON MUCH SHORTER TIMESCALES AND IN LABORATORY DEFINED TASKS (AS HAS BEEN SHOWN TO DATE). SUCH A DISCOVERY WOULD ARGUE THAT EXPECTATIONS BUILT OVER MINUTES IN REAL-WORLD CONDITIONS ARE ULTIMATELY FED INTO SLOWLY RAMPING NEURONAL SIGNALS SO AS TO GUIDE NATURAL DECISION-MAKING. OUR SECOND AIM IS TO DISCOVER REWARD-PREDICTION ERROR SIGNALS IN FRUIT FLIES ACTIVELY PERFORMING A TRIAL-BY-TRIAL CONDITIONING TASK AND TO DEFINE A CIRCUIT MECHANISM THROUGH WHICH SUCH SIGNALS ALLOW BRAINS TO FORM QUANTITATIVELY PRECISE EXPECTATIONS––UPDATED ON A TRIAL-BY-TRIAL BASIS––ON THE LIKELIHOOD OF REWARDS ARRIVING OR NOT ARRIVING IN THE NEAR FUTURE. SUCH DISCOVERIES IN A GENETICALLY TRACTABLE MODEL WILL INFORM OUR THINKING ON HOW OUR BRAINS GENERATE EXPECTATIONS THAT ALLOW FOR FLEXIBLE, ADAPTIVE BEHAVIORS, ULTIMATELY INFORMING NEW THERAPEUTIC APPROACHES TO NEUROLOGICAL CONDITIONS IN WHICH FLEXIBILITY IS IMPAIRED, SUCH AS OBSESSIVE-COMPULSIVE DISORDER.

MOLECULAR MECHANISMS UNDERLYING TRNA-FRAGMENT REGULATION OF CANCER

TMEM41B: A PAN-FLAVIVIRUS AND PAN-CORONAVIRUS HOST FACTOR WITH ANTIVIRAL POTENTIAL - PROJECT SUMMARY ARTHROPOD-BORNE FLAVIVIRUSES AND RESPIRATORY-TRANSMITTED CORONAVIRUSES HAVE THE POTENTIAL TO CAUSE SEVERE EPIDEMICS AND PANDEMICS. ONE STRATEGY TO PREPARE FOR AND RESPOND TO VIRAL OUTBREAKS IS TO DEVELOP DRUGS THAT TARGET HOST FACTORS VIRUSES REQUIRE TO COMPLETE THEIR LIFECYCLES. THROUGH A SERIES OF CRISPR/CAS9 GENE DISRUPTION SCREENS, WE IDENTIFIED TRANSMEMBRANE PROTEIN 41B (TMEM41B) AND THE CLOSELY RELATED VACUOLE MEMBRANE PROTEIN 1 (VMP1) AS CRITICAL PAN-FLAVIVIRUS AND PAN-CORONAVIRUS HOST FACTORS. BOTH PROTEINS ARE HIGHLY CONSERVED LIPID SCRAMBLASES WITH ROLES IN AUTOPHAGY. OUR CURRENT MODEL IS THAT VIRUSES FROM BOTH THE FLAVIVIRDAE AND CORONAVIRIDAE FAMILIES HIJACK TMEM41B AND VMP1 FOR THEIR ABILITY TO REMODEL ER MEMBRANES AND INDUCE MEMBRANE CURVATURE TO ESTABLISH MEMBRANE-PROTECTED VIRAL RNA REPLICATION ORGANELLES. OUR OVERALL GOAL FOR THIS PROPOSAL IS TO UNDERSTAND HOW, ON A MECHANISTIC LEVEL, BOTH PROTEINS SUPPORT FLAVIVIRUS AND CORONAVIRUS INFECTION. OUR PREVIOUS WORK INDICATES THAT TMEM41B IS REQUIRED AT A POST-ENTRY STEP AT OR PRIOR TO VIRAL RNA REPLICATION. IN AIM 1, WE WILL INTERROGATE EARLY EVENTS OF THE VIRUS LIFECYCLE INCLUDING PRIMARY TRANSLATION, POLYPROTEIN PROCESSING, AND REPLICATION ORGANELLE FORMATION IN WT, TMEM41B AND VMP1 KNOCKOUT (KO) CELLS TO DETERMINE HOW FAR THE FLAVIVIRUS AND CORONAVIRUS LIFECYCLES PROGRESS IN THE ABSENCE OF EITHER PROTEIN. WE PREVIOUSLY SHOWED THAT LACK OF TMEM41B AND VMP1, INDUCES A HEIGHTENED INNATE IMMUNE RESPONSE UPON FLAVIVIRUS INFECTION. WE HYPOTHESIZE THAT BOTH PROTEINS ARE RECRUITED TO SITES OF VIRAL RNA REPLICATION, AND THAT IN THEIR ABSENCE, RNA REPLICATION INITIATES AND VIRAL DOUBLE STRANDED RNA (DSRNA) IS PRODUCED. HOWEVER, WITHOUT A PROPER REPLICATION ORGANELLE DSRNA IS EXPOSED AND TRIGGERS AN INNATE IMMUNE RESPONSE. ALTERNATIVELY, GIVEN TMEM41B’S AND VMP1’S LIPID SCRAMBLASE ACTIVITY AND FUNCTION IN LIPID HOMEOSTASIS, THEIR ABSENCE MAY INDUCE ER STRESS, WHICH TRIGGERS AN UNFOLDED PROTEIN RESPONSE (UPR) THAT IN SYNERGY WITH DSRNA MAY CAUSE A HEIGHTENED INNATE IMMUNE RESPONSE. IN AIM 2, WE WILL TEST VIRUS INFECTION IN DOUBLE KO CELLS THAT LACK EITHER PROTEIN IN ADDITION TO GENES THAT ARE ESSENTIAL FOR PATHOGEN SENSING, IFN SIGNALING, AND UPR ACTIVATION. WE WILL FURTHER CONDUCT RNASEQ EXPERIMENTS TO INVESTIGATE LACK OF TMEM41B IN STEM CELLS AND STEM CELL-DERIVED PRIMARY-LIKE CELLS REPRESENTING DIFFERENT TISSUE LINEAGES IN THE ABSENCE AND PRESENCE OF VIRAL REPLICATION. LASTLY, IN AIM 3, WILL USE A PANEL OF PHENOTYPIC AND MECHANISTIC ASSAYS TO CHARACTERIZE NATURALLY OCCURRING SNPS IN TMEM41B THAT WE PREVIOUSLY FOUND TO IMPACT FLAVIVIRUS REPLICATION, AND SEVERAL REPORTED VMP1 LOSS-OF- FUNCTION MUTANTS. WE WILL FURTHER TAKE A DEEP MUTATIONAL SCANNING APPROACH TO COMPREHENSIVELY CHARACTERIZE TMEM41B AND VMP1 AND DETERMINE IF ANY DOMAINS OR AMINO ACIDS ARE DIFFERENTIALLY REQUIRED FOR THEIR CELLULAR AND PROVIRAL FUNCTIONS. THIS FUNCTIONAL CHARACTERIZATION WILL IDENTIFY MUTANTS THAT CAN BE STUDIED IN DETAIL IN MECHANISTIC ASSAYS AND MAY IDENTIFY AMINO ACIDS OR INTERFACES IN BOTH PROTEINS THAT CAN BE TARGETED TO PREVENT VIRUS INFECTION WITH MINIMAL DISRUPTION TO CELLULAR BIOLOGY.

REPRESENTATION AND MODULATION OF SOCIAL INFORMATION IN THE ANT CHEMOSENSORY SYSTEM - PROJECT SUMMARY SOCIAL INSECTS SHOW ROBUST AND COMPLEX BEHAVIORS, AND HAVE SERVED AS IMPORTANT STUDY SYSTEMS IN ETHOLOGY FOR DECADES. HOWEVER, BECAUSE THEY ARE NOT GENETICALLY TRACTABLE, RESEARCHERS HAVE NOT BEEN ABLE TO STUDY THESE BEHAVIORS AT THE LEVEL OF BRAIN CIRCUITRY WITH CUTTING-EDGE NEUROGENETIC TOOLS. THE PROPOSED WORK WILL PIONEER SUCH TOOLS IN THE CLONAL RAIDER ANT OOCERAEA BIROI, A SPECIES THAT UNIQUELY COMBINES EXPERIMENTAL AMENABILITY WITH THE FASCINATING BEHAVIOR OF SOCIAL INSECTS. IT WILL THEN ADDRESS AN IMPORTANT BIOLOGICAL QUESTION: HOW DOES VARIATION IN NEURAL RESPONSIVENESS GIVE RISE TO CONSISTENT DIFFERENCES IN HOW INDIVIDUALS RESPOND TO SOCIAL AND ENVIRONMENTAL STIMULI? O. BIROI IS PARTICULARLY SUITABLE TO STUDY THIS QUESTION FOR A NUMBER OF REASONS. FIRST, THE ANTS REPRODUCE ASEXUALLY AND CLONALLY, IMPLYING THAT BEHAVIORAL DIFFERENCES ARISE FROM PHENOTYPIC PLASTICITY, RATHER THAN GENETIC DIFFERENCES. SECOND, UNLIKE IN CONVENTIONAL MODEL SYSTEMS LIKE DROSOPHILA OR MICE, DIFFERENCES IN BEHAVIORAL PROPENSITIES ARE ADAPTIVE BECAUSE THEY GIVE RISE TO STABLE DIVISION OF LABOR IN A COLONY CONTEXT. ACCORDINGLY, THESE DIFFERENCES ARE ROBUST AND PREDICTABLE, AND THEY HAVE RECEIVED A LOT OF THEORETICAL AND EMPIRICAL ATTENTION AT THE BEHAVIORAL LEVEL. GIVEN THAT ANTS COMMUNICATE ALMOST EXCLUSIVELY VIA PHEROMONES, WE WILL FOCUS ON THE ANTENNAL LOBE, THE PRIMARY PROCESSING AREA OF CHEMOSENSORY INFORMATION IN THE INSECT BRAIN, ANALOGOUS TO THE MAMMALIAN OLFACTORY BULB. IN AIM 1, WE WILL GENERATE TRANSGENIC LINES EXPRESSING THE GENETICALLY ENCODED CALCIUM INDICATOR GCAMP IN THE ANTENNAL LOBE TO ENABLE LIVE IMAGING OF NEURAL ACTIVITY WITH TWO-PHOTON MICROSCOPY. WE WILL ALSO GENERATE LINES EXPRESSING THE PHOTOACTIVATABLE FLUORESCENT PROTEIN CAMPARI2, ENABLING STABLE LABELING OF NEURONS ACTIVE IN FREELY BEHAVING ANIMALS. IN AIM 2, WE WILL USE THESE TOOLS TO CREATE A FUNCTIONALLY ANNOTATED MAP OF CHEMOSENSORY REPRESENTATION IN THE ANT ANTENNAL LOBE. WE WILL ALSO USE SINGLE-CELL RNA-SEQUENCING OF LABELLED NEURONS TO IDENTIFY ODORANT RECEPTORS RESPONDING TO PHEROMONES. WE WILL THEN USE THE PROMOTERS OF THESE RECEPTORS TO GENERATE ADDITIONAL, NARROWLY TARGETED TRANSGENIC LINES. IN AIM 3, WE WILL STUDY HOW DIFFERENCES IN NEURAL REPRESENTATION AND SENSITIVITY CORRELATE WITH PLASTIC DIFFERENCES IN BEHAVIORAL RESPONSES TO IDENTICAL SOCIAL STIMULI. BASED ON THESE DATA, WE WILL BUILD A PREDICTIVE THEORETICAL MODEL OF DIVISION OF LABOR IN INSECT SOCIETIES. ON A FUNDAMENTAL LEVEL, OUR RESULTS ON THE MODULATION OF SENSORY PERCEPTION WILL ALSO INFORM OUR UNDERSTANDING OF HUMAN DISORDERS INVOLVING ABNORMAL SENSORY SENSITIVITY, SUCH AS AUTISM AND SCHIZOPHRENIA. FINALLY, WE WILL MAKE THE TOOLS AND PROTOCOLS DEVELOPED UNDER THIS PROPOSAL AVAILABLE TO THE SCIENTIFIC COMMUNITY, GREATLY ADVANCING THE FIELD OF SOCIAL INSECT NEUROSCIENCE AND OPENING UP A VAST NEW EXPERIMENTAL SPACE. THE ROBUST AND EXPANSIVE BEHAVIORAL REPERTOIRE OF SOCIAL INSECTS COMBINED WITH THE SIMPLICITY OF A COMPACT INVERTEBRATE NERVOUS SYSTEM ALLOWS O. BIROI TO FILL AN IMPORTANT NICHE IN NEUROSCIENCE.

