Rockefeller University

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

TRANSFORMING CLINICAL AND TRANSLATIONAL RESEARCH AND EDUCATION AT ROCKEFELLER

TRANSFORMING TRANSLATIONAL SCIENCE AND EDUCATION TO BENEFIT HUMAN HEALTH

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

INTEGRIN ALPHAIIBBETA3 STRUCTURE, ACTIVATION, AND LIGAND BINDING

TREATMENT OF ADDICTIONS-BIOLOGICAL CORRELATES

DRUGS OF ABUSE -- ROLE OF PROTEIN PHOSPHORYLATION

MID-SCALE RI-1 (M1: DA): DESIGNING SCALABLE COMMUNITY-DRIVEN INFRASTRUCTURE TO SUPPORT BIODIVERSITY GENOMICS (SCIBG). -THE FULL INVENTORY OF LIVING BEINGS ON EARTH, UNDERPINS HUMAN HEALTH, ECONOMIC PROSPERITY, ENVIRONMENTAL RESILIENCE, AND NATIONAL SECURITY. THE GENETIC DIFFERENCES FOUND ACROSS PLANTS, ANIMALS, FUNGI, AND OTHER ORGANISMS DRIVES DISCOVERIES THAT ADVANCE BIOTECHNOLOGY, AGRICULTURE, FOOD SECURITY, BIOSECURITY, AND THE DEVELOPMENT OF FUTURE MEDICINES AND THERAPEUTICS, WHILE SUPPORTING THE RAPIDLY GROWING U.S. BIOECONOMY. WHILE OTHER NATIONS AND INTERNATIONAL INITIATIVES HAVE INVESTED IN COORDINATED BIODIVERSITY GENOMICS EFFORTS, THE UNITED STATES CURRENTLY LACKS A COORDINATED, NATIONAL INFRASTRUCTURE FOR PRODUCING HIGH-QUALITY REFERENCE GENOMES AT THE SCALE NEEDED TO MEET GROWING SCIENTIFIC DEMAND AND REMAIN GLOBALLY COMPETITIVE IN GENOMICS. THIS PROJECT WILL DESIGN THE SCALABLE COMMUNITY INFRASTRUCTURE FOR BIODIVERSITY GENOMICS (SCIBG), ESTABLISHING THE FOUNDATION FOR A SHARED NATIONAL RESOURCE THAT EXPANDS ACCESS TO ADVANCED GENOMICS CAPABILITIES, STRENGTHENS THE NATION'S RESEARCH INFRASTRUCTURE, SUPPORTS WORKFORCE DEVELOPMENT, AND ACCELERATES DISCOVERIES THAT ADVANCE SCIENCE WHILE ENHANCING ECONOMIC COMPETITIVENESS, PUBLIC HEALTH, BIOSECURITY, AND ECOSYSTEM RESILIENCE. THIS AWARD ADDRESSES NSF PRIORITIES IN ARTIFICIAL INTELLIGENCE AND BIOTECHNOLOGY. SCIBG WILL DESIGN AN INTEGRATED, END-TO-END INFRASTRUCTURE FOR GENERATING HIGH-QUALITY, REFERENCE GENOME RESOURCES REPRESENTING U.S. BIODIVERSITY. THE PROJECT WILL DEVELOP SCALABLE LABORATORY PROTOCOLS, INTEROPERABLE COMPUTATIONAL WORKFLOWS, AUTOMATED DATA MANAGEMENT SYSTEMS, COMMUNITY GOVERNANCE MODELS, AND MECHANISMS FOR BROAD RESEARCHER ACCESS THROUGH A COMMUNITY ACCESS SEQUENCING PROGRAM. IT WILL ALSO DEVELOP ADVANCED COMPUTATIONAL METHODS, INCLUDING ARTIFICIAL INTELLIGENCE TOOLS, TO AUTOMATE GENOME ASSEMBLY AND CURATION, OVERCOMING MAJOR BOTTLENECKS THAT CURRENTLY LIMIT THE SPEED, SCALABILITY, AND ACCURACY OF BIODIVERSITY GENOMICS. THE RESULTING DESIGN AND PROTOTYPE WILL PAVE THE WAY FOR FUTURE IMPLEMENTATION OF A SUSTAINABLE, COMMUNITY-DRIVEN NATIONAL PLATFORM THAT SUPPORTS THE EARTH BIOGENOME PROJECT, ENABLES THE FUTURE PRODUCTION OF THOUSANDS OF HIGH-QUALITY REFERENCE GENOMES ANNUALLY, AND PROVIDES OPENLY AVAILABLE SOFTWARE, STANDARDS, TRAINING RESOURCES, AND GENOMIC DATA FOR THE BROADER SCIENTIFIC COMMUNITY. 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 PLANNED FOR THIS AWARD.

REGULATION OF EPIDERMAL DEVELOPMENT AND DIFFERENTIATION

MOLECULAR MECHANISMS OF ANTIBODY-MEDIATED IMMUNOTHERAPIES

NEW TOOLS FOR EXPLORING THE DYNAMIC INTERACTOME

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

GENOME INSTABILITY IN CANCER: TELOMERES AND DNA REPAIR

NOVEL APPROACHES FOR VACCINE DESIGN

CELL ADHESION AND CYTOSKELETAL DYNAMICS IN SKIN

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.

NATIONAL RESOURCE FOR MASS SPECTROM OF BIOLOGICAL MACROMOLECULES

IDENTIFICATION OF CELL TYPE-SPECIFIC ACTIONS OF ANTIPSYCHOTIC DRUGS

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

MECHANISM OF THE TELOMERIC PROLIFERATION LIMIT

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.

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.

