Granule exocytosis and neutrophil activation

胞吐作用在中性粒细胞介导各种功能反应中的假定作用 尚未直接测试激活。我们将使用一种新的方法， 含有不同SNARE蛋白的卷曲螺旋结构域;为了检验阻断SNARE蛋白的作用的假设， 不同颗粒亚群的胞吐作用将阻止特异性中性粒细胞表型变化， 可用于预防体内嗜中性粒细胞介导的组织损伤。受指导者的具体目标 本阶段的建议是：1）确定TAT融合蛋白含有SNARE的能力 SNAP-23、突触融合蛋白和VAMP的结构域抑制四种中性粒细胞颗粒中的每一种的胞吐作用 子集这一目标的培训目标是发展分子生物学和细胞生物学技能， 申请人在新的水平上解决实验问题，并将其应用于独立阶段， 提议2)为了确定体内施用TAT-SNARE融合蛋白的最有效方式， 为了确定这些融合蛋白对急性嗜中性粒细胞依赖性炎症模型的作用， 大鼠这个目标的训练目标是学习嗜中性粒细胞依赖性炎症的动物模型， 建立TAT融合蛋白在体内抑制嗜中性粒细胞胞吐的能力。3)定义其他蛋白质 通过蛋白质组学分析与SNARE蛋白相互作用。这一目标的培训目标是发展 进行蛋白质组分析的能力。在辅导阶段获得的最先进技术 将大大提高专业知识，使PI成为一名成功的独立研究者。 对于独立阶段，具体目标是：1）确定特定的功能反应 2）确定抑制中性粒细胞胞吐作用的机制， 中性粒细胞胞吐作用在体内阻断炎症反应;和3）研究分子机制， 刺激胞吐作用的机制。申请人将接受的培训将使她独一无二 在经验，兴趣和最先进的技术相结合，并将最大限度地发挥她的潜力， 成为一名独立的NIH资助的调查员。

The postulated role of exocytosis in mediating various functional responses 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 specific objectives of the mentored phase of this proposal are: 1) To determine the ability of TAT-fusion proteins containing the SNARE domains of SNAP-23, syntaxins, and VAMPs to inhibit exocytosis of each of the four neutrophil granule subsets. The training goal of this aim is to develop molecular biology and cell biology skills that will allow the applicant to address experimental questions at new levels and apply them in the independent phase of this proposal. 2) To determine the most effective way to administer the TAT-SNARE fusion proteins in vivo and to determine the effect of these fusion proteins on models of acute neutrophil-dependent inflammation in rats. The training goals of this aim are to learn animal models of neutrophil-dependent inflammation, and to establish the ability of TAT-fusion proteins to inhibit neutrophil exocytosis in vivo. 3) Define other proteins that interact with SNARE proteins by proteomic analysis. The training goal of this aim is to develop the ability to perform proteomic analysis. The state-of-the-art techniques acquired during the mentored phase will greatly increase the expertise that 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 will receive will 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.