HIGH-SPEED VOLUMETRIC IMAGING OF NEURONAL NETWORK ACTIVITY AT DEPTH USING MULTIPLEXED SCANNED TEMPORAL FOCUSING (MUST)

TOWARDS A MOLECULAR UNDERSTANDING OF PERSISTENT TUBERCULOSIS INFECTION

CHIPPEWA CREE TRIBE ROUTE 6 PLANNING GRANT UNDER THE REBUILDING AMERICAN INFRASTRUCTURE AND EQUITY (RAISE) PROGRAM

DISCOVERY OF GPCR-ACTIVE NATURAL PRODUCTS AND THEIR BIOSYNTHETIC GENES FROM THE HUMAN ASSOCIATED BACTERIA

CHIPPEWA CREE TRIBE AMERICAN INDIAN VOCATIONAL REHABILITATION SERVICES (AIVRS) PROJECT

MOLECULAR PATHWAYS CONNECTING SLEEP, STRESS, METABOLISM AND LONGEVITY

HOST PROTEIN TARGETS OF HIV-1 VPR IN GENE EXPRESSION, CELL CYCLE AND INNATE IMMUNITY

THIS AGREEMENT PROVIDES FUNDING FOR THE OPERATION OF THE CHIPPEWA CREE TRIBE'S CONTINUING ENVIRONMENTAL PROGRAMS WHILE GIVING IT GREATER FLEXIBILITY TO ADDRESS ITS HIGHEST ENVIRONMENTAL PRIORITIES, IMPROVE ENVIRONMENTAL PERFORMANCE, ACHIEVE ADMINISTRATIVE SAVINGS AND STRENGTHEN THE PARTNERSHIP BETWEEN THE CHIPPEWA CREE TRIBE AND EPA. THIS AGREEMENT FUNDS TRIBAL PROGRAMS FOR WATER QUALITY, LAND REVITALIZATION, DATA COLLECTION, POLLUTION CONTROL, BROWNFIELDS TRIBAL RESPONSE, AND ENVIRONMENTAL PROGRAM DEVELOPMENT.

FUNCTIONAL MAPPING OF ENTERIC-ASSOCIATED NEURONS

ASO AND SHRNA FOR TARGETING THE ONCOGENIC TRANSCRIPT DRIVING FIBROLAMELLAR HEPATOCELLULAR CARCINOMA

IN VIVO IMAGING OF NEUROACTIVITY IN THE DEEP FORWARD SCATTERING REGIME USING SPECKLE IDENTIFICATION AND DEMIXING (SPID) MICROSCOPY

AMERICAN INDIAN VOCATIONAL REHABILITATION SERVICES

DEVELOPMENT OF SMALL MOLECULE CGAS INHIBITORS FOR REPRESSION OF DSDNA-TRIGGERED INTERFERON EXPRESSION

ELUCIDATING THE GATING MECHANISMS OF BACTERIAL MECHANOSENSITIVE CHANNELS - MECHANOSENSITIVE (MS) CHANNELS SENSE AND RESPOND TO MECHANICAL FORCES BY OPENING AN ION-CONDUCTING PATHWAY. MS CHANNELS ARE FOUND IN ALL KINGDOMS OF LIFE, AND IN HUMANS PLAY ESSENTIAL ROLES IN A NUMBER OF SENSORY PROCESSES, INCLUDING HEARING, THE SENSE OF TOUCH, BALANCE AND REGULATION OF BLOOD PRESSURE. THE FIRST MS CHANNELS LIKELY EVOLVED IN EARLY PROKARYOTES AS PROTECTION FROM HYPOOSMOTIC STRESS. BECAUSE BACTERIAL MS CHANNELS ARE UBIQUITOUSLY EXPRESSED IN BACTERIA, BUT NOT IN HUMANS, AND BECAUSE THEIR UNCONTROLLED OPENING HAS A DELETERIOUS AND OFTEN LETHAL EFFECT ON THE BACTERIA, PRESUMABLY DUE TO THE LOSS OF IMPORTANT METABOLITES, BACTERIAL MS CHANNELS ARE INTRIGUING TARGETS FOR DEVELOPING NOVEL ANTIBIOTICS. BACTERIA EXPRESS TWO TYPES OF MS CHANNELS, MS CHANNELS OF LARGE CONDUCTANCE (MSCL) AND MS CHANNELS OF SMALL CONDUCTANCE (MSCS). MEMBERS OF THE MSCL FAMILY ARE HIGHLY CONSERVED AND MSCL HAS BECOME A PARADIGM FOR THE UNDERSTANDING OF MS CHANNELS BECAUSE OF ITS SIMPLICITY AND AMENABILITY TO DIFFERENT EXPERIMENTAL APPROACHES. MSCS CHANNELS ARE MORE DIVERSE, AND BACTERIA OFTEN EXPRESS MORE THAN ONE PARALOG. BOTH BACTERIAL MS CHANNELS ARE GATED BASED ON THE ‘FORCE-FROM- LIPIDS’ PRINCIPLE AND RESPOND TO THE TRANSMEMBRANE PRESSURE PROFILE OF THE SURROUNDING MEMBRANE. HOWEVER, EVEN THOUGH STRUCTURES ARE AVAILABLE FOR MSCL AND MSCS IN DIFFERENT FUNCTIONAL STATES, THE MECHANISM BY WHICH MEMBRANE TENSION OPENS THESE CHANNELS HAS REMAINED ENIGMATIC. WE HAVE RECENTLY DETERMINED CRYO-ELECTRON MICROSCOPY (CRYO-EM) STRUCTURES OF MSCS IN DIFFERENT MEMBRANE ENVIRONMENTS, PROVIDED BY NANODISCS, INCLUDING ONE MIMICKING A MEMBRANE UNDER TENSION. THE STRUCTURES, COMPLEMENTED BY MOLECULAR DYNAMICS (MD) SIMULATIONS AND ELECTROPHYSIOLOGICAL STUDIES, ALLOWED US TO VISUALIZE THE CHANNEL IN DIFFERENT FUNCTIONAL STATES AND TO DEDUCE WHAT ROLES LIPIDS ASSOCIATED WITH MSCS PLAY IN MECHANOSENSATION. WE WILL CONTINUE TO USE A COMBINATION OF SINGLE-PARTICLE CRYO-EM, PATCH-CLAMP ELECTROPHYSIOLOGY AND MD SIMULATIONS TO STUDY THE STRUCTURE AND GATING OF BACTERIAL MS CHANNELS. IN AIM 1, WE WILL CONTINUE TO EXPLORE THE FUNCTION OF LIPIDS IN MSCS FUNCTION, IN PARTICULAR WHETHER IT ADOPTS A DEFINED OPEN CONFORMATION IN A NATIVE LIPID ENVIRONMENT, HOW MODULATORS AFFECT MSCS BY CHANGING ITS LIPID ENVIRONMENT, AND WHETHER 16-CARBON ACYL CHAINS PLAY A SPECIFIC ROLE IN MSCS GATING. IN AIM 2, WE WILL EXPAND OUR STUDIES TO BACTERIAL CYCLIC NUCLEOTIDE-GATED (BCNG) CHANNELS TO ELUCIDATE HOW THE MSCS FOLD WAS ADAPTED TO MAKE THE CHANNEL RESPOND TO CAMP BINDING RATHER THAN MEMBRANE TENSION. AIM 3 WILL FOCUS ON MSCL. WE WILL DETERMINE THE STRUCTURE OF MSCL IN A NATIVE LIPID ENVIRONMENT TO CONFIRM (OR DISPROVE) THE EXISTENCE OF LIPID-FILLED NANO-POCKETS THAT WERE SUGGESTED TO PLAY A CRITICAL ROLE IN GATING. FINALLY, WE WILL DETERMINE THE STRUCTURE OF MSCL OPENED BY DIFFERENT EFFECTORS TO VISUALIZE THE STRUCTURE OF THIS CHANNEL IN THE OPEN STATE AND TO TEST OUR HYPOTHESIS THAT DIFFERENT EFFECTORS RESULT IN OPEN CONFORMATIONS WITH DIFFERENT PORE DIAMETERS. THE RESULTS OF THESE STUDIES WILL NOT ONLY PROVIDE NEW INSIGHTS INTO THE GATING MECHANISM OF BACTERIAL MS CHANNELS, BUT ALSO HELP IN EXPLOITING THESE CHANNELS FOR BIOMEDICAL APPLICATIONS.

ENERGY COMMUNITY REVITALIZATION PROGRAM: BIPARTISAN INFRASTRUCTURE LAW (BIL) SEC. 40601(D) ORPHANED WELL TRIBAL PROGRAMAWARD PURPOSE: FUNDING THROUGH THE PROGRAM MAY BE UTILIZED TO PLUG, REMEDIATE OR RECLAIM ORPHANED WELLS ON TRIBAL LAND, RESTORE SOIL AND HABITAT IN THE DEGRADED AREA, DECOMMISSION OR REMOVE ASSOCIATED INFRASTRUCTURE, IDENTIFY AND CHARACTERIZE ADDITIONAL UNDOCUMENTED WELLS ON TRIBAL LAND AND SET UP WELL-PLUGGING CAPACITY WHERE NOT ALREADY ESTABLISHED. SECTION 40601(D) OF THE BIL CREATES AN ORPHANED WELL SITE PLUGGING, REMEDIATION, AND RECLAMATION GRANT PROGRAM WITHIN THE DEPARTMENT OF INTERIOR TO ADDRESS ORPHANED WELLS AND WELL SITES ON TRIBAL LANDS.ACTIVITIES TO BE PERFORMED: THIS GRANT FUNDS CHIPPEWA CREE TRIBE TO PERFORM THE FOLLOWING ACTIVITIES: PLUG, REMEDIATE, OR RECLAIM AN ORPHANED WELL ON TRIBAL LAND REMEDIATE SOIL AND RESTORE HABITAT THAT HAS BEEN DUE TO THE PRESENCE OF AN ORPHANED WELL OR ASSOCIATED PIPELINES, FACILITIES, OR INFRASTRUCTURE ON TRIBAL LAND REMEDIATE TRIBAL LAND ADJACENT TO ORPHANED WELLS AND DECOMMISSION OR REMOVE ASSOCIATED PIPELINES, FACILITIES, AND INFRASTRUCTURE PROVIDE AN ACCOUNTING OF THE COST OF PLUGGING, REMEDIATION, AND RECLAMATION FOR EACH ORPHANED WELL SITE ON TRIBAL LAND IDENTIFY AND CHARACTERIZE UNDOCUMENTED ORPHANED WELLS ON TRIBAL LAND AND DEVELOP OR ADMINISTER A TRIBAL PROGRAM TO CARRY OUT ANY ACTIVITIES DESCRIBED IN THIS SECTION. ACTIVITIES FUNDED BY THIS GRANT MUST COMPLY WITH THE UNIFORM GUIDANCE FOR GRANTS AND AGREEMENTS (2 CFR 200), BUILD AMERICA BUY AMERICA, DAVIS-BACON ACT, AND BIL, AND INCLUDE ALL COSTS NECESSARY TO CONDUCT AND MANAGE THE APPROVED PROJECT, SUCH AS EQUIPMENT AND CONTRACTED SERVICES. EXPECTED DELIVERABLES OR OUTCOMES: 1. PLUG, REMEDIATE, OR RECLAIM AN ORPHANED WELL ON TRIBAL LAND.2. DECREASE THE AMOUNT OF METHANE EMISSIONS FROM ORPHANED WELLS.3. ENHANCE THE PROTECTION OF PUBLIC HEALTH, SAFETY, AND PROPERTY FROM ADVERSE EFFECTS OF OIL DRILLING PRACTICES FROM ORPHANED WELLS ON TRIBAL LANDS.4. LOCATE, ASSESS, CHARACTERIZE AND OR INVENTORY UNDOCUMENTED ORPHANED WELLS ON TRIBAL LANDS.5. RESTORE LAND AND WATER RESOURCES, THE ENVIRONMENT, AND PROPERTY FROM OIL DRILLING ACTIVITIES, FACILITIES, AND ASSOCIATED PIPELINES FROM ORPHANED WELLS ON TRIBAL LANDS.6. DEVELOP OR ADMINISTER A TRIBAL ORPHANED WELLS PROGRAM.INTENDED BENEFICIARIES: THE GENERAL PUBLIC BENEFITS DIRECTLY AND INDIRECTLY FROM THE PROPOSED PROJECT THAT ADDRESSES LONG OVERDUE INFRASTRUCTURE AND ENVIRONMENTAL IMPROVEMENTS TO STRENGTHEN OUR RESILIENCE TO THE CHANGING CLIMATE AND TO MITIGATE THE EFFECTS OF LEGACY POLLUTION. THIS INVESTMENT ON TRIBAL LANDS ACROSS THE COUNTRY WILL GROW THE ECONOMY SUSTAINABLY AND EQUITABLY FOR DECADES TO COME. INFORMATION AND DATA COLLECTED BY THIS PROJECT WILL BE MADE AVAILABLE AS APPLICABLE. THE GENERAL PUBLIC WILL BENEFIT BY REMOVING HAZARDOUS LEGACY FACILITIES, INCLUDING IN DISPROPORTIONATELY IMPACTED COMMUNITIES WHERE WORK WILL BE PRIORITIZED.SUBRECIPIENT ACTIVITIES: N A