STRUCTURE-FUNCTION MAPPING OF THE NUCLEAR PORE COMPLEX

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

THE MOLECULAR BASIS OF SELECTIVE DOPAMINERGIC CELL LOSS IN PARKINSON'S DISEASE AND PD RELATED DEPRESSION

INSTITUTIONAL CAREER DEVELOPMENT CORE

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

EPITOPE-FOCUSED VACCINE STRATEGIES AGAINST ZIKA VIRUS

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

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.

INNATE FUNCTIONS OF DENDRITIC CELLS IN AUTOREACTIVITY

POTASSIUM CHANNEL GATING

DEFINITION OF SERUM RIBONUCLEOPROTEIN COMPOSITION AND ITS REGULATION AND FUNCTION

SKIN STEM CELLS: PURIFICATION AND CHARACTERIZATION

INTESTINAL REGULATION OF THPOK EXPRESSION AND CD4 HELPER T CELL FUNCTION

REGULATION OF MITOTIC CHROMOSOMES - REVISION - 2

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.

IDENTIFICATION OF CELL-TYPE SPECIFIC TARGETS FOR THE TREATMENT OF PARKINSON'S DISEASE AND PD-RELATED DEPRESSION

ENZYMOLOGY AND FUNCTION(S) OF HISTONE PHOSPHORYLATION

MOLECULAR DEFINITION OF BRAIN CIRCUITS CONTROLLING ADDICTION

SIGNALING TRANSDUCTION AND ALZHEIMER'S DISEASE

NEW TOOLS FOR EXPLORING THE DYNAMIC INTERACTOME (RENEWAL)

DECIPHERING THE MECHANISMS OF DOPAMINERGIC NEURODEGENERATION IN PARKINSON'S DISEASE USING INDUCED PLURIPOTENT STEM CELLS

ROLE OF MGLUR5/NORBIN IN DYSKINESIA ASSOCIATED WITH PARKINSON'S DISEASE

NATL RESOURCE FOR MASS SPECTROM OF BIOLOG MACROMOLECULES

CHEMICAL BIOLOGY OF CELL DIVISION

GENERATION OF IMMUNOLOGICAL MEMORY BY CRISPR-CAS SYSTEMS

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

A NOVEL P11 PATHWAY AS A THERAPEUTIC TARGET FOR PARKINSON'S DISEASE

GENETICS AND CELL BIOLOGY

ROLE OF FIBRINOGEN IN ALZHEIMER'S DISEASE

DYNAMICS OF ANTIGEN-DRIVEN SELECTION IN GERMINAL CENTERS

DETERMINANTS OF INFECTIOUS HIV-1 PARTICLE PRODUCTION

MODELING HUMAN HEPATOTROPIC INFECTIONS IN COMPLEX TISSUE ORGANOIDS

INTESTINAL SURVEILLANCE BY INTRAEPITHELIAL LYMPHOCYTES

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

MAPPING THE MECHANISMS OF PROTEIN SYNTHESIS-DEPENDENT SYNAPTIC PLASTICITY

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

ROLE OF THE CONTACT SYSTEM IN ALZHEIMER'S DISEASE

SUBSYSTEM PROPOSAL FOR PRE-SHOWER & SHOWER-MAXIMUM DETECTORS

PROPERTIES OF MOUSE DENDRITIC CELLS AND MACROPHAGES

MOTOR PROTEIN DYNAMICS AND MITOTIC MECHANISMS

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.

DISCOVERY AND MECHANISM OF ANTIRETROVIRAL FACTORS

USING EVOLUTIONARY VARIATION TO PROBE THE NEURAL BASIS FOR BEHAVIOR

B CELL CLONAL SELECTION IN GUT-ASSOCIATED GERMINAL CENTERS

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.

DISCOVERY OF ANTIBIOTICS FROM SOIL MICROBIOMES USING METAGENOMICS

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

CELL ADHESION AND CYTOSKELETAL DYNAMICS IN SKIN

GENOME-WIDE DISSECTION OF MENDELIAN SUSCEPTIBILITY TO MYCOBACTERIAL DISEASE

TRANSLATIONAL AND EPIGENETIC PROFILING OF CELL TYPES ASSOCIATED WITH ADDICTION

MECHANISMS OF ANTIBODY-DEPENDENT ENHANCEMENT OF DENGUE DISEASE

MOLECULAR MECHANISMS UNDERLYING INSECT ODORANT RECEPTOR FUNCTION AND MODULATION

TRANSCRIPTIONAL REGULATION BY SMARCA3 IN CHOLECYSTOKININ-EXPRESSING NEURONS

SIGNALING MECHANISMS UNDERLYING NEUROLEPTIC DRUG ACTIONS

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

POSTTRANSCRIPTIONAL REGULATION BY MRNA-BINDING SHUTTLING AND TRANSPORT PROTEINS

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

IDENTIFICATION AND CHARACTERIZATION OF CELLULAR FACTORS INVOLVED IN HCV ENTRY

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

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

ROLE OF THE TPA/PLASMIN SYSTEM IN ALZHEIMERS DISEASE

THERAPEUTIC TARGETING OF BREAST CANCER WITH METASTASIS SUPPRESSOR MICRORNAS

INTERDISCIPLINARY STUDIES OF SLEEP AND CIRCADIAN RHYTHMS

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.

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.

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

EVOLUTION AND ACQUISTION OF DRUG RESISTANCE IN MRSA

NUCLEAR RECEPTOR COACTIVATOR FUNCTIONS AND MECHANISMS

NEURO-IMMUNE INTERACTIONS AT THE INTESTINAL SURFACE

BACTERIAL RNAP SIGMA FACTOR STRUCTURE AND FUNCTION

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.