Molecular mechanisms of megakaryocyte differentiation and maturation during inflammation

炎症过程中巨核细胞分化和成熟的分子机制

• 批准号: 9221704
• 负责人: Kellie Rae Machlus
• 金额: $ 15.97万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-14 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9221704
• 关键词: Acute; Affect; Appointment; Biochemistry; Biogenesis; Biological Process; Blood; Blood Coagulation Factor; Blood Platelet Disorders; Blood Platelets; Bone Marrow; CCR5 gene; Cells; Cellular biology; Clinical; Confocal Microscopy; Data; Development; Development Plans; Disease; Doctor of Philosophy; Electron Microscopy; Environment; Equipment; Fellowship; Fluorescence; Functional disorder; Goals; Health; Hematopoiesis; Hematopoietic stem cells; Hemorrhage; Hemostatic Agents; Hemostatic function; Hospitals; Human; Immune response; In Vitro; Infection; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Infusion procedures; Institutes; Joints; Laboratories; Lead; Life; Link; MARCKS gene; Mediating; Mediator of activation protein; Megakaryocytes; Membrane; Mentors; Mentorship; Modeling; Molecular; Molecular Biology Techniques; Molecular and Cellular Biology; Morbidity - disease rate; Mus; Myelogenous; National Institute of Diabetes and Digestive and Kidney Diseases; North Carolina; Pathologic; Pathway interactions; Patients; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Physiological; Plasma; Platelet Count measurement; Ploidies; Postdoctoral Fellow; Primary Cell Cultures; Process; Production; Protein Kinase C; Proteins; RANTES; Regulation; Research; Research Personnel; Risk; Role; Scientist; Signal Pathway; Signal Transduction; Stem cells; Stress; System; Techniques; Therapeutic; Thrombocytopenia; Thrombophilia; Thrombopoiesis; Thrombosis; Time; Training; Training Programs; Transgenic Mice; United States; Universities; Up-Regulation; Woman; Work; Writing; arm; career; career development; cytokine; experience; improved; in vivo; in vivo Model; insight; interest; live cell imaging; medical schools; meetings; mid-career faculty; mimetics; mortality; mouse model; new therapeutic target; precursor cell; pro-apoptotic protein; progenitor; programs; receptor; research study; seven-transmembrane G-protein-coupled receptor; skills; stem cell differentiation; targeted treatment; thrombocytosis

PROJECT SUMMARY/ABSTRACT Candidate. My Ph.D. thesis, under the direction of Dr. Alisa Wolberg (Associate Professor, University of North Carolina at Chapel Hill), entailed the application of cellular and molecular biology, confocal microscopy, biochemistry, and murine models of thrombosis to identify the role of elevated plasma coagulation factor levels (hypercoagulability) in the pathophysiology of thrombotic disorders. 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, high-content, and electron microscopy, live cell imaging, retroviral infection, cell culture of primary megakaryocytes, and transgenic mouse models to study megakaryocyte maturation and platelet production. These projects have provided me with the necessary expertise to meet my career goals by familiarizing me with cell biological processes and signaling pathways that orchestrate hematopoietic stem cell differentiation and megakaryocyte maturation. Environment. Dr. Italiano’s laboratory has offered unequalled access to an extensive network of exceptionally talented megakaryocyte and platelet 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 megakaryocyte biologist and microscopist. The opportunity to train at an institute that is world-renowned for its megakaryocyte research has allowed me to establish meaningful collaborative relationships with experts 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. My interests lay in investigating the mechanisms of megakaryocyte differentiation for the purpose of understanding how and why platelets are made, and ultimately developing targeted therapies to enhance or repress megakaryocyte differentiation and maturation. The ability to control megakaryocyte maturation in vivo will result in the ability to regulate platelet count in thrombocytopenia and thrombocytosis. My short-term goal is to investigate the role of the cytokine CCL5 and its receptor CCR5 in hematopoiesis and megakaryocyte maturation, for which a research plan comprising three specific aims is proposed. I hypothesize that in times of inflammation, the cytokine CCL5 signals through its receptor, CCR5. This may work to 1) increase the number of hematopoietic stem cells that differentiate into megakaryocytes and/or 2) enhance megakaryocyte maturation through increased pro-survival signaling. In Aim 1 I will determine the role of the CCL5/CCR5 axis in hematopoietic stem cell differentiation. These experiments will examine the effect of CCL5 on hematopoietic stem cells in vitro and determine the mechanism by which CCL5 results in skewing of hematopoietic stem cells along the myeloid lineage. In Aim 2 I will define the role of the CCL5/CCR5 axis in terminal megakaryocyte maturation. Specifically, I will focus on the role of BAD phosphorylation in augmenting megakaryocyte ploidy and proplatelet formation. Experiments proposed in this aim will determine the mechanism by which CCL5 signaling through CCR5 results in BAD phosphorylation. In addition, I will define the signaling pathway that connects CCR5 activation to BAD phosphorylation and pro-survival signaling. In Aim 3 I will examine the mechanism by which the CCL5/CCR5 axis affects hematopoietic stem cell differentiation and megakaryocyte development in vivo. I will accomplish this using multiple murine models including infusion of CCL5 directly and a model of inflammatory bowel disease. Using these models, I will determine if CCL5 affects hematopoietic stem cell differentiation along the myeloid lineage and augments megakaryocyte maturation through BAD phosphorylation in vivo. By studying the CCL5/CCR5 pathway, I will gain insights into the mechanisms that drive megakaryocyte differentiation and maturation in both hemostatic and pathologic conditions. Research career development plan. The goals described represent a mentored departure from my primary supervisor, whose research is focused on developing bio-mimetic systems to generate human platelets for infusion. The specific aims listed in this application do not overlap with those of my mentor, and I have received permission to take them with me to my own research lab. In addition, my co-mentor Dr. Berliner, will provide support for in vitro and in vivo hematopoiesis studies, which will allow me to further diverge from Dr. Italiano’s work and gain additional skills and expertise. The preliminary data derived from Aims 1 and 2 in this fellowship will allow me to launch an independent research program, which I anticipate happening in year 3. These data will support my ultimate career goal to improve the management of thrombocytosis and thrombocytopenia. I will do this by becoming a successful academic scientist whose research is focused on understanding how and why megakaryocytes are made in both health and disease. By understanding the mechanisms that drive megakaryocyte maturation, I will be able to manipulate these pathways and therefore develop new, transformative therapeutics.