FUNCTIONS OF HUMAN RAD51 AND ITS PARALOGS DURING DNA INTERSTRAND CROSSLINK REPAIR

RURAL INNOVATION FUND

INDIAN COMMUNITY DEVELOPMENT BLOCK GRANT (ICDBG)

MOTOR COMPOSITIONALITY IN THE CONTROL OF FACIAL MOVEMENTS

MOLECULAR AND CELLULAR BASIS OF EPIDERMODYSPLASIA VERRUCIFORMIS

A RENEWABLE AND GENETICALLY TRACTABLE HUMAN STEM CELL-DERIVED MULTICELLULAR PLATFORM FOR THE STUDY OF FIBROTIC LIVER DISEASES - PROJECT SUMMARY FIBROTIC LIVER DISEASE IS A GROWING PUBLIC HEALTH CONCERN SIGNIFICANTLY IMPACTING THE GLOBAL POPULATION. FIBROTIC LIVER DISEASE IS OFTEN THE RESULT OF SUSTAINED INSULTS; CHRONIC VIRAL HEPATITIS B AND C AS WELL AS NAFLD ARE THE MOST PREVALENT CAUSES. WHILE ANTIVIRAL REGIMENS FOR HEPATITIS B AND C HAVE DECREASED THE VIRAL CIRRHOSIS BURDEN, OTHER CAUSES LIKE NAFLD ARE INCREASINGLY PREVALENT, NOW AFFECTING 14-27% OF INDIVIDUALS IN DEVELOPED COUNTRIES. DESPITE THIS HIGH DISEASE BURDEN, THERE ARE CURRENTLY NO APPROVED THERAPIES. RATHER, THE LACK OF CONSENSUS ON OPTIMAL DRUG TARGETS OR STRATEGIES REFLECTS A GAP IN OUR MECHANISTIC UNDERSTANDING OF DISEASE DRIVERS. SIMILARLY, THE UTILITY AND CLINICAL RELEVANCE OF ANIMAL MODELS IS EQUALLY CONTROVERSIAL. A MULTITUDE OF MODELS ARE CURRENTLY USED, BUT EACH RECAPITULATES ONLY ISOLATED ASPECTS OF HUMAN PATHOPHYSIOLOGY. THERE IS AN UNMET NEED FOR HUMAN-RELEVANT SYSTEMS THAT RECAPITULATE KEY ELEMENTS OF FIBROTIC LIVER DISEASE TO ENABLE MECHANISTIC DISSECTION. THE MEASURABLE IMPROVEMENT IN LIVER FIBROSIS IN SOME BUT NOT ALL PATIENTS FOLLOWING SUCCESSFUL ANTIVIRAL TREATMENT REINFORCES THE NEED FOR BETTER UNDERSTANDING OF FIBROGENESIS AT A MOLECULAR LEVEL TO AID DEVELOPMENT OF NEW TREATMENTS TO PREVENT FIBROSIS OR ENCOURAGE REGRESSION. FOR NAFLD, DESPITE IDENTIFICATION OF INCREASING NUMBERS OF SUSCEPTIBILITY-ASSOCIATED GENETIC VARIANTS AND LIFESTYLE-DEPENDENT RISK FACTORS, THE MECHANISMS BY WHICH THEY INDIVIDUALLY OR SYNERGISTICALLY CONTRIBUTE TO DISEASE PROGRESSION REMAIN LARGELY UNCLEAR. THE PRIMARY RESEARCH GOAL OF THIS PROPOSAL IS TO EXPLOIT A UNIQUE RENEWABLE AND GENETICALLY MANIPULATABLE HUMAN PLURIPOTENT STEM CELL (HPSC)-DERIVED MULTICELLULAR CULTURE SYSTEM TO ADDRESS THE AFOREMENTIONED GAPS. IN THIS MULTICELLULAR SYSTEM, WE COCULTURE HPSC-DERIVED HEPATOCYTES, HEPATIC STELLATE CELLS (HSCS), AND MACROPHAGES IN A MANNER THAT RECAPITULATES THE COMPLEXITY OF LIVER PHYSIOLOGY IN BOTH HEALTH AND DISEASE. MODELING HEPATITIS VIRUS INFECTION AND NAFLD IN THE MULTICELLULAR CULTURES, WE FOUND THAT BOTH HCV INFECTION AND A LIPOTOXIC MILIEU INDUCED INFLAMMATORY SIGNALS AND STELLATE CELL ACTIVATION. A LIPOTOXIC MILIEU ALSO TRIGGERED OTHER FEATURES OF NAFLD CLINICAL PHENOTYPES. ELIMINATING HCV REVERSED FIBROSIS-LIKE PHENOTYPES AND TREATING WITH OBETICHOLIC ACID SHOWED IMPROVEMENT IN NAFLD-LIKE FEATURES, AS OBSERVED IN THE CLINIC. ACROSS THREE AIMS, USING CELL AND MOLECULAR APPROACHES, WE CAPITALIZE ON THE UNIQUE FEATURES OF THIS NOVEL PLATFORM TO ADDRESS QUESTIONS THAT CANNOT BE ADEQUATELY ANSWERED WITH ANY EXISTING EX VIVO HUMAN-RELEVANT SYSTEM, INCLUDING THE ROLES OF CYTOKINES IN HSC ACTIVATION (AIM 1), THE MECHANISMS OF REVERSION AFTER ACTIVATION (AIM 2) AND THE ROLE OF GENETICS AND LIFESTYLE-ASSOCIATED RISK FACTORS IN NAFLD (AIM 3). UNDERSTANDING THE MOLECULAR MECHANISMS OF STELLATE CELL ACTIVATION AND FIBROSIS DEVELOPMENT ARE CRITICAL FOR DEVELOPING DIAGNOSTICS AND DESIGNING NEW TREATMENTS TO BLOCK FIBROSIS OR PROMOTE REGRESSION. IN ADDITION, ELUCIDATING THE MECHANISMS OF HOW GENETIC VARIANTS AND RISK FACTORS CONTRIBUTE TO LIVER DISEASE PROGRESSION MAY OFFER THE OPPORTUNITY TO CRAFT TARGETED ANTIFIBROTIC INTERVENTIONS OPTIMIZED FOR PARTICULAR PATIENT SUBGROUPS.

THE ROLE OF MITOCHONDRIAL GLUTATHIONE HOMEOSTASIS IN TUMOR FORMATION - PROJECT SUMMARY CANCER CELLS REQUIRE SUBSTANTIAL ANTIOXIDANT CAPACITY TO OVERCOME TOXIC EFFECTS OF REACTIVE OXYGEN SPECIES (ROS). DESPITE THEIR EXCLUSIVE CYTOSOLIC PRODUCTION, CELLULAR ANTIOXIDANTS ARE ALSO ABUNDANTLY PRESENT IN ORGANELLES, PARTICULARLY IN MITOCHONDRIA. IN PARTICULAR, MITOCHONDRIA, AS THE SOURCE OF REACTIVE OXYGEN SPECIES (ROS), REQUIRE SUBSTANTIAL AVAILABILITY OF ANTIOXIDANTS TO PROTECT ITS CRITICAL REDOX FUNCTIONS. WHILE PREVIOUS WORK SUGGESTS A LINK BETWEEN MITOCHONDRIAL REDOX METABOLISM AND TUMOR GROWTH, MOLECULAR MECHANISMS INVOLVED IN MAINTAINING MITOCHONDRIAL REDOX HOMEOSTASIS AND THE ROLE OF MITOCHONDRIAL ANTIOXIDANTS IN TUMOR PROGRESSION ARE POORLY UNDERSTOOD. AMONG ENDOGENOUS MITOCHONDRIAL ANTIOXIDANTS, GLUTATHIONE (GSH) IS THE DOMINANT SMALL MOLECULE THIOL, EXISTING IN MILLIMOLAR CONCENTRATIONS. GSH IS COMMONLY UPREGULATED IN CANCER CELLS, ENABLING METASTATIC COLONIZATION AND RESISTANCE TO CHEMOTHERAPEUTIC DRUGS. IN OUR PREVIOUS WORK, USING BIOCHEMICAL AND PROTEOMICS METHODS, WE IDENTIFIED SLC25A39, A MITOCHONDRIAL MEMBRANE CARRIER OF UNKNOWN FUNCTION, TO MEDIATE GSH IMPORT INTO MITOCHONDRIA OF CANCER CELLS. SLC25A39 LOSS STRONGLY REDUCES MITOCHONDRIAL GSH LEVELS AND ITS IMPORT, WITHOUT IMPACTING THOSE IN THE CYTOSOL. OUR PRELIMINARY WORK SUGGESTS THAT SLC25A39 EXPRESSION IS ASSOCIATED WITH POOR PROGNOSIS AND DECREASED SURVIVAL OF BREAST CANCER PATIENTS, AND THAT SLC25A39 IS NECESSARY FOR BREAST CANCER INVASION AND METASTASIS. THIS FINDING GAVE US THE OPPORTUNITY, FOR THE FIRST TIME, TO UNCOUPLE MITOCHONDRIAL REDOX METABOLISM FROM THAT OF CYTOSOL DURING TUMOR PROGRESSION. BUILDING UPON THIS EVIDENCE, IN THIS PROPOSAL, WE WILL TEST THE HYPOTHESIS THAT MITOCHONDRIAL GSH IMPORT BY SLC25A39 ENABLES METASTATIC COLONIZATION OF BREAST CANCER CELLS. TO ADDRESS THIS, USING BIOCHEMICAL AND GENETIC EXPERIMENTS, WE WILL FIRST IDENTIFY THE PRECISE MECHANISM BY WHICH MITOCHONDRIAL GSH ENABLES BREAST CANCER CELLS TO METASTASIZE TO LUNG. WE WILL THEN TEST THE ROLE OF MITOCHONDRIAL GSH IMPORT BY SLC25A39 IN TUMOR FORMATION AND METASTASIS USING MOUSE AND HUMAN BREAST CANCER MODELS. FINALLY, SLC25A39 PROTEIN LEVELS SUBSTANTIALLY INCREASE DURING LUNG COLONIZATION, INDICATING A STRONG SELECTIVE PRESSURE TO INDUCE MITOCHONDRIAL GSH UPTAKE DURING METASTASIS. THEREFORE, WE WILL DETERMINE HOW MITOCHONDRIAL GSH AVAILABILITY AND LUNG ENVIRONMENT REGULATE SLC25A39 PROTEIN LEVELS IN BREAST CANCER CELLS. THIS PROPOSAL WILL REVEAL THE ROLE OF MITOCHONDRIAL GSH HOMEOSTASIS IN BREAST CANCER PROGRESSION AND WILL IDENTIFY A COMPARTMENTALIZED SENSING PATHWAY ESSENTIAL FOR METASTATIC COLONIZATION.

