LONG-DURATION SPACE FLIGHT IMPOSES NUMEROUS PHYSIOLOGICAL CHALLENGES TO THE HUMAN BODY SUCH AS BONE LOSS DYSREGULATION OF THE IMMUNE SYSTEM AND RADIATION EXPOSURE. ALTHOUGH CONSIDERABLE PROGRESS HAS BEEN MADE IN DELINEATING THESE BIOLOGICAL PHENOMENA ONE AREA OF RESEARCH THAT HAS RECEIVED LITTLE ATTENTION IS THE IMPACT THAT SPACE FLIGHT HAS ON THOSE COMMENSAL NON-PATHOGENIC MICROBES THAT ASSOCIATE WITH HUMAN TISSUES. TO ADDRESS THIS GAP IN KNOWLEDGE WE PROPOSE TO EXAMINE HOW MICROGRAVITY ALTERS NORMAL BACTERIAINDUCED DEVELOPMENT IN ANIMAL TISSUES. SPECIFICALLY WE PROPOSE TO ELUCIDATE THE PATHWAYS ASSOCIATED WITH BACTERIA-INDUCED APOPTOSIS A TYPE OF PROGRAMMED CELL DEATH THAT IS CRITICAL IN MAINTAINING TISSUE HOMEOSTASIS AND THE IMMUNE RESPONSE OF ANIMALS. TO ADDRESS THIS GOAL THE MONOSPECIFIC SYMBIOSIS BETWEEN THE BOBTAIL SQUID EUPRYMNA SCOLOPES AND ITS LUMINESCENT PARTNER VIBRIO FISCHERI WILL BE USED AS A MODEL SYSTEM TO EXAMINE THE EFFECTS OF MODELED MICROGRAVITY (I.E. HARV VESSELS) ON BACTERIA-INDUCED APOPTOSIS IN THE HOST ANIMAL. THE CENTRAL HYPOTHESIS OF THIS PROJECT IS THAT MICROGRAVITY ALTERS THE EXTRINSIC AND INTRINSIC SIGNALING PATHWAYS ASSOCIATED WITH BACTERIAINDUCED APOPTOSIS IN THE HOST ANIMAL. OUR PREVIOUS STUDIES HAVE SHOWN THAT MODELED MICROGRAVITY CAUSES AN ACCELERATION OF BACTERIAINDUCED APOPTOSIS IN THE HOST HOWEVER THE MECHANISMS UNDERLYING THE DEVELOPMENTAL ACCELERATION IS UNKNOWN. TO ADDRESS THIS ISSUE WE PROPOSE THE FOLLOWING SPECIFIC OBJECTIVES: OBJECTIVE 1. DELINEATE THE IMPACT OF MODELED MICROGRAVITY ON THE SHEDDING OF APOPTOSIS-INDUCING LIPOPOLYSACCHARIDE BY THE BACTERIAL SYMBIONT. WE HYPOTHESIZE THAT THE ACCELERATION OF CELL DEATH IN THE HOST TISSUES IS DUE TO THE INCREASED SHEDDING OF BACTERIAL LIPOPOLYSACCHARIDE (LPS) THEREBY TRIGGERING AN UPREGULATION OF GENES ASSOCIATED WITH THE EXTRINSIC APOPTOSIS PATHWAY. TO ADDRESS THIS OBJECTIVE WE WILL USE A THREE-PRONGED APPROACH TO EXAMINE THE CONTROLS ON LPS-SHEDDING AND IDENTIFY THE HOST MOLECULAR RESPONSE TO THE SHEDDING USING NANOSTRING WESTERN BLOTTING AND IMMUNOCYTOCHEMISTRY UNDER MODELED MICROGRAVITY CONDITIONS. OBJECTIVE 2. ELUCIDATE CHANGES IN ACTIVATION AND MEDIATION OF THE EXTRINSIC AND INTRINSIC APOPTOSIS PATHWAYS ASSOCIATED WITH BACTERIAINDUCED APOPTOSIS IN MICROGRAVITY. BY DATA MINING CURRENTLY AVAILABLE TRANSCRIPTOMES WE WILL MONITOR THE DIFFERENTIAL EXPRESSION OF POTENTIAL TARGET LIGANDS RECEPTORS ADAPTOR PROTEINS AND INHIBITORS OF APOPTOSIS. OBJECTIVE 3. EXAMINE THE ONSET OF CASPASE ACTIVITY DURING ACCELERATED APOPTOSIS IN HOST LIGHT ORGAN UNDER SIMULATED MICROGRAVITY CONDITIONS AND ATTEMPT TO MODULATE ITS ONSET. WE WILL EXAMINE THE DIFFERENTIAL EXPRESSION OF CASPASES WITHIN THE LIGHT ORGAN UNDER MODELED MICROGRAVITY CONDITIONS AND ANTICIPATE THAT THE CASPASES ARE HIGHLY INTEGRATED WITH THE INNATE IMMUNE RESPONSE OF THE HOST. ADDITIONALLY WE WILL ATTEMPT TO MODULATE THE PROTEASE ACTIVITY USING A RANGE OF INHIBITORS TO POTENTIALLY MITIGATE THE MICROGRAVITYINDUCED EFFECTS ON BACTERIA-INDUCED APOPTOSIS IN THE HOST TISSUES. SIGNIFICANCE OF PROJECT - THE PROPOSED WORK HAS SEVERAL IMPLICATIONS FOR THE FIELD OF SPACE BIOLOGY AND DIRECTLY RELATES TO NASA SPACE BIOLOGY SCIENCE PLAN AND DECADAL SURVEY SPECIFICALLY THE MICROBIOLOGY (MB) AND CELL MOLECULAR BIOLOGY (CMB) PROGRAM ELEMENTS. FIRST BY EXAMINING HOW THE NORMAL BACTERIA-INDUCED DEVELOPMENT OF ANIMALS IS MEDIATED UNDER MICROGRAVITY CONDITIONS WE CAN BEGIN TO DETERMINE THE SENSITIVITY OF THESE ASSOCIATIONS TO PERTURBATIONS IN THE SPACE ENVIRONMENT. SECONDLY BY REMOVING GRAVITY AS A CONSTANT WE CAN ADDRESS THE QUESTION OF WHETHER GRAVITY OBSCURES ASPECTS OF BACTERIA-INDUCED ANIMAL DEVELOPMENT THAT MAY OTHERWISE GO UNDETECTED UNDER TERRESTRIAL CONDITIONS.