Exploring the roles of Gi and S1Pr1 in endothelial barrier function in vivo

项目摘要/摘要。这个职业发展奖的目标是让Wilsbacher博士 培养成功成为学术中心独立内科医生兼科学家所需的技能。 Wilsbacher博士在西北大学医学科学家培训计划中获得医学博士和博士学位 在那里，她研究了驱动昼夜节律的遗传和分子机制。她 在加州大学旧金山分校接受内科和心脏病学方面的培训。威尔斯巴赫医生的 目前的研究目标是研究正常生理过程中血管屏障功能的机制 和疾病，她的长期职业目标是弥合昼夜生理和血管内皮细胞之间的差距 有助于增进对心血管生物学和疾病调控机制的理解。 培训计划包括世界级G蛋白研究人员肖恩·考夫林博士的指导。 偶联受体(GPCR)信号在止血、血栓形成和血管完整性中的作用 培训年轻调查人员使其独立的记录。将由一名专家提供其他指导 领导GPCR信号传递(Mark von Zastrow博士)、炎症(Donald博士)的科学家小组 和蛋白质化学(詹姆斯·威尔斯博士)。优秀的研究设施和设备随手可得 可用。培训计划还包括高级教学课程，重点是细胞信号， 膜运输、蛋白质相互作用和显微镜；出席系列研讨会；参与和 在地方和国家会议上发表演讲；指导手稿和赠款的准备工作。 治疗或预防炎症需要彻底了解内皮细胞的机制 屏障功能。这项研究计划旨在揭示调节血管完整性的信号机制， 特别是通过鞘氨醇-1-磷酸受体1(S1PR1)和GI信号转导。目标1调查角色 内皮细胞GI和S1PR1信号在屏障功能中的作用。具体地说，表达基因的老鼠模型 内皮细胞中的百日咳毒素(抑制GI)，在内皮中表达百日咳不敏感的GI，或 有条件地删除成人内皮细胞中的S1PR1将检测血管通透性的变化。AIM 2用途 一种新的GPCR激活检测系统用于探测S1PR1在体内何时何地被激活 发炎。新的记者系统，转录报道gpr激活，是一个创新的 该工具将广泛适用于不同组织中的其他GPCR。目的3探讨凹陷是否会影响 S1PR1-GI偶联，以及S1PR1在小窝中的定位是否影响arrestin介导的内化。 威尔斯巴赫博士的职业发展奖提案包括一位很有前途的候选人，杰出的 培训环境、严格的培训计划和令人振奋的研究提案，其中包括创新的新 这是一个工具，侧重于与人类健康和疾病直接相关的主题。在完成这项工作后 作为获奖者，Wilsbacher博士应该具备作为一名独立调查员取得成功所需的技能。

PROJECT SUMMARY/ABSTRACT. The goal of this Career Development Award is for Dr. Wilsbacher to develop the skills necessary to successfully become an independent physician-scientist at an academic center. Dr. Wilsbacher earned her M.D. and Ph.D. degrees in the Medical Scientist Training Program at Northwestern University, where she investigated genetic and molecular mechanisms that drive circadian rhythms. She trained in Internal Medicine and Cardiology at the University of California, San Francisco. Dr. Wilsbacher's current research objective is to investigate mechanisms of vascular barrier function during normal physiology and disease, and her long-term career goal is to bridge the gap between circadian physiology and endothelial function to help advance understanding of mechanisms governing cardiovascular biology and disease. The training plan includes mentorship by Dr. Shaun Coughlin, a world-class investigator of G protein- coupled receptor (GPCR) signaling in hemostasis, thrombosis, and vascular integrity who has an established record of training young investigators to independence. Additional guidance will be provided from an expert group of scientists who are leaders in GPCR signaling (Dr. Mark von Zastrow), inflammation (Dr. Donald McDonald), and protein chemistry (Dr. James Wells). Outstanding research facilities and equipment are readily available. The training plan also incorporates advanced didactic coursework that focuses on cell signaling, membrane trafficking, protein interactions, and microscopy; attendance at seminar series; participation and presentation in local and national conferences; and mentored guidance with manuscript and grant preparation. Treating or preventing inflammation requires thorough understanding of the mechanisms of endothelial barrier function. The research proposal aims to uncover signaling mechanisms that regulate vascular integrity, particularly through sphingosine-1-phosphate receptor 1 (S1Pr1) and Gi signaling. Aim 1 investigates the role of endothelial Gi and S1Pr1 signaling in barrier function. Specifically, genetic mouse models that express pertussis toxin (which inhibits Gi) in endothelium, express a pertussis-insensitive Gi in endothelium, or conditionally delete S1Pr1 in adult endothelium will be tested for changes in vascular permeability. Aim 2 uses a new GPCR activation detection system to probe when and where S1Pr1 is activated in vivo during inflammation. The new reporter system, which transcriptionally reports on GPCR activation, is an innovative tool that will be widely applicable to other GPCRs in diverse tissues. Aim 3 probes whether caveolae influence S1Pr1-Gi coupling, and whether S1Pr1 localization in caveolae affects ¿arrestin-mediated internalization. Dr. Wilsbacher's Career Development Award proposal comprises a promising candidate, outstanding training environment, rigorous training plan, and exciting research proposal that includes an innovative new tool and focuses on a topic with direct relevance to human health and disease. At the completion of this Award, Dr. Wilsbacher should have the skills necessary to succeed as an independent investigator.