BIOLOGICAL ROLES AND MEDIATOR-DEPENDENT TRANSCRIPTION MECHANISMS OF RNA POLYMERASE II(G)

UNRAVELING THE MOLECULAR CONTROL OF A PRO-METASTATIC REGULATORY NETWORK IN MELANOMA

ALTERED COMMUNICATION BETWEEN THE NUCLEUS AND THE MITOCHONDRIA UNDER ONCOGENIC STATES

ZAP ISOFORMS IN INNATE IMMUNITY-THE LONG, THE SHORT AND THE EXTRA-LONG OF IT ALL

MECHANISMS UNDERLYING DIVERSE EFFECTS OF LOW-DOSE EMBRYONIC ETHANOL ON DEVELOPMENT AND FUNCTION OF HYPOCRETIN/OREXIN NEURONS

EXCESS ETHANOL DRINKING AFTER PRENATAL EXPOSURE TO ETHANOL: CHEMOKINE SIGNALING AND OREXIGENIC NEUROPEPTIDES

DISSECTING THE DUAL ROLE OF DOPAMINE IN CONTEXT-DEPENDENT AND LEARNED BEHAVIORS

REGULATION OF METASTATIC PROGRESSION BY AN ENDOTHELIAL-DERIVED FACTOR

REHABILITATION SERVICES - AMERICAN INDIANS WITH DISABILITIES - AMERICAN INDIANS WITH DISABILITIES

MODERNIZATION OF RESEARCH LABORATORY SPACE AT THE ROCKY MOUNTAIN BIOLOGICAL LABORATORY

MOLECULAR PHENOTYPING OF CORTICAL CELL TYPES IN MULTIPLE RODENT MODELS OF ALS

LINKER CELL DEATH REGULATION IN C. ELEGANS - OUR LONG-TERM GOAL IS TO UNDERSTAND THE MOLECULAR BASIS OF A NOVEL MORPHOLOGICALLY-CONSERVED NON-APOPTOTIC DEVELOPMENTAL CELL-DEATH PROGRAM WE UNCOVERED, AND TO DETERMINE ITS ROLES IN MAMMALIAN DEVELOPMENT AND DISEASE. PROGRAMMED CELL DEATH IS A MAJOR CELL FATE. APOPTOSIS, AN EXTENSIVELY STUDIED CELL DEATH PROCESS, REQUIRES CASPASE PROTEASES AND IS ACCOMPANIED BY CHROMATIN COMPACTION AND CYTOPLASMIC SHRINKAGE. SURPRISINGLY, MICE LACKING APOPTOTIC EFFECTORS SURVIVE TO ADULTHOOD. THESE OBSERVATIONS SUGGEST THAT NON- APOPTOTIC CELL DEATH MAY PLAY KEY ROLES IN ANIMAL DEVELOPMENT. ALTHOUGH GENES PROMOTING NECROTIC CELL DEATH HAVE BEEN DESCRIBED, THESE ARE NOT REQUIRED FOR DEVELOPMENT. THUS, WHETHER ALTERNATIVE DEVELOPMENTAL CELL DEATH PATHWAYS EXIST, AND IF SO, WHAT MOLECULAR MECHANISMS GOVERN THEIR EXECUTION, IS A MAJOR OUTSTANDING QUESTION. OUR STUDIES OF THE C. ELEGANS LINKER CELL PROVIDE DIRECT EVIDENCE THAT CASPASE-INDEPENDENT NON- APOPTOTIC CELL DEATH PATHWAYS OPERATE DURING ANIMAL DEVELOPMENT. LINKER CELL DEATH OCCURS IN THE ABSENCE OF C. ELEGANS CASPASES, AND OTHER APOPTOSIS GENES ARE ALSO NOT REQUIRED, NOR ARE GENES IMPLICATED IN AUTOPHAGY OR NECROSIS. THE MORPHOLOGY OF A DYING LINKER CELL IS CHARACTERIZED BY LACK OF CHROMATIN CONDENSATION, A CRENELLATED NUCLEUS, AND SWELLING OF CYTOPLASMIC ORGANELLES. REMARKABLY, CELL DEATH WITH SIMILAR FEATURES (LINKER CELL-TYPE DEATH, LCD) ALSO OCCURS IN VERTEBRATES, AND IS CHARACTERISTIC OF NEURONAL DEGENERATION IN POLYGLUTAMINE DISEASES. WE RECENTLY DESCRIBED A PATHWAY GOVERNING C. ELEGANS LCD. THIS IS THE FIRST SUCH FRAMEWORK FOR A NON-APOPTOTIC DEVELOPMENTAL CELL-DEATH PROGRAM. LCD IS CONTROLLED BY WNT SIGNALS THAT FUNCTION IN PARALLEL WITH A DEVELOPMENTAL-TIMING AND A MAPKK PATHWAY TO CONTROL NON-CANONICAL ACTIVITY OF HSF-1, A CONSERVED HEAT-SHOCK TRANSCRIPTION FACTOR. LET-70/UBE2D2, ENCODING A CONSERVED E2 UBIQUITIN- CONJUGATING ENZYME, IS A KEY TARGET OF HSF-1. THE E3 COMPONENTS CUL-3/CULLIN, RBX-1, BTBD-2, AND EBAX-1 FUNCTION WITH LET-70/UBE2D2 FOR LCD. OUR RECENT EVIDENCE SUGGESTS THAT HISTONE METHYLATION MAY BE A TARGET OF THIS PATHWAY, LIKELY RESULTING IN GENOME-WIDE CHROMATIN OPENING, ALLOWING NUCLEASE ACCESS AND DNA DEGRADATION. LCD PATHWAY COMPONENTS PROMOTE VERTEBRATE CELL-DEGENERATIVE PROCESSES. PQN-41, A GLUTAMINE- RICH PROTEIN, IS REMINISCENT OF POLYQ PROTEINS CAUSING NEURODEGENERATION. AND TIR-1/SARM AND BTBD-2 PROMOTE DISTAL AXON DEGENERATION FOLLOWING AXOTOMY, SUPPORTING CONSERVED CELL DISMANTLING ROLES. WE RECENTLY SHOWED THAT TREATMENT OF MAMMALIAN CELLS WITH THE KINASE INHIBITOR STAUROSPORIN (STS) CAUSES LCD LIKE DEATH. HERE WE WILL BUILD ON THESE STUDIES TO UNCOVER LCD PATHWAY TARGETS, AND STUDY RELEVANCE TO MAMMALS. WE WILL: (1) INVESTIGATE THE ROLE OF SAMS-4, A BTBD-2 TARGET, AND NUC-1, A DNASEII ENZYME, IN LCD, AND TEST AN HYPOTHESIZED PATHWAY FOR THESE IN CHROMATIN MODIFICATION AND DNA DEGRADATION. (2) IDENTIFY EBAX-1 TARGET GENES AND ASSESS ROLES IN LCD CONTROL. (3) CHARACTERIZE STS-INDUCED DEATH IN MAMMALIAN CELLS, DEFINE CONSERVATION WITH C. ELEGANS LCD, AND IDENTIFY RELEVANT GENES.

NEUROPEPTIDE REGULATION OF MOSQUITO HOST-SEEKING BEHAVIOR

MICROPATTERN DIFFERENTIATION AND MORPHOGENESIS OF THE HUMAN ECTODERM

LANDOWNER COLLABORATIVE STRATEGIES FOR NONLETHAL PREDATOR COTROL

ANTI-HIGH MOLECULAR WEIGHT KININOGEN ANTIBODY FOR ALZHEIMER'S DISEASE DIAGNOSIS AND THERAPY

SYNERGISTIC NANOBODIES FOR PANDEMIC PREPAREDNESS - PROJECT SUMMARY BETACORONAVIRUSES (BETA-COVS), INCLUDING SARS-COV-1, MERS-COV, AND SARS-COV-2, HAVE RESHAPED OUR UNDERSTANDING OF PANDEMIC PREPAREDNESS. THESE VIRUSES DEMONSTRATE A REMARKABLE ABILITY TO MUTATE AND EVADE DEFENSES, CONTINUING TO INFECT POPULATIONS WORLDWIDE DESPITE EXTENSIVE VACCINATION EFFORTS AND ANTIVIRAL THERAPIES. THE CHAMELEON-LIKE NATURE OF SARS-COV-2, PARTICULARLY ITS MODIFICATIONS TO THE SPIKE PROTEIN, CONSISTENTLY OUTPACES EXISTING COUNTERMEASURES, NECESSITATING NEW STRATEGIES. THIS PROPOSAL INTRODUCES A PIONEERING CLASS OF NANOBODIES (NBS), ENGINEERED FROM THE IMMUNE SYSTEM OF LLAMAS, DESIGNED TO PROVIDE COMPREHENSIVE PROTECTION AGAINST ALL BETA-COVS. THESE BIOLOGICS NOT ONLY ADVANCE TREATMENT BUT ALSO SIGNIFY A PIVOTAL STEP IN PANDEMIC PREPAREDNESS, EQUIPPING US TO OUTPACE THE RELENTLESS EVOLUTION OF BETA-COVS. OUR INNOVATION LIES IN DEVELOPING MULTIVALENT, SYNERGISTIC COMBINATIONS OF BROAD-SPECTRUM, HIGH-EFFICACY NBS. BY HARNESSING THESE COMBINATIONS, WE AMPLIFY THEIR EFFICACY AND SCOPE, CONCURRENTLY INCREASING THEIR RESISTANCE TO VIRAL MUTATIONS. ADMINISTERED INTRANASALLY OR DIRECTLY TO THE LUNGS, THESE NBS SERVE AS BOTH PROPHYLACTICS AND THERAPEUTIC AGENTS. OUR FIRST AIM IS TO STRATEGICALLY EXPAND UPON OUR PROVEN REPERTOIRES TO IDENTIFY, ISOLATE, AND CHARACTERIZE A MUCH LARGER AND MORE DIVERSE REPERTOIRE OF NBS THAT COLLECTIVELY ARE STRONGLY NEUTRALIZING ACROSS THE BETA-COVS. WE WILL USE CUTTING-EDGE METHODS TO PRODUCE DIVERSE NBS FROM LLAMAS EXPOSED TO SPIKE PROTEINS OF VARIOUS BETA- COVS, SELECTING THOSE WITH HIGH AFFINITY, SPECIFICITY, AND STABILITY. WE AIM TO DISCOVER SYNERGISTIC, ESCAPE-RESISTANT NB PAIRS THROUGH COMBINATION TESTING AND STRUCTURAL ANALYSIS. IN OUR SECOND AIM, WE WILL OPTIMIZE CRITICAL PARAMETERS IMPORTANT FOR DEVELOPING BROADLY NEUTRALIZING NB COMBINATIONS AND DERIVATIVES FOR HUMAN USE. WE WILL EVALUATE THE IN VIVO SYNERGISTIC POTENTIAL OF NBS TARGETING MAJOR THREATS LIKE MERS-COV AND SARS-COV-2, AND ENGINEER NBS TO OPTIMIZE THEIR PROPERTIES AND EFFICACY IN PREPARATION FOR CLINICAL TRIALS. DEPLOYING THESE PRE- PROGRAMMED NBS AT AN OUTBREAK'S ONSET WILL PROTECT FIRST RESPONDERS AND MEDICAL PERSONNEL, REDUCE HOSPITAL SURGES, LIMIT TRANSMISSION, AND BUY TIME FOR NEW VACCINE DEVELOPMENT AND ROLLOUT. THEY WILL ALSO PROVIDE CRUCIAL SUPPORT TO IMMUNOCOMPROMISED INDIVIDUALS, SAFEGUARDING THE MOST VULNERABLE FROM THE START. WE HYPOTHESIZE THAT OUR SYNERGISTIC NB COMBINATIONS WILL INTRODUCE NEW BETA-COV NEUTRALIZATION METHODS, EFFECTIVELY PREVENT AND TREAT INFECTIONS, AND MAINTAIN EFFICACY AGAINST EMERGING BETA-COV THREATS.

