The postulated role of exocytosis in mediating various functional response associated with neutrophil activation has not been directly tested. We will use a novel approach; generating TAT-fusion proteins containing the coiled-coil domains of different SNARE proteins; to test the hypothesis that blocking exocytosis of distinct granule subsets will prevent specific neutrophil phenotypic changes and this strategy may be used to prevent neutrophil-mediated tissue damage in vivo. The two years of mentored phase of this ^ward allowed the applicant to generate TAT-SNAP-23 and TAT-Syntaxin-4 fusion proteins and test the ability of these fusion proteins to block neutrophil granule exocytosis both in vitro and in vivo. Additionally, the state-of-the-art techniques acquired during the mentored phase of the award allowed the applicant to increase her expertise in molecular biology techniques, animal models of neutrophil-dependent inflammation, and perform proteomic analysis; which will allow the PI to become a successful independent investigator. For the independent phase the specific objectives are: 1) To determine the specific functional responses that are regulated by neutrophil granule exocytosis; 2) To define the mechanisms by which inhibition of neutrophil exocytosis in vivo blocks the inflammatory response; and 3) To investigate the molecular mechanisms by which exocytosis is stimulated. The training the applicant received through out her K99 phase make her unique in the combination of experience, interest, and state-of-the-art technology and will maximize her potential to become an independent NIH-funded investigator.
Neutrophils participate in a number of acute and chronic human illnesses, including ischemia/reperfusion injury, vasculitis, and inflammatory arthritis. The new knowledge developed from this proposal will have a significant impact on the development of novel therapeutic strategies for these illnesses.
