Examination of the Final Stages of Platelet Production

描述(申请人提供)：考试最后阶段的血小板生产候选人。我的博士论文在Dana Devine博士(不列颠哥伦比亚大学教授，加拿大血液服务部副总裁)的指导下，应用蛋白质组学、分子生物学和生物化学来确定参与调节与储存相关的血小板(PLT)恶化的机制。在丹娜的实验室里，我密切合作 与加拿大血液服务局、临床医生和血液研究中心的研究人员合作，改进血液PLT的收集、处理和存储。我在Joseph Italiano博士的实验室(哈佛大学副教授)进行的博士后研究为我的技能增加了许多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，以研究PLT的产生。这些项目为我提供了实现职业目标所需的专业知识，使我熟悉了细胞骨架机制、细胞外基质(ECM)成分和剪切力等环境因素的贡献，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，可以接触到才华横溢的PLT研究人员的广泛网络，他们的投入和经验帮助指导了我的研究，并使我能够显著扩大我的分析、管理、写作和演讲技能。意大利亚诺博士还为我提供了一系列高度专业化的设备，为我提供了许多与我的研究目标相辅相成的分子生物学技术，并为我提供了专注的指导，使我成为一名有成就的PLT生物学家和显微镜专家。有机会在一家以PLT工作而闻名世界的研究所接受培训，使我能够与世界各地的PLT研究人员建立有意义的合作关系。我在哈佛医学院和布里格姆妇女医院的联合任职使我有机会参加各种课程、内部培训计划、部门研讨会以及职业发展和教育计划，这些计划使我成为一名更好的科学家，并支持了我走向独立调查人员的职业道路。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。我的兴趣在于研究PLT的形成机制，以开发针对血小板减少症的靶向治疗。控制巨核细胞(MK)体外扩增和成熟为PLT的能力将成为PLT输注的重要来源。我的短期目标是调查PLT生产的最后阶段，为此提出了一个包括三个具体目标的研究计划。巨噬细胞集落刺激因子释放长的分支延伸，称为ProPLT，进入正弦血管，经过反复的排泄，产生循环的PLT。目标1将检查单个PLT从ProPLT中释放的细胞骨架机制。这些实验将涉及微管和基于丝状肌动蛋白的力量，这些力量推动活细胞和渗透性细胞系统中的ProPLT延伸和PLT释放。目标2将模拟巨噬细胞如何建立极性，并将ProPLT导向正弦血管，以将PLT输送到循环中。目标3将解决环境因素，如细胞外基质的相互作用和持续的血液流动在产生功能性PLT和调节其释放速率/程度方面的贡献。研究职业发展计划。对目标1的研究将在指导阶段(K99)开始，目标1.3继续进入独立阶段(R00)。目标1.1和1.2中描述的目标与我的主要导师的研究目标在指导下背道而驰，我的主要导师的研究目标将与我的工作并行，并专注于启动ProPLT产生的蛋白质翻译/降解途径，以及基于血影蛋白的膜骨架在PLT形成中的作用。对目标2的研究也将在指导阶段开始，并进入独立阶段的第一年，以弥合细胞骨架和环境决定因素之间的差距，这些决定因素包括PLT延长和PLT释放。来自AIMS 1和2的初步数据将使我能够在第3年启动一项独立的研究计划，以概括体外骨髓和血窦血管微环境，并研究它们在终末PLT产生中的作用(AIMS 3.1和3.2)。对Aim 3.3的研究将在最初的生物芯片开发之后的第三年开始，并将随着生物芯片设计的每一次新进展，检查培养衍生的PLT的质量。这些数据将支持我的长期职业目标，即(1)开发生物模拟系统，以产生可用于临床的可供输注的人类PLT，以及(2)建立具有代表性的人体骨髓和周围血管的体外模型，用于测试药物和开发针对血小板减少症的靶向治疗。这份申请中列出的具体目标或研究目标与我的导师们的目标都没有重叠，我已经得到许可，可以把它们带到我自己的研究实验室。

DESCRIPTION (provided by applicant): 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.