Examination of the Final Stages of Platelet Production

血小板生产最后阶段的检查

• 批准号: 8510070
• 负责人: JONATHAN N THON
• 金额: $ 13.27万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8510070
• 关键词: 3-Dimensional; Actins; Address; Aplastic Anemia; Appointment; Authorization documentation; Automobile Driving; Biochemistry; Biological; Birth; Blood; Blood Circulation; Blood Platelet Disorders; Blood Platelets; Blood Vessels; Blood flow; Bone Marrow; British Columbia; Caliber; Cell Culture Techniques; Cells; Cellular biology; Clinical; Collection; Cytoskeleton; Data; Degradation Pathway; Deterioration; Development; Development Plans; Doctor of Philosophy; Dynein ATPase; Dysmyelopoietic Syndromes; Electron Microscopy; Endothelial Cells; Environment; Environmental Risk Factor; Equipment; Excision; Extracellular Matrix; Extracellular Matrix Proteins; F-Actin; Fluorescence Microscopy; Functional disorder; Goals; HIV; Hemorrhage; Hemostatic function; Hospitals; Human; Immune; Immune response; In Vitro; Individual; Infection; Infusion procedures; Inherited; Institutes; Integrins; Joints; Laboratories; Lead; Life; Mechanics; Mediating; Megakaryocytes; Membrane; Mentors; Mentorship; Microtubules; Modeling; Molecular; Molecular Biology; Molecular Biology Techniques; Operative Surgical Procedures; Outcome; Pathway interactions; Patients; Pattern; Phase; Platelet Count measurement; Platelet Transfusion; Play; Postdoctoral Fellow; Pregnancy; Process; Production; Property; Proteins; Proteomics; Public Health; Recruitment Activity; Regulation; Relative (related person); Research; Research Personnel; Resistance; Risk; Role; Running; Scientist; Services; Shapes; Side; Signal Pathway; Signal Transduction; Site; Slide; Source; Spectrin; Staging; Surface; System; Techniques; Thrombocytopenia; Thrombocytopenic Purpura; Thrombosis; Training; Training Programs; Transgenic Mice; Translations; United States; Universities; Vascular Endothelial Cell; Venous; Virus Diseases; Woman; Work; Writing; arm; base; biochip; career; career development; cellular imaging; chemotherapy; design; drug testing; experience; improved; in vitro Model; in vivo; interest; medical schools; meetings; membrane skeleton; mimetics; mouse model; polymerization; professor; programs; protein complex; receptor; research study; skills

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.

描述（由申请人提供）：血小板生产候选者最后阶段的检查。我的博士学位在 Dana Devine 博士（不列颠哥伦比亚大学教授；加拿大血液服务中心副总裁）的指导下，论文需要应用蛋白质组学、分子生物学和生物化学来确定参与调节血小板储存相关恶化的机制（PLT）。在达纳的实验室里我密切合作 与加拿大血液服务中心、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、处理和储存。我在 Joseph Italiano 博士（哈佛大学副教授）实验室的博士后研究为我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研究 PLT 生产的模型。这些项目让我熟悉细胞骨架力学、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，为我提供了实现职业目标所需的专业知识。环境。 Italiano 博士的实验室为我提供了无与伦比的机会，可以接触到由才华横溢的 PLT 研究人员组成的广泛网络，他们的投入和经验帮助指导了我的研究，并使我能够显着扩展我的分析、管理、写作和演讲技能。 Italiano 博士还为我提供了一系列高度专业化的设备，为我配备了许多与我的研究目标相辅相成的分子生物学技术，并为我提供了专门的指导，使我能够成为一名出色的 PLT 生物学家和显微镜学家。在一家因 PLT 工作而闻名的研究所接受培训的机会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莱根妇女医院的联合任命使我有机会参加大量课程、内部培训计划、部门研讨会以及职业发展和教育计划，这些使我成为一名更好的科学家，并支持我走向独立的职业轨迹研究者。研究。血液 PLT 在止血和血栓形成的病理生理学中发挥着重要作用。我的兴趣在于研究 PLT 形成的机制，以开发血小板减少症的靶向疗法。控制体外巨核细胞 (MK) 扩增和成熟为 PLT 的能力将成为输注 PLT 的重要来源。我的短期目标是研究 PLT 生产的最后阶段，为此提出了一个包含三个具体目标的研究计划。 MK 将称为 proPLT 的长分支延伸物释放到正弦血管中，这些血管经历反复脱落以产生循环 PLT。目标 1 将检查从 proPLT 中释放单个 PLT 的细胞骨架力学。这些实验将研究微管和基于丝状肌动蛋白的力，这些力在活细胞和透化细胞系统内为 proPLT 延伸和 PLT 释放提供动力。目标 2 将模拟 MK 如何建立极性并将 proPLT 引导至正弦血管以将 PLT 输送到循环中。目标 3 将解决环境因素（例如 ECM 相互作用和连续血流）在生成功能性 PLT 和调节其释放速率/程度方面的贡献。研究职业发展规划。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 中描述的目标代表了与我的主要导师的研究目标的指导背离，该目标将与我的工作并行，重点关注启动 proPLT 生产的蛋白质翻译/降解途径，以及基于血影蛋白的膜的作用PLT形成的骨架。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 proPLT 延伸和 PLT 释放的细胞骨架和环境决定因素之间的差距。来自目标 1 和 2 的初步数据将使我能够在第 3 年启动一项独立研究计划，以在体外概括骨髓和正弦血管微环境，并研究它们在最终 PLT 生产中的作用（目标 3.1 和 3.2）。目标 3.3 的研究将在初始生物芯片开发后的第 3 年开始，并将随着生物芯片设计的每一项新进展检查培养物衍生的 PLT 的质量。这些数据将支持我的长期职业目标：(1) 开发仿生系统，以生成可用数量的临床上可行的人类 PLT 用于输注，以及 (2) 建立具有代表性的人骨髓和周围脉管系统的离体模型。测试药物并开发血小板减少症的靶向疗法。本申请中列出的具体目的和研究目标均与我的导师的目标不重叠，并且我已获得许可将他们带到我自己的研究实验室。