Examination of the Final Stages of Platelet Production

项目摘要/摘要--检查血小板生产的最后阶段 候选人。我的博士论文，在Dana Devine博士的指导下(不列颠哥伦比亚大学教授； 加拿大血液服务部副总裁)，需要应用蛋白质组学、分子生物学和 生物化学，以确定参与调节与储存有关的血小板(PLT)退化的机制。 在Dana的实验室里，我与加拿大血液服务中心、临床医生和中心的研究人员密切合作 血液研究，以改进血液血小板的收集、加工和储存。我的博士后研究 约瑟夫·意大利博士的实验室(哈佛大学副教授)为我的剧目增加了一个 一些专门的细胞生物学技术，包括荧光和电子显微镜，活细胞 成像、逆转录病毒感染、细胞培养和转基因小鼠模型来研究PLT的产生。这些 项目为我提供了必要的专业知识，通过让我熟悉 细胞骨架力学，环境因素的贡献，如细胞外基质(ECM)成分 和剪切力，以及协调PLT形成和调节其功能的信号通路。 环境意大利亚诺博士的实验室提供了无与伦比的机会，可以接触到非常广泛的 有才华的PLT研究人员，他们的意见和经验帮助指导了我的研究，并使我能够 显著扩展我的分析、管理、写作和演讲技能。意大利亚诺博士也做出了 为我提供了一系列高度专业化的设备，用一些分子生物学武装了我 与我的研究目标相辅相成的技术，并为我提供了专门的指导， 使我成为一名有成就的PLT生物学家和显微镜学家。在学院接受培训的机会 它以其PLT工作而闻名世界，使我能够与 世界各地的PLT研究人员。我在哈佛医学院和布里格姆女子学院的联合任命 医院为我提供了大量的课程，内部培训计划，部门研讨会， 职业发展和教育项目让我成为一名更好的科学家，并支持我的 独立调查员的职业轨迹。 研究。血小板在止血和血栓形成的病理生理中起着重要的作用。我的 人们的兴趣在于研究血小板形成的机制，以开发靶向治疗。 治疗血小板减少症。巨核细胞(MK)体外扩增成熟为PLT的能力 将产生用于输液的PLT的重要来源。我的短期目标是调查最后阶段 为此，提出了一项包含三个具体目标的研究计划。MKS发布时间长 称为ProPLT的分支延伸成正弦血管，经过反复abc排出以产生 循环中的血小板。目标1将检查单个PLT释放的细胞骨架机制 PROPLTS。这些实验将涉及微管和以丝状肌动蛋白为基础的力量 在活细胞和通透性细胞系统中，ProPLT延伸和PLT释放。AIM 2将成为典范 巨噬细胞集落刺激因子如何建立极性并将PLT导向正弦血管，从而将PLT输送到 发行量。目标3将解决环境因素的贡献，如ECM相互作用和 产生功能性PLT并调节其释放速率/程度的持续血流。 研究职业发展计划。对目标1的研究将在指导(K99)阶段开始， 目标1.3继续进入独立(R00)阶段。目标1.1和1.2中描述的目标代表 与我的主要导师的研究目标背道而驰，这将与我的工作和 重点关注启动ProPLT产生的蛋白质翻译/降解途径，以及幽灵蛋白的作用- 基于PLT形成的膜骨架。对AIM 2的研究也将在指导期间开始 阶段并进入独立阶段的第一年，以弥合细胞骨架和 PLT延长和PLT释放的环境决定因素。来自AIMS 1的初步数据 和第二年将允许我在第三年启动一个独立的研究计划，以概括骨髓和 体外正弦血管微环境，并研究它们在终末PLT产生中的作用(目标3.1和 3.2)。在最初的生物芯片开发之后，对Aim 3.3的研究将于第三年开始，并将审查 随着生物芯片设计的每一次新进展，培养衍生的PLT的质量。这些数据将支持我的 长期职业目标：(1)开发生物模拟系统以产生临床上可用的数量 (2)建立具有代表性的人骨髓和骨髓的体外模型。 周围血管系统，用于测试药物和开发有针对性的治疗 血小板减少症。本申请书中列出的具体目标或研究目标均不与之重叠 我的导师，我已经得到许可，带他们去我自己的研究实验室。

PROJECT SUMMARY/ABSTRACT - Examination of the Final Stages of Platelet Production Candidate. My Ph.D. thesis, under the direction of Dr. Dana Devine (Professor, University of British Columbia; Vice President, Canadian Blood Services), entailed the application of proteomics, molecular biology, and biochemistry to identify mechanisms involved in regulating the storage-related deterioration of platelets (PLTs). In Dana's lab I worked closely with Canadian Blood Services, clinicians, and investigators at the Centre for Blood Research to improve the collection, processing, and storage of blood PLTs. My post-doctoral research in Dr. Joseph Italiano's laboratory (Associate Professor, Harvard University), has added to my repertoire a number of specialized cell biology techniques including fluorescence and electron microscopy, live cell imaging, retroviral infection, cell culture, and transgenic mouse models to study PLT production. These projects have provided me with the necessary expertise to meet my career goals by familiarizing me with the cytoskeletal mechanics, contribution of environmental factors such as extracellular matrix (ECM) components and shear forces, and the signaling pathways that orchestrate PLT formation and regulate their function. Environment. Dr. Italiano's laboratory has offered unequalled access to an extensive network of exceptionally talented PLT researchers whose input and experience have helped guide my research and allowed me to markedly expand my arsenal of analytical, management, writing, and oratory skills. Dr. Italiano has also made available to me a range of highly specialized equipment, armed me with a number of molecular biology techniques that are complementary to my research goals, and provided me with dedicated mentorship that has enabled me to become an accomplished PLT biologist and