Examination of the Final Stages of Platelet Production

项目总结/摘要-血小板生成最后阶段的检查 候选人我的博士论文，根据博士Dana迪瓦恩（教授，不列颠哥伦比亚省大学的指导; 副总裁，加拿大血液服务），需要应用蛋白质组学，分子生物学， 生物化学来鉴定涉及调节血小板（PLT）的储存相关劣化的机制。 在Dana的实验室里，我与加拿大血液服务中心的临床医生和研究人员密切合作， 血液研究旨在改善血液PLT的采集、处理和储存。我的博士后研究在 博士Joseph Italiano的实验室（哈佛大学副教授）为我的曲目增加了一个 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产生。这些 项目为我提供了必要的专业知识，通过熟悉我的职业目标， 细胞骨架力学，环境因素如细胞外基质（ECM）成分的作用 和剪切力，以及协调PLT形成和调节其功能的信号通路。 环境Italiano博士的实验室提供了无与伦比的访问到一个广泛的网络， 有才华的PLT研究人员，他们的投入和经验帮助指导了我的研究，并使我能够 显著地扩展了我的分析、管理、写作和演讲技能。伊塔利亚诺博士还使 我可以使用一系列高度专业化的设备，用一些分子生物学知识武装我， 这些技术与我的研究目标相辅相成，并为我提供了专门的指导， 使我成为一个有成就的血小板生物学家和显微镜学家。在研究所接受培训的机会 这是世界著名的PLT工作，使我能够建立有意义的合作关系， 全世界的PLT研究人员。我在哈佛医学院和布里格姆妇女学院的联合任命 医院为我提供了大量的课程，内部培训计划，部门研讨会， 职业发展和教育计划，使我成为一个更好的科学家，并支持我的 走向独立调查员的职业轨迹。 research.血液PLT在止血和血栓形成的病理生理学中起重要作用。我 研究血小板形成的机制，以开发靶向治疗方法， 血小板减少症控制体外巨核细胞（MK）扩增和成熟为PLT的能力 将导致用于输注的PLT的重要来源。我的短期目标是调查 的PLT生产，其中包括三个具体目标的研究计划提出。MK长释放 称为proPLT的分支延伸进入窦状血管，其经历重复的脱落以产生 循环血小板目的1将检查单个PLT从细胞骨架释放的细胞骨架机制。 proPLT。这些实验将解决微管，以及基于肌动蛋白的力量， proPLT延伸和PLT释放在活细胞中和在透化细胞系统内。目标2将模型 MK如何建立极性并将proPLT导向窦状血管以将PLT输送到窦状血管中 流通目标3将解决环境因素的贡献，如ECM相互作用， 在产生功能性PLT和调节其释放速率/程度中的连续血流。 研究职业发展计划。关于目标1的研究将在指导阶段（K99）开始， 目标1.3继续进入独立（R00）阶段。目标1.1和1.2中所述的目标是 指导偏离我的主要主管的研究目标，这将与我的工作并行， 关注启动proPLT产生的蛋白质翻译/降解途径，以及血影蛋白的作用， 基于膜骨架的血小板形成。关于目标2的研究也将在接受指导期间开始。 阶段，并进入独立阶段的第一年，以弥合细胞骨架和细胞骨架之间的差距。 proPLT延伸和PLT释放的环境决定因素。来自目标1的初步数据 2将允许我在第三年启动一个独立的研究项目，以概括骨髓， 体外正弦血管微环境，并研究它们在终末PLT产生中的作用（目的3.1和 3.2）。目标3.3的研究将在第三年开始，在初步生物芯片开发之后， 随着生物芯片设计的每一个新进展，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，以产生可用数量的临床 用于输注的活的人PLT，和（2）建立人骨髓的代表性离体模型， 为了测试药物和开发针对性治疗， 血小板减少症本申请中所列的具体目的和研究目标均不与那些 我的导师们，我已经得到许可，把他们带到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.