项目概要/摘要 候选人。我的博士学位论文，在 Alisa Wolberg 博士（北大学副教授）的指导下 Carolina at Chapel Hill），需要应用细胞和分子生物学、共焦显微镜、 生物化学和小鼠血栓形成模型，以确定血浆凝血因子水平升高的作用 （高凝状态）在血栓性疾病的病理生理学中。我在约瑟夫博士的博士后研究 Italiano 的实验室（哈佛大学副教授）为我添加了许多 专门的细胞生物学技术，包括荧光、高内涵和电子显微镜、活细胞 成像、逆转录病毒感染、原代巨核细胞的细胞培养和转基因小鼠模型进行研究 巨核细胞成熟和血小板产生。这些项目为我提供了必要的 通过熟悉细胞生物过程和信号通路来实现我的职业目标的专业知识 协调造血干细胞分化和巨核细胞成熟。 环境。 Italiano 博士的实验室提供了无与伦比的访问广泛网络的机会 才华横溢的巨核细胞和血小板研究人员，他们的投入和经验帮助指导了我的研究 并让我显着扩展了我的分析、管理、写作和演讲技能。博士。 Italiano 还为我提供了一系列高度专业化的设备，为我配备了许多 分子生物学技术与我的研究目标相辅相成，并为我提供了专门的 指导使我成为一名出色的巨核细胞生物学家和显微镜学家。这 在一家因巨核细胞研究而闻名的研究所接受培训的机会使我能够 与世界各地的专家建立有意义的合作关系。我在哈佛的联合任命 医学院和布莱根妇女医院为我提供了多种课程、内部课程 培训计划、部门研讨会以及职业发展和教育计划，使 我成为了一名更好的科学家，并支持我走向独立调查员的职业轨迹。 研究。我的兴趣在于研究巨核细胞分化的机制，目的是 了解血小板的产生方式和原因，并最终开发靶向疗法来增强或 抑制巨核细胞的分化和成熟。体内控制巨核细胞成熟的能力 将导致血小板减少症和血小板增多症中调节血小板计数的能力。我的短期目标 是研究细胞因子CCL5及其受体CCR5在造血和巨核细胞中的作用 成熟，为此提出了一项包括三个具体目标的研究计划。我假设在 炎症时，细胞因子 CCL5 通过其受体 CCR5 发出信号。这可能有助于 1) 增加数量 分化为巨核细胞的造血干细胞和/或 2) 增强巨核细胞 通过增加促生存信号而成熟。在目标 1 中，我将确定 CCL5/CCR5 轴的作用 在造血干细胞分化中。这些实验将检验 CCL5 对造血功能的影响 体外干细胞并确定 CCL5 导致造血干细胞倾斜的机制 沿着骨髓谱系。在目标 2 中，我将定义 CCL5/CCR5 轴在终末巨核细胞中的作用 成熟。具体来说，我将重点关注 BAD 磷酸化