microscopist. The opportunity to train at an institute that is world-renowned for its PLT work has allowed me to establish meaningful collaborative relationships with PLT researchers worldwide. My joint appointment at Harvard Medical School and Brigham and Women's Hospital has afforded me access to a multitude of courses, internal training programs, departmental seminars, and career development and educational programs that have made me a better scientist, and supported my career trajectory toward independent investigator. Research. Blood PLTs play an essential role in hemostasis and the pathophysiology of thrombosis. My interests lay in investigating the mechanisms of PLT formation for the purpose of developing targeted therapies for thrombocytopenia. The ability to control in vitro megakaryocyte (MK) expansion and maturation into PLTs will result in an important source of PLTs for infusion. My short-term goals are to investigate the final stages of PLT production, for which a research plan comprising three specific aims is proposed. MKs release long branched extensions called proPLTs into sinusoidal blood vessels that undergo repeated abcissions to yield circulating PLTs. Aim 1 will examine the cytoskeletal mechanics by which individual PLTs are released from proPLTs. These experiments will address the microtubule, and filamentous-actin-based forces that power proPLT extension and PLT release both in live cells and within a permeabilized cell system. Aim 2 will model how MKs establish polarity and direct proPLTs toward sinusoidal blood vessels to deliver PLTs into the circulation. Aim 3 will resolve the contribution of environmental factors such as ECM interactions and continuous blood flow in generating functional PLTs and regulating their rate/extent of release. Research career development plan. Research on aim 1 will commence during the mentored (K99) phase, with aim 1.3 continuing into the independent (R00) phase. The goals described in aims 1.1 and 1.2 represent a mentored departure from the research aims of my primary supervisor, which will run parallel to my work and focus on protein translation/degradation pathways that initiate proPLT production, and the role of the spectrin- based membrane skeleton on PLT formation. Research on aim 2 will also commence during the mentored phase and proceed into the first year of the independent phase, to bridge the gap between the cytoskeletal and environmental determinants of proPLT extension and PLT release. The preliminary data deriving from aims 1 and 2 will allow me to launch an independent research program in year 3 to recapitulate the bone marrow and sinusoidal blood vessel microenvironments in vitro and study their role in terminal PLT production (aim 3.1 and 3.2). Research on aim 3.3 will commence in year 3, after initial biochip development, and will examine the quality of culture-derived PLTs with each new advancement in biochip design. These data will support my long-term career goals of (1) developing bio-mimetic systems to generate useable numbers of clinically viable human PLTs for infusion, and (2) establishing representative ex vivo models of human bone marrow and surrounding vasculature for the purposes of testing drugs and developing targeted therapies for thrombocytopenia. Neither the specific aims nor the research goals listed in this application overlap with those of my mentors, and I have recieved permission to take them with me to my own research lab.