Macromolecular Engineering

摘要 所有项目都建议采用新的工程试剂，使操纵和询问 在精确的时空控制下，独立的信号通路正如每个项目中所描述的， 核心C提供的试剂将用于确定调节肺恢复的过程 内皮屏障这些工具的开发和优化将需要大量的努力， 在分子工程核心C中描述。核心C的主要功能是：1）开发 为个别项目中的每个特定问题定制的分子工具，2）评估新的 试剂并建立其在原代人和小鼠肺微血管中应用的方案 内皮细胞，以及3）提供工具应用、故障排除方面的帮助，以及 结果分析。其目的是简化新技术在该计划中的应用 项目，并允许参与者专注于需要这些试剂的建议问题。的 核心C将开发和使用的特定试剂包括：1）控制 每个项目鉴定的蛋白质的定位和相互作用; 2）活性调节试剂 活细胞中选定的蛋白质，并有针对性地激活这些蛋白质在特定的复合物， 亚细胞位置;和3）用于光介导的小细胞的时空调节的试剂。 GTP酶Rac 1和Cdc 42以及活细胞中的其他GTP酶（根据需要）。Core C还将生成和 优化用于在原代内皮细胞和动物模型中表达工程蛋白的试剂， 并开发新的试剂来操纵和询问蛋白质相互作用和细胞信号 根据需要开发的工具将能够操纵单个信号通路， 评估它们在调节内皮屏障恢复和恢复完整性中的作用， 粘附连接。

Abstract All Projects propose to employ novel engineered reagents that enable manipulation and interrogation of individual signaling pathways with precise spatial and temporal control. As described in each Project, reagents provided by Core C will be used to define the processes that regulate the recovery of the lung endothelial barrier. Development and optimization of these tools will require significant effort as described in the Molecular Engineering Core C. The central functions of Core C will be 1) to develop molecular tools customized for each specific question in individual Projects, 2) to evaluate the new reagents and establish protocols for their application in primary human and mouse lung microvessel endothelial cells, and 3) to provide assistance with the application of the tools, troubleshooting, and analysis of the results. The intent will be to simplify application of new technologies in the Program Project and allow participants to focus on the proposed questions requiring these reagents. The specific reagents that will be developed and employed by Core C include: 1) new tools to control localization and interaction of proteins identified by each Project; 2) reagents for regulation of activity of selected proteins in living cells, and targeted activation of these proteins in specific complexes and subcellular locations; and 3) reagents for light-mediated spatio-temporal regulation of the small GTPases Rac1 and Cdc42 and other GTPases in living cells as needed. Core C will also generate and optimize reagents for expression of engineered proteins in primary endothelial cells and animal models, and develop new reagents for manipulation and interrogation of protein interactions and cell signaling as needed. The tools developed will enable manipulation of individual signaling pathways and assessment of their roles in the regulation of endothelial barrier recovery and restoration of the integrity of adherens junctions.