Granule exocytosis and neutrophil activation

胞吐作用在调节与中性粒细胞相关的各种功能反应中的作用 激活还没有经过直接测试。我们将使用一种新的方法；产生TAT-融合蛋白 包含不同SNARE蛋白的卷曲卷曲结构域；以检验阻断 不同的颗粒亚群的胞吐作用将防止特定的中性粒细胞表型改变和这一策略 可用于预防体内中性粒细胞介导的组织损伤。被辅导者的具体目标 这项建议的阶段是：1)确定含有SNARE的TAT-融合蛋白的能力 SNAP-23、合成素和VAMPS结构域抑制四种中性粒细胞颗粒的胞吐作用 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，使 申请者在新的水平上解决试验性问题并将其应用于本课程的独立阶段 求婚。2)确定体内应用TAT-SNARE融合蛋白的最有效途径 目的：确定这些融合蛋白对急性中性粒细胞依赖炎症模型的影响。 老鼠。这一目标的训练目标是学习中性粒细胞依赖炎症的动物模型，并 建立TAT融合蛋白在体内抑制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 进行蛋白质组学分析的能力。在指导阶段获得的最先进的技术 将大大增加专业知识，使私家侦探成为一名成功的独立调查员。 对于独立阶段，具体目标是：1)确定特定的功能反应 通过中性粒细胞胞吐作用进行调节；2)确定抑制 体内中性粒细胞胞吐阻断炎症反应；3)研究 胞吐作用被刺激的机制。申请者将接受的培训将使她变得独特 结合了经验、兴趣和最先进的技术，并将最大限度地发挥她的潜力 成为美国国立卫生研究院资助的独立调查员。

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.