SPACER ACQUISITION DURING THE TYPE III-A CRISPR-CAS IMMUNE RESPONSE - PROJECT SUMMARY CRISPR-CAS SYSTEMS PROVIDE BACTERIA AND ARCHAEA WITH ADAPTIVE IMMUNITY AGAINST THEIR VIRUSES (PHAGES). THE HALLMARK OF THE CRISPR-CAS IMMUNE RESPONSE IS THE ACQUISITION OF A “MEMORY” OF INFECTION IN THE FORM OF A SHORT DNA SEQUENCE FROM THE INVADING PHAGE GENOME. THIS SEQUENCE, KNOWN AS “SPACER”, IS INTEGRATED INTO THE CRISPR LOCUS OF THE HOST AND THEN TRANSCRIBED AND PROCESSED INTO A CRISPR RNA (CRRNA) GUIDE. CRISPR-ASSOCIATED (CAS) EFFECTORS USE THE CRRNA TO RECOGNIZE THE NUCLEIC ACIDS OF THE INVADING PHAGE THROUGH BASE-PAIR COMPLEMENTARITY AND TRIGGER DIFFERENT DEFENSE STRATEGIES. FOR TYPE III CRISPR SYSTEMS, COMMONLY PRESENT IN THE HUMAN PATHOGEN STAPHYLOCOCCUS AUREUS, TARGET RECOGNITION LEADS TO THE DORMANCY OF THE INFECTED CELL, AN EVENT THAT PREVENTS VIRAL REPLICATION AND PROPAGATION. HERE, WE PROPOSE TO INVESTIGATE A CENTRAL, YET UNANSWERED, QUESTION ABOUT THIS MECHANISM: IF THERE ARE SPACERS THAT TRIGGER A GROWTH ARREST IN THE HOST, HOW ARE THEY MAINTAINED IN THE BACTERIAL COMMUNITY AFTER THEY ARE ACQUIRED? OUR CENTRAL HYPOTHESIS IS THAT THE DEGRADATION OF THE PHAGE DNA EVENTUALLY ELIMINATES THE VIRAL GENOME FROM THE HOST, ENABLING GROWTH AND THE FIXATION OF THE SPACER IN THE POPULATION. TO INVESTIGATE THIS, WE WILL EXPLORE SEVERAL ASPECTS OF THE CSM6-MEDIATED RESPONSE REQUIRED FOR THE DEFENSE MEDIATED BY TYPE III CRISPR SPACERS THAT MATCH LATE- EXPRESSED VIRAL GENES. FIRST, WE WILL DEFINE WHETHER DORMANT CELLS EVENTUALLY DIE OR ARE ABLE TO EXIT THIS STATE, SURVIVE INFECTION AND CONTINUE GROWING. SECOND, WE WILL DETERMINE WHETHER SPACERS THAT MATCH LATE-EXPRESSED PHAGE GENES CAN PROVIDE A SELECTIVE ADVANTAGE TO THE CELL THAT HARBORS THEM, EVEN WHEN THEY TRIGGER HOST DORMANCY. THIRD, WE WILL DETERMINE IF THESE SPACERS ARE ACTUALLY ACQUIRED DURING INFECTION. IN ALL THESE EXPERIMENTS WE WILL TEST OUR CENTRAL HYPOTHESIS BY USING MUTANT STAPHYLOCOCCI LACKING IN THE EXPRESSION OF SEVERAL NUCLEASES TO DETERMINE IF THEY ARE REQUIRED FOR THE FIXATION OF DORMANCY-TRIGGERING SPACERS. FINALLY, WE WILL USE A TRANSPOSON LIBRARY OF MUTANTS TO INVESTIGATE, IN AN UNBIASED MANNER, THE IMPACT OF HOST GENES THAT COULD BE INVOLVED IN THE EXIT FROM DORMANCY. OUR PROPOSED EXPERIMENTS, AIMED AT UNDERSTANDING HOW SPACERS FROM DORMANCY-INDUCING CRISPR SYSTEMS ARE FIXED IN THE HOST POPULATION, WILL FILL IN A FUNDAMENTAL KNOWLEDGE GAP IN OUR UNDERSTANDING OF THE HALLMARK FEATURE OF CRISPR IMMUNITY: THE GENERATION OF A MEMORY OF INFECTION. IN ADDITION, BY DIRECTLY ADDRESSING A FUNDAMENTAL MECHANISM OF PHAGE DEFENSE OF STAPHYLOCOCCI, OUR PROPOSAL CAN FACILITATE THE SUCCESS OF PHAGE THERAPIES FOR THE TREATMENT OF STAPHYLOCOCCAL DISEASE. IN A MORE INDIRECT MANNER, THE CHARACTERIZATION OF THE MOLECULAR MECHANISMS OF TYPE III CRISPR SYSTEMS CAN LEAD TO AVENUES TO REPURPOSE THESE IMMUNE SYSTEMS FOR GENE EDITING, PARTICULARLY FOR THE DEVELOPMENT OF GENE THERAPIES TO TREAT GENETIC DISEASES.

CCDD-2026 - CHILD CARE AND DEVELOPMENT BLOCK GRANT DISCRETIONARY

GENETICS AND CELL BIOLOGY TRAINING PROGRAM - PROJECT SUMMARY: THIS IS A NEW APPLICATION TO SUPPORT A PRE-DOCTORAL TRAINING PROGRAM IN GENETICS AND CELL BIOLOGY AT THE ROCKEFELLER UNIVERSITY, AN INSTITUTION WITH A RICH HISTORY IN THESE AREAS. THE MISSION OF THE PROGRAM IS TO PROVIDE A DIVERSE POOL OF TRAINEES WITH THE INSTRUCTION, EXPERIENCE, AND CAREER DEVELOPMENT SUPPORT NEEDED FOR SUCCESSFUL CAREERS IN GENETICS AND CELL BIOLOGY. OBJECTIVES OF THE PROGRAM INCLUDE RECRUITING OUTSTANDING GRADUATE TRAINEES FROM DIVERSE BACKGROUNDS TO THE TRAINING PROGRAM AND PROVIDING THEM WITH COMPREHENSIVE DIDACTIC TRAINING, STATE-OF-THE-ART RESEARCH SKILLS, AND PROFESSIONAL SKILLS NEEDED FOR CAREERS IN THE BIOMEDICAL SCIENCE WORKFORCE. INCORPORATED INTO THE TRAINING PLAN ARE RECURRING TRAINING IN RESPONSIBLE CONDUCT OF RESEARCH AND MECHANISMS TO INCREASE RETENTION AND REDUCE TIME-TO-DEGREE. WE PROPOSE TO SUPPORT 12 PRE-DOCTORAL TRAINEES PER YEAR IN THEIR SECOND AND THIRD YEAR OF TRAINING: 6 IN YEAR TWO AND 6 IN YEAR THREE. THE APPLICANT POOL IS OUTSTANDING, INCLUDING MANY STUDENTS WITH ACCOMPLISHED UNDERGRADUATE RECORDS, EXTENSIVE RESEARCH EXPERIENCE AND A STRONG INTEREST IN GENETICS AND CELL BIOLOGY. THE 49 FACULTY TRAINERS ARE ACCOMPLISHED SCIENTISTS, INCLUDING 3 NOBEL LAUREATES AND 19 MEMBERS OF THE US NATIONAL ACADEMY OF SCIENCES, WITH A SHARED INTEREST AND EXPERIENCE IN GRADUATE EDUCATION. THE INTERDISCIPLINARY NATURE OF THE PROGRAM ENCOURAGES TRAINEES TO PERFORM COLLABORATIVE WORK IN VARIOUS AREAS WITH DIFFERENT FACULTY. TRAINEES WOULD BE MENTORED BY THE PROGRAM DIRECTORS; A PROGRAM ADVISORY COMMITTEE OF SELECTED FACULTY FOR GENERAL CURRICULUM AND RESEARCH ADVICE; AND A FACULTY ADVISORY COMMITTEE, SPECIFICALLY DESIGNED FOR EACH TRAINEE TO PROVIDE DETAILED EXPERIMENTAL GUIDANCE. AN EXTERNAL ADVISORY COMMITTEE WILL EVALUATE THE EFFECTIVENESS OF THE PROGRAM AND PROVIDE ADVICE ON NEW INITIATIVES. OUR PAST TRAINEES HAVE BEEN HIGHLY SUCCESSFUL, WITH MOST CONTINUING IN BIOMEDICAL RESEARCH CAREERS. UPON COMPLETION, WE EXPECT TRAINEES TO HAVE DIVERSE SCIENTIFIC KNOWLEDGE, TECHNICAL EXPERTISE, AND PROFESSIONAL DEVELOPMENT SKILLS NECESSARY TO BECOME LEADERS IN THEIR CHOSEN FIELD.

GRADUATE RESEARCH FELLOWSHIP PROGRAM (GRFP) -THE NATIONAL SCIENCE FOUNDATION (NSF) GRADUATE RESEARCH FELLOWSHIP PROGRAM (GRFP) IS A HIGHLY COMPETITIVE, FEDERAL FELLOWSHIP PROGRAM. GRFP HELPS ENSURE THE VITALITY AND DIVERSITY OF THE SCIENTIFIC AND ENGINEERING WORKFORCE OF THE UNITED STATES. THE PROGRAM RECOGNIZES AND SUPPORTS OUTSTANDING GRADUATE STUDENTS WHO ARE PURSUING RESEARCH-BASED MASTER'S AND DOCTORAL DEGREES IN SCIENCE, TECHNOLOGY, ENGINEERING, AND MATHEMATICS (STEM) AND IN STEM EDUCATION. THE GRFP PROVIDES THREE YEARS OF FINANCIAL SUPPORT FOR THE GRADUATE EDUCATION OF INDIVIDUALS WHO HAVE DEMONSTRATED THEIR POTENTIAL FOR SIGNIFICANT RESEARCH ACHIEVEMENTS IN STEM AND STEM EDUCATION. THIS AWARD SUPPORTS THE NSF GRADUATE FELLOWS PURSUING GRADUATE EDUCATION AT THIS GRFP INSTITUTION. 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.

