Examination of the Final Stages of Platelet Production

血小板生产最后阶段的检查

• 批准号: 9023825
• 负责人: JONATHAN N THON
• 金额: $ 24.9万
• 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 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9023825
• 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; targeted treatment

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.

项目摘要/摘要 - 血小板生产的最后阶段检查 候选人。我的博士学位论文，在Dana Devine博士（不列颠哥伦比亚省大学教授）的指导下； 加拿大血液服务副总裁），需要应用蛋白质组学，分子生物学和 生物化学确定与调节血小板（PLTS）储存相关的恶化的机制。 在达纳（Dana）的实验室中，我与加拿大血液服务部，临床医生和调查人员密切合作。 血液研究以改善血液PLT的收集，加工和储存。我的博士后研究 约瑟夫·伊塔利亚诺（Joseph Italiano）博士的实验室（哈佛大学副教授）已增加了我的曲目 专业细胞生物学技术的数量，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研究PLT产生。这些 项目为我提供了必要的专业知识来实现​​我的职业目标，从而使我熟悉 细胞骨架力学，环境因素（例如细胞外基质（ECM）组件）的贡献 和剪切力，以及编排PLT形成并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权限 才华横溢的PLT研究人员，他们的投入和经验有助于指导我的研究并允许我 明显地扩大了我的分析，管理，写作和演讲技巧的武器库。 Italiano博士也做了 我可以提供一系列高度专业的设备，并为我提供了许多分子生物学 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 使我成为一名出色的PLT生物学家和显微镜家。在研究所训练的机会 这是因为其PLT的工作而享誉世界的，这使我能够与 全球PLT研究人员。我在哈佛医学院和杨百翰和妇女的联合任命 医院为我提供了许多课程，内部培训计划，部门研讨会， 以及职业发展和教育计划，使我成为更好的科学家，并支持我 对独立研究者的职业轨迹。 研究。血液PLT在止血和血栓形成的病理生理中起着至关重要的作用。我的 利益是研究PLT形成的机制，目的是开发目标疗法 用于血小板减少症。控制体外巨核细胞（MK）扩展和成熟到PLT的能力 将导致输注的重要来源。我的短期目标是调查最后阶段 在PLT生产中，提出了一个包括三个特定目标的研究计划。 MKS释放很长 分支的扩展名称为正弦血管，这些延伸物重复出现以产生 循环的PLT。 AIM 1将检查单个PLT从中释放的细胞骨架力学 建议。这些实验将解决微管和基于丝状 - 肌动蛋白的力量 在活细胞和通透性细胞系统中释放Proplt延伸和PLT。 AIM 2将建模 MK如何建立极性并直接朝着正弦血管递送PLT进入 循环。 AIM 3将解决环境因素的贡献，例如ECM相互作用和 在产生功能性PLT并调节其释放速度/程度的连续血流中。 研究职业发展计划。 AIM 1的研究将在指导阶段开始， AIM 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 与我的主要主管的研究目标有指导的偏离，这将与我的工作平行， 关注蛋白质翻译/降解途径，该途径启动推荐产生，以及谱蛋白的作用 基于PLT形成的膜骨架。 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 Proplt扩展和PLT释放的环境决定因素。源自目标1的初步数据 2将使我能够在第三年启动独立的研究计划，以概括骨髓和 正弦血管体外微环境并研究其在末端PLT产生中的作用（AIM 3.1和 3.2）。 AIM 3.3的研究将在最初的生物芯片开发后，将于3年级开始，并将检查 文化衍生的PLT质量以及生物芯片设计的每一个新进步。这些数据将支持我 （1）开发生物模拟系统的长期职业目标，以生成可用数量的临床数量 可行的人类PLT进行输注，（2）建立人类骨髓的代表性离体模型 围绕脉管系统，目的是测试药物并开发靶向疗法 血小板减少症。本申请中的具体目的或本应用程序中列出的研究目标均不与那些重叠 在我的导师中，我已经获得了将他们带到我自己的研究实验室的许可。