BIOCHEMISTRY OF EUKARYOTIC REPLICATION FORK AND DNA REPAIR - PROJECT SUMMARY DNA REPLICATION IS PERFORMED BY NUMEROUS PROTEINS THAT ACT AS A DYNAMIC MACHINE, TERMED A REPLISOME. THE CORE COMPONENTS OF THE EUKARYOTIC REPLISOME CONSIST OF 1) AN 11 SUBUNIT “CMG” HELICASE THAT SEPARATES THE PARENTAL DNA STRANDS, 2) THE LEADING AND LAGGING STRAND DNA POLYMERASES (POL), POL D AND POL E, RESPECTIVELY,3) THE PCNA SLIDING CLAMP THAT ENCIRCLES DNA AND TETHERS BOTH POLS TO DNA FOR HIGH PROCESSIVITY, 4) THE RFC CLAMP LOADER PENTAMER THAT LOADS PCNA ONTO DNA AND 5) POL A-PRIMASE THAT MAKES A HYBRID RNA- DNA PRIMED SITE FOR THE POLS TO INITIATE DNA SYNTHESIS. IN ADDITION TO THESE “CORE” COMPONENTS, THERE ARE SEVERAL ANCILLARY PROTEINS INCLUDING RPA, TOF1, MEC1, CSM3, FACT, MCM10, CTF4, CTF18-RFC. THERE ARE A HOST OF PROTEINS THAT ASSEMBLE TWO CMGS AROUND DUPLEX DNA AT ORIGINS. THE CMG DIMER UNWINDS THE CLOSED DUPLEX IN AN UNKNOWN REACTION AND SCAFFOLDS ENZYMES TO ASSEMBLE REPLISOMES. WE HAVE PURIFIED THESE PROTEINS IN THE YEAST (SACCHAROMYCES CEREVISIAE; S.C.) SYSTEM. IN THIS PROPOSAL, WE WILL EXTEND OUR STUDIES ON THE STRUCTURE/FUNCTION OF THE EUKARYOTIC REPLISOME. WE WILL USE BIOCHEMICAL AND SINGLE-MOLECULE METHODS TO DETERMINE IF PCNA ACCUMULATES ON THE LAGGING STRAND AS EXPECTED, AND WHETHER PCNA MAY PERIODICALLY BE LEFT ON THE LEADING STRAND FOR MISMATCH REPAIR AND ASSEMBLY OF NAÏVE NUCLEOSOMES. WE HAVE SOLVED NUMEROUS STRUCTURES WITH OUR COLLABORATOR, HUILIN LI (VANANDEL INSTITUTE, MI), AND HAVE MANY MORE STRUCTURES IN PROGRESS AND PLANNED. WE HAVE PURIFIED THE SEVERAL FACTORS OF THE ATR DNA DAMAGE CHECKPOINT SIGNALING SYSTEM OF WHICH MANY REPLISOME PROTEINS ARE TARGETS OF THIS PATHWAY. WE PLAN BIOCHEMICAL STUDIES THAT WILL CLARIFY TARGETS AND THEIR EFFECT ON REPLISOMES. WE WILL DETERMINE THE MECHANISM OF NUCLEOSOME INHERITANCE DURING REPLICATION. IN METAZOANS, EPIGENETIC INHERITANCE OF NUCLEOSOMES, GONE AWRY, CAN LEAD TO CANCER AND OTHER DISEASES. IN YEAST, CELL STUDIES HAVE SHOWN THAT A MCM2 HISTONE BINDING MUTANT PREVENTS EPIGENETIC TRANSFER TO LAGGING STRANDS, AND POL E LACKING THE DPB3/4 SUBUNITS DOES NOT TRANSFER EPIGENETIC MARKS TO THE LEADING STRAND. WE PLAN TO VISUALIZE NUCLEOSOME TRANSFER DURING REPLICATION IN REAL TIME USING SINGLE-MOLECULE STUDIES WITH OUR NEWLY ACQUIRED Q TRAP) IN COLLABORATION WITH DR. SHIXIN LIU (ROCKEFELLER UNIVERSITY)). WE HAVE VARIOUS NUCLEOSOME MOBILITY FACTORS AND YEAST NUCLEOSOMES HAVING DIFFERENT FLUORESCENTLY TAGGED HISTONES FOR THESE STUDIES. REPLICATION OCCURS IN NUCLEAR FOCI, HAVING “REPLICATION FACTORIES” WITH MANY DNA REPLICATION FORKS. OUR RECENT BIOCHEMICAL AND STRUCTURAL STUDIES HAVE DEFINED THE COMPOSITION AND ATOMIC STRUCTURE OF THE MOST BASIC UNIT OF A REPLICATION FACTORY, A DIMERIC REPLISOME. WE WILL EMPLOY SUPER RESOLUTION MICROSCOPY TO VALIDATE IF OUR RECONSTITUTED FACTORY IS THE SAME AS THAT INSIDE CELLS. WE HAVE INSIGHT INTO HOW DUPLEX DNA AT ORIGINS IS OPENED INTO SINGLE STRANDS FROM OUR RECENT FINDING THAT THE TWIN CMG HELICASES ENCIRCLING DUPLEX DNA AT AN ORIGIN ARE DIRECTED INWARD, OPPOSITE THE “OUTWARD” DIRECTION THOUGHT FOR A DECADE. WE FIND THAT TWO INWARD DIRECTED CMG CAN SHEAR DNA APART. WE HAVE PLANS TO FURTHER THIS LINE OF INVESTIGATION.

MECHANOSENSING VIA CYTOSKELETAL STRAIN

MOLECULAR PHENOTYPING OF CORTICAL CELL TYPES IN ALS-RELATED NEURODEGENERATION - THE COMBINED DEGENERATION OF BOTH “LOWER” MOTOR NEURONS IN THE BRAINSTEM AND SPINAL CORD AND “UPPER” MOTOR NEURONS (UMNS) IN THE CEREBRAL CORTEX IS AN IMPORTANT HALLMARK OF ALS. ALMOST ALL CASES OF ALS ARE EVENTUALLY FATAL, AND THE RAPID PROGRESSION OF THE DISEASE MAKES IT PARTICULARLY TERRIBLE, WITH OVER 80% OF PATIENTS DYING WITHIN FIVE YEARS OF DIAGNOSIS. NO CURE EXISTS FOR ALS AND THE ONLY AVAILABLE TREATMENTS SLOW DISEASE PROGRESSION BY MERELY A FEW MONTHS. THEREFORE, A GREAT NEED EXISTS FOR MORE EFFECTIVE AND SPECIFIC THERAPIES THAT CAN STOP OR EVEN REVERSE NEURODEGENERATION. INNOVATION FOR SUCH THERAPIES WILL ONLY ARISE FROM A BETTER UNDERSTANDING OF THE MOLECULAR MECHANISMS UNDERLYING THE PATHOLOGICAL PROCESS, ESPECIALLY SINCE MOST GENES LINKED TO ALS ARE UBIQUITOUSLY EXPRESSED YET ONLY SPECIFIC POPULATIONS OF CELLS DEGENERATE. UNDERSTANDING WHY CERTAIN CELLS ARE UNIQUELY VULNERABLE AND MAPPING CELL TYPE SPECIFIC PATHWAYS THAT ARE DYSREGULATED DURING DISEASE ARE CRUCIAL MILESTONES. THIS HAS POSED A CONSIDERABLE CHALLENGE FOR THE SPINAL CORD PROJECTING UMNS SINCE THEY ARE DIFFICULT TO DISTINGUISH FROM OTHER PYRAMIDAL CELL TYPES AND ARE THEREFORE OFTEN OVERLOOKED IN PRECLINICAL STUDIES. BECAUSE OF THIS, THE BASIS FOR THEIR SELECTIVE VULNERABILITY TO ALS-CAUSING MUTATIONS HAS REMAINED A MYSTERY. THE PROPOSED STUDY AIMS TO OVERCOME THIS BY BUILDING ON RECENT WORK THAT IDENTIFIED TWO HIGHLY SIMILAR YET MOLECULARLY DISTINCT SUBPOPULATIONS OF PROJECTION NEURONS IN LAYER 5B OF MOTOR CORTEX, WHERE UMNS RESIDE. THESE POPULATIONS HAVE OVERLAPPING PROJECTIONS TO PONS, BUT NON-OVERLAPPING PROJECTIONS TO THE SPINAL CORD OR THALAMUS. EXAMINING THESE CELLS IN PRECLINICAL MODELS OF ALS REVEALED THAT THE CORTICOSPINAL PROJECTING NEURONS (CSTNS) WERE VULNERABLE TO DEGENERATION, WHILE THE CORTICOPONTINE-ONLY POPULATION (CPN) DID NOT DEGENERATE. THE SELECTIVE VULNERABILITY OF THE CSTNS WAS LIKELY DUE TO DYSREGULATION OF MITOCHONDRIAL FUNCTION SINCE A DRAMATIC UPREGULATION OF GENES RELATED TO OXIDATIVE PHOSPHORYLATION AND MITOPHAGY WAS OBSERVED AT SYMPTOMATIC STAGES OF DISEASE. AIM 1 OF THIS GRANT WILL EMPLOY AN INTEGRATIVE MULTI-OMICS APPROACH TO ADDRESS WHETHER DIFFERENCES IN THE PROPERTIES OF MITOCHONDRIA BETWEEN CSTNS AND CPNS DRIVE DIFFERENTIAL RESPONSES TO DISEASE USING A NOVEL, VIRAL-BASED STRATEGY TO ISOLATE CELL TYPE SPECIFIC MITOCHONDRIA DURING DISEASE PROGRESSION IN TWO PRECLINICAL ALS MODELS, SOD1G93A AND FUSP525L. AIM 2 FOCUSES ON DISSECTING THE CELLULAR ROLE OF IDENTIFIED CANDIDATE GENES THAT ARE ENRICHED IN CSTNS AND CELL TYPE-SPECIFIC BIOENERGETIC PATHWAYS, LINKING THEM TO MITOCHONDRIAL FUNCTION AND DISEASE VULNERABILITY. TO INCREASE THE TRANSLATIONAL SIGNIFICANCE OF THIS WORK, AIM 3 WILL LEVERAGE NOVEL MARKERS FOR CSTNS AND CPNS FOR A DETAILED ANATOMICAL ANALYSIS OF POSTMORTEM TISSUE FROM ALS PATIENTS TO PERFORM TRANSCRIPTIONAL PROFILING ON CELL TYPE SPECIFIC NUCLEI ISOLATED BY FLUORESCENCE ACTIVATED NUCLEAR SORTING (FANS) FROM POSTMORTEM PATIENT TISSUE. RESULTS FROM THIS STUDY WILL YIELD NOVEL MECHANISMS UNDERLYING SELECTIVE VULNERABILITY OF UMNS IN ALS.

SINGLE VIRIONS TO STUDY ASSEMBLY OF HIV-1

CSC6-2021

INFERRING THE RULES OF ANTIBODY EVOLUTION USING REPLICATED GERMINAL CENTERS - PROJECT SUMMARY GERMINAL CENTERS (GCS) ARE THE SITE OF AFFINITY MATURATION, A PROTOTYPICAL DARWINIAN PROCESS THAT IS REQUIRED TO GENERATE THE POTENT ANTIBODIES THAT PROTECT AGAINST INFECTIOUS DISEASE. OVER THE PAST DECADES, WORK BY SEVERAL GROUPS, INCLUDING OUR OWN, HAS LED TO A COMPREHENSIVE GENERAL UNDERSTANDING OF THE CELLULAR AND MOLECULAR MECHANISMS THAT DRIVE EVOLUTION IN THE GC. HOWEVER, THIS MECHANISTIC UNDERSTANDING HAS YET TO REACH SUFFICIENT GRANULARITY TO ALLOW FOR ACCURATE PREDICTION OF THE OUTCOMES OF GC EVOLUTION AND EFFICIENT GUIDANCE OF GC B CELLS ALONG PREDETERMINED MUTATIONAL TRAJECTORIES. SEVERAL POPULATION-LEVEL PHENOMENA OBSERVED IN GCS REMAIN POORLY UNDERSTOOD, INCLUDING THE APPARENT STOCHASTICITY OF “CLONAL BURSTS,” PROLIFERATIVE SPREES IN WHICH THE ENTIRE GC IS TAKEN OVER BY THE DESCENDANTS OF A SINGLE CELL IN A MATTER OF A FEW DAYS, THE CONTINUOUS PRESENCE OF B CELLS WITH LOW AFFINITIES IN GCS EVEN AT LATE STAGES OF THE REACTION, AND THE APPARENT INABILITY OF GCS TO FIND CERTAIN SOMATIC MUTATIONS DESPITE THEIR BENEFIT IN TERMS OF AFFINITY. GREATER UNDERSTANDING OF THESE APPARENTLY ABERRANT EVOLUTIONARY PATHWAYS MAY IMPROVE OUR ABILITY TO PREDICT AND POTENTIALLY CONTROL THE OUTPUT OF THE GC REACTION. IN THIS APPLICATION, WE DEVELOP A FULL TOOLSET CONSISTING OF A MOUSE MONOCLONAL FOR IG GENES ENCODING AN ANTIBODY TO A CLASSIC PROTEIN ANTIGEN, A DEEP MUTATIONAL SCAN (DMS) EXPERIMENT IN WHICH THE EFFECTS ON AFFINITY OF EVERY POSSIBLE MUTATION IN BOTH CHAINS OF THIS IG ARE MEASURED EXPERIMENTALLY, AND A COMPUTATIONAL FRAMEWORK THAT ALLOWS US TO ASSIGN AFFINITIES TO ANY IG SEQUENCE THAT WE RECOVER FROM THESE MONOCLONAL GC B CELLS. WE WILL USE THESE TOOLS TO CONDUCT REPLICATED EVOLUTION EXPERIMENTS ON HUNDREDS OF GCS, RECONSTRUCTING THE EVOLUTIONARY PATHS AND AFFINITIES OF THOUSANDS OF B CELLS IN THE COURSE OF GC MATURATION. WE PROPOSE TO USE THIS APPROACH TO GAIN INSIGHT INTO HOW B CELL EVOLUTION PLAYS OUT WITHIN GCS IN VIVO, FOCUSING ON THE INTERPLAY BETWEEN REPRODUCIBILITY AND STOCHASTICITY IN EVOLUTION. UNDERSTANDING SUCH EVOLUTIONARY ASPECTS WILL BE IMPORTANT FOR THE FIELD’S EFFORTS TO GUIDE B CELL CLONES THROUGH DEFINED AFFINITY MATURATION TRAJECTORIES THROUGH VACCINATION.

BREACHING THE SPECIES BARRIER: TOWARDS AN IMMUNOCOMPETENT HBV-SUSCEPTIBLE MOUSE MODEL - PROJECT SUMMARY CHRONIC HEPATITIS B VIRUS (HBV) INFECTION REMAINS AN ENDURING GLOBAL PUBLIC HEALTH CHALLENGE, AFFECTING APPROXIMATELY 300 MILLION INDIVIDUALS, DESPITE PROPHYLACTIC VACCINES. THE CURRENT FDA-APPROVED ANTI-HBV DRUGS SUPPRESS VIRAL REPLICATION BUT ARE UNABLE TO ELIMINATE THE VIRAL COVALENTLY CLOSED CIRCULAR DNA (CCCDNA) GENOME WITHIN INFECTED HEPATOCYTES. RESTORING THE IMMUNE RESPONSE HOLDS PROMISE AS A POTENTIAL AVENUE TOWARD A CURE; HOWEVER, T-CELL EXHAUSTION COMMONLY OBSERVED IN CHRONIC HBV-INFECTED PATIENTS IMPEDES EFFECTIVE VIRAL CLEARANCE. REGRETTABLY, PROGRESS TOWARDS CURATIVE TREATMENTS HAS BEEN STYMIED BY THE SCARCITY OF APPROPRIATE IMMUNOCOMPETENT ANIMAL MODELS SUSCEPTIBLE TO HBV. HBV EXHIBITS HIGH SPECIES SPECIFICITY, INFECTING ONLY HUMANS AND CHIMPANZEES. MICE, WIDELY USED FOR MODELING HUMAN DISEASES DUE TO THEIR WELL-CHARACTERIZED IMMUNE SYSTEM, HIGH REPRODUCTIVE CAPABILITY, AND SHORT GESTATION PERIOD, ARE NOT NATURALLY SUSCEPTIBLE TO HBV INFECTION, EVEN AFTER EXPRESSING HUMAN SODIUM TAUROCHOLATE CO-TRANSPORTING POLYPEPTIDE, THE HBV ENTRY RECEPTOR. THE PRIMARY OBSTACLE TO HBV INFECTION IN MURINE HEPATOCYTES IS THE INABILITY TO ESTABLISH CCCDNA, WHICH IS ESSENTIAL FOR HBV INFECTION AND PERSISTENCE. ALTHOUGH HBV CAN INFECT MICE XENOTRANSPLANTED WITH HUMAN HEPATOCYTES, THESE MODELS ARE IMMUNODEFICIENT. HUMANIZED MICE ENGRAFTED WITH BOTH HUMAN HEPATOCYTES AND HUMAN IMMUNE CELLS COULD SURMOUNT THIS DEFICIENCY, BUT THEIR IMMUNE RESPONSE TENDS TO BE WEAK AND CONSTRAINED. IN THIS STUDY, WE AIM TO IDENTIFY THE HOST AND VIRAL DETERMINANTS CAPABLE OF BREACHING THE SPECIES BARRIER FOR HBV INFECTION IN MURINE HEPATOCYTES AND THEREBY DEVELOP HBV-SUSCEPTIBLE MOUSE MODELS. IN AIM 1, WE WILL TAKE A HOST-CENTRIC APPROACH TO DETERMINE IF ALTERATIONS IN THE MURINE HEPATOCYTE CELLULAR ENVIRONMENT CAN LEAD TO HBV CCCDNA FORMATION. SPECIFICALLY, WE WILL TEST WHETHER GENETIC DIVERSITY ACROSS MOUSE STRAINS OR DIVERSITY INDUCED BY FUNCTIONAL GENOMICS APPROACHES RENDERS MURINE HEPATOCYTES MORE PERMISSIVE TO HBV INFECTION AND REPLICATION. IN AIM 2, WE WILL TAKE A VIRUS-CENTRIC APPROACH TO DETERMINE THE ROLE OF NUCLEOCAPSID UNCOATING IN CCCDNA FORMATION WITHIN MURINE HEPATOCYTES. WE WILL ALSO EXPLOIT THE POWER OF VIRAL DIVERSITY TO A DEGREE NEVER ATTEMPTED WITH HBV TO SELECT VIRAL VARIANTS CAPABLE OF FORMING CCCDNA IN MOUSE HEPATOCYTES. THE PROPOSED EXPERIMENTS HAVE HIGH REWARD POTENTIAL. THERE IS AN INHERENT RISK; HOWEVER, THE RISKS ARE MORE THAN JUSTIFIED BECAUSE A SIMPLE MOUSE MODEL WOULD BE ACCESSIBLE TO SCIENTISTS AROUND THE GLOBE AND ACCELERATE RESEARCH. WE DESIGNED MOST EXPERIMENTS SO THAT REGARDLESS OF THE OUTCOME, THE RESULTS WILL PROVIDE VALUABLE NOVEL INSIGHTS INTO HBV HOST TROPISM AND DEVELOP NEW TECHNOLOGIES. WE EXPECT THESE EFFORTS WILL ULTIMATELY LEAD TO THE CREATION OF A FULLY HBV-SUSCEPTIBLE IMMUNOCOMPETENT MOUSE MODEL THAT IS SUITABLE FOR DEVELOPING THERAPEUTIC STRATEGIES, INCLUDING IMMUNE PERTURBATIONS, TO PROMOTE A FUNCTIONAL CURE.

2021 TANF

MOLECULAR AND FUNCTIONAL DISSECTION OF DISTINCT MRNA EXPORT PATHWAYS

STUDYING THE CELLULAR ECOLOGY OF ORGAN FORMATION USING A NOVEL TISSUE RECONSTITUTION SYSTEM - ABSTRACT: OUR ORGANS FUNCTION VIA REPETITIVE MORPHOLOGICAL STRUCTURES LIKE FOLLICLES, VERTEBRA, AND VILLI THAT RIVAL THE ORDERLINESS ACHIEVED BY MODERN MANUFACTURING. IN RECENT DECADES, THE GENERATION OF THESE PERIODIC STRUCTURES HAS BEEN PRIMARILY ASCRIBED TO PRE-EXISTING GENE EXPRESSION PATTERNS. IN THE DEVELOPING SKIN, HOWEVER, RECENT STUDIES SUGGEST THAT THE CONCEPT OF A MOLECULAR BLUEPRINT BE SHED IN ORDER TO CONSIDER MECHANISMS WHERE CELLS SELF-ORGANIZE THROUGH PHYSICAL INTERACTIONS. SELF-ORGANIZATION MECHANISMS ARE ESPECIALLY UNCHARTED IN THE COLLECTIVES OF FIBROBLASTS THAT MAKE UP MESENCHYMAL TISSUES. IN OUR LATEST WORK, WE FIND THAT THE SELF- ORGANIZATION OF FIBROBLASTS EMBEDDED IN EXTRACELLULAR MATRIX (ECM) IS SUFFICIENT TO ROBUSTLY GENERATE THE ORDERED STRUCTURES OF THE SKIN: A GRID OF PRE-FOLLICLE AGGREGATES. THESE RESULTS HIGHLIGHT THE PATTERN-GENERATING POWER OF THE MESENCHYME, WHERE THE FORMATION OF CELL-ECM SUPRA-STRUCTURES MAY PROVE TO BE A BROADLY-USED TOOL TO EFFICIENTLY AND ROBUSTLY INITIATE ORDERED TISSUE STRUCTURES. A CENTRAL GAP THAT REMAINS IS DISSECTING HOW THE BIOPHYSICAL FEATURES OF INDIVIDUAL CELLS IMPACT THE DYNAMICS OF CELL-CELL COORDINATION TO ENABLE THE STRUCTURING OF ORGANS. IN OUR PROPOSED STUDIES OF SUCH CELLULAR ECOLOGY, WE AIM TO UNDERSTAND HOW CELLS CONVERT ENERGY INJECTED AT THE MOLECULAR SCALE TO COUPLE MOTION, ORGANIZE FORCE, AND COMMUNICATE DURING TISSUE MORPHOGENESIS. THIS INQUIRY IS MADE POSSIBLE BY A NOVEL COLLECTIVE CELL BEHAVIORAL PLATFORM THAT SUCCESSFULLY CAPTURES THE SELF-ORGANIZING PROCESS THAT SKIN PROGENITORS UNDERGO AS THEY COALESCE INTO AN ORDERED AND STRUCTURALLY LINKED TISSUE. WE WILL INVESTIGATE HOW BIOPHYSICAL FEATURES IMPACT SELF-ORGANIZATION AND THE CELL-CELL LINKAGES THAT EMERGE AS A RESULT. BASED ON OUR RECENT FINDINGS, WE PROPOSE TO INVESTIGATE BIOELECTRICAL SIGNALING TO DETERMINE WHETHER CALCIUM OSCILLATORY BEHAVIOR CAN SERVE AS A MEANS TO MAKE MECHANICAL COUPLING OF CELLS MORE ROBUST. WE WILL ALSO PROBE THE ENERGETIC FLOWS OCCURRING ACROSS THE CELL COLLECTIVE AS THEY SELF-ORGANIZE IN ORDER TO DISCOVER WHICH METABOLIC PATHWAYS SERVE TO GUIDE THE ENERGY FLOWS REQUIRED FOR CELLS TO EXPRESS THEIR MECHANICAL BEHAVIOR. UNDERSTANDING HOW PHYSICAL ENTITIES SUCH AS MECHANICS, ELECTRICITY, AND ENERGY ARE CO-REGULATED DURING MESENCHYMAL TISSUE SELF-ORGANIZATION FORMATION WILL OFFER NEW PATHWAYS FOR TISSUE DESIGN AND RECONSTITUTION AS WELL AS PRESENT NEW AVENUES FOR DRUG DEVELOPMENT.

MECHANISTIC STUDIES OF TRANSCRIPTION INITIATION AND ELONGATION FUNCTIONS OF AN RNA POLYMERASE II VARIANT, POL II(G), THAT IS IMPLICATED IN DEVELOPMENT AND CANCER - EUKARYOTIC RNA POLYMERASE II (POL II) PLAYS A PIVOTAL ROLE IN TRANSCRIPTION. NORMAL PHYSIOLOGICAL PROCESSES DEPEND UPON PRECISE TRANSCRIPTIONAL CONTROLS, WHEREAS TRANSCRIPTIONAL DYSREGULATION IS THE BASIS OF NUMEROUS PATHOLOGIES THAT INCLUDE CANCER. POL II RECRUITMENT TO SPECIFIC PROMOTERS IS REGULATED BY MULTIPLE COFACTORS THAT INCLUDE THE MULTI-SUBUNIT MEDIATOR, WHICH DIRECTLY BINDS BOTH TO ENHANCER/PROMOTER-BOUND TRANSCRIPTIONAL ACTIVATORS AND TO POL II TO FACILITATE GENE ACTIVATION. FOLLOWING INITIATION AND PROMOTER ESCAPE, POL II REMAINS SUBJECT TO REGULATION BY MULTIPLE ELONGATION FACTORS, ACTING EITHER AT POL II PAUSE-RELEASE OR PRODUCTIVE ELONGATION STEPS. POL II(G) IS A RECENTLY DESCRIBED FORM OF POL II THAT CONTAINS THE TIGHTLY ASSOCIATED, METAZOAN-SPECIFIC GDOWN1 POLYPEPTIDE ALONG WITH THE NORMAL 12 SUBUNITS. OUR GENETIC- BASED STUDIES OF POL II(G) HAVE DEMONSTRATED THAT GDOWN1 IS ESSENTIAL FOR EARLY EMBRYONIC DEVELOPMENT AND FOR CELL-SPECIFIC TRANSCRIPTION IN QUIESCENT HEPATOCYTES, IN WHICH HEAVY LOCALIZATION TO GENE BODIES OF HIGHLY EXPRESSED LIVER-SPECIFIC GENES (E.G., ALBUMIN) IS INDICATIVE OF ELONGATION FUNCTIONS AND IN WHICH ABLATION LEADS TO DOWNREGULATION OF BOTH LIVER-SPECIFIC AND LIPID METABOLISM GENES, CELL CYCLE RE-ENTRY AND (IN THE ABSENCE OF P53) A PREMALIGNANT TYPE OF TRANSFORMATION. STUDIES IN HEPATOCARCINOMA AND BREAST CANCER CELLS HAVE ALSO INDICATED A KEY ROLE FOR GDOWN1 IN CELL GROWTH AND IN EXPRESSION OF LIPID METABOLISM GENES, WHICH ARE GENERALLY IMPORTANT FOR MAINTENANCE OF CANCER CELL GROWTH. OUR BIOCHEMICAL STUDIES HAVE REVEALED THAT THE POL II-ASSOCIATED GDOWN1 CONDITIONALLY REPRESSES BASAL (ACTIVATOR- AND MEDIATOR-INDEPENDENT) TRANSCRIPTION INITIATION BY PREVENTING ASSOCIATION OF TFIIB AND TFIIF WITH POL II, THEREBY ESTABLISHING A POTENTIAL CHECKPOINT AND ELICITING A STRONG REQUIREMENT FOR ACTIVATOR-BOUND MEDIATOR TO OVERCOME REPRESSION. OUR STRUCTURAL STUDIES HAVE DEFINED GDOWN1 INTERACTION SITES ON POL II AND PROVIDED CLUES REGARDING MEDIATOR INTERACTIONS THAT MIGHT FACILITATE ITS REVERSAL OF THE CONDITIONALLY REPRESSED INITIATION CAPACITY OF POL II(G), ALTHOUGH THE UNDERLYING MECHANISM REMAINS UNCLEAR. WITH THE GENERAL OBJECTIVE OF UNDERSTANDING THE MOLECULAR MECHANISMS OF ACTION OF POL II(G) IN CONJUNCTION WITH ITS ROLES IN BREAST CANCER AND HEPATOCARCINOMA CELLS, ESPECIALLY ON GDOWN1-REGULATED CELL-SPECIFIC AND LIPID METABOLISM GENES, AS A POTENTIAL BASIS FOR NEW CANCER THERAPEUTICS, OUR SPECIFIC AIMS ARE: (I) TO INVESTIGATE THE MECHANISMS UNDERLYING MEDIATOR-DEPENDENT TRANSCRIPTION INITIATION AND POST-INITIATION EVENTS BY POL II(G), INCLUDING CONCOMITANT, NEWLY DESCRIBED INTERACTIONS WITH GENERAL TRANSCRIPTION FACTORS AND ELONGATION FACTOR TFIIS, USING POWERFUL IN VITRO TRANSCRIPTION AND IMMOBILIZED TEMPLATE ASSAYS AND CX-MS AND CRYO-EM STRUCTURAL ANALYSES OF INTERACTING COMPLEXES AND (II) TO INVESTIGATE GDOWN1 FUNCTIONS IN HEPATOCARCINOMA CELLS IN PROMOTER-PROXIMAL PAUSING, PAUSE RELEASE AND TRANSCRIPTIONAL PROCESSIVITY USING (A) A MULTIOMICS CELL-BASED APPROACH IN CONJUNCTION WITH ACUTE DEGRADATION OF GDOWN1 AND (B) BIOCHEMICAL (IN VITRO RECONSTITUTION OF THESE PROCESSES WITH PURIFIED FACTORS AND RECOMBINANT CHROMATIN TEMPLATES) AND STRUCTURAL (CX-MS AND CRYO-EM) ANALYSES OF POL II(G) ELONGATION FACTOR COMPLEXES.

STRUCTURAL MECHANISMS OF CYTOSKELETAL FORCE-SENSING - PROJECT SUMMARY CELLS IN THE BODY PERCEIVE CUES FROM THEIR LOCAL ENVIRONMENT, WHICH CONTROL CELLULAR BEHAVIOR THROUGH A COORDINATED SERIES OF MOLECULAR EVENTS KNOWN AS SIGNALING. SIGNALING IS CRITICALLY IMPORTANT FOR TELLING A CELL IF IT SHOULD GROW AND DIVIDE, MIGRATE TO A DIFFERENT PART OF THE BODY, OR COMMIT SUICIDE IF IT HAS COMPLETED ITS FUNCTION OR BEEN IRREPARABLY DAMAGED. FREQUENTLY, SIGNALING PROCESSES ARE FOUND TO BE WORKING INCORRECTLY IN DISEASED CELLS. FOR INSTANCE, CANCER CELLS DIVIDE AND MIGRATE OUT OF CONTROL AND IGNORE CUES WHICH SHOULD KEEP THEM IN CHECK. SIGNALS COME IN MULTIPLE FORMS. SPECIFIC MOLECULES BIND AND ACTIVATE COGNATE RECEPTOR PROTEINS IN THE CELL, KNOWN AS “CHEMICAL SIGNALING”, WHICH IS BROADLY WELL-UNDERSTOOD. PHYSICAL FORCES AND THE RIGIDITY OF A CELL’S ENVIRONMENT ALSO ELICIT SPECIFIC CELL BEHAVIORS, BUT WE HAVE A COMPARATIVELY POOR UNDERSTANDING OF HOW PROTEINS TRANSMIT THESE “MECHANICAL SIGNALS”. A SIGNIFICANT FRACTION OF SUCCESSFUL DRUGS TARGET PROTEIN MOLECULES WHICH OPERATE IN CHEMICAL SIGNALING. THE DEVELOPMENT OF MANY SUCH TREATMENTS WAS STIMULATED BY DETERMINING THE DETAILED THREE-DIMENSIONAL CHEMICAL STRUCTURES OF THE INTERACTIONS BETWEEN RECEPTOR PROTEINS AND THE MOLECULES WHICH ACTIVATE THEM, FACILITATING THE DESIGN OF DRUGS WHICH PRECISELY INTERVENE IN THESE PROCESSES. DESPITE ITS IMPORTANCE, EFFORTS TO THERAPEUTICALLY TARGET MECHANICAL SIGNALING HAVE BEEN LIMITED. THE LONG-TERM GOAL OF THIS RESEARCH PROJECT IS TO VISUALIZE HOW FORCES MODULATE THE THREE-DIMENSIONAL STRUCTURE OF MECHANICAL SIGNALING PROTEINS TO ACTIVATE THEM, IN ORDER TO FACILITATE THE DEVELOPMENT OF DRUGS THAT BLOCK THESE CHANGES. THIS PROPOSAL IS SPECIFICALLY FOCUSED ON UNDERSTANDING HOW CELLULAR POLYMERS (“FILAMENTS”) COMPOSED OF THE PROTEIN ACTIN COORDINATE MECHANICAL SIGNALING. THE CELL CONTAINS MANY NETWORKS COMPOSED OF ACTIN FILAMENTS, MYOSIN MOLECULAR MOTOR PROTEINS, AND HUNDREDS OF OTHER BINDING PARTNERS, WHICH COLLECTIVELY GENERATE AND TRANSMIT DIVERSE FORCES. WE HYPOTHESIZE THAT SPECIFIC TYPES OF FORCES CAUSE DISTINCT PHYSICAL REARRANGEMENTS IN ACTIN FILAMENTS, WHICH CAN BE DETECTED BY OTHER PROTEINS IN THE CELL THROUGH DIRECT BINDING INTERACTIONS. WE WILL IDENTIFY PROTEINS WHICH BIND ACTIN IN A FORCE-SENSITIVE MANNER (AIM 1), FOCUSING SPECIFICALLY ON DELINEATING THE PRECISE REGIONS OF THE PROTEINS WHICH CONFER FORCE-SENSITIVITY. WE WILL NEXT VISUALIZE HOW SIDE-WISE BENDING FORCES (AIM 2) AND LENGTH-WISE TENSILE AND COMPRESSIVE FORCES GENERATED BY MYOSIN MOTOR PROTEINS (AIM 3) IMPACT ACTIN FILAMENT STRUCTURE, HYPOTHESIZING THESE FORCE REGIMES PRODUCE DISTINCT REARRANGEMENTS WHICH CAN BE DISCRIMINATED BY BINDING PARTNERS. IN PURSUIT OF THESE AIMS, WE ARE DEVELOPING SAMPLE PREPARATION AND COMPUTATIONAL IMAGE ANALYSIS APPROACHES TO VISUALIZE THE THREE-DIMENSIONAL STRUCTURE OF ACTIN POLYMERS IN THE PRESENCE OF MECHANICAL FORCES WITH CRYO-ELECTRON MICROSCOPY (CRYO-EM). IN ADDITION TO PROVIDING BASIC INSIGHTS INTO HOW FORCES ARE PERCEIVED BY CELLS THROUGH CHANGES IN PROTEIN STRUCTURE, OUR STUDIES WILL GUIDE THE DEVELOPMENT OF PRECISE MOLECULAR INTERVENTIONS INTO MECHANICAL SIGNALING PROCESSES GOVERNED BY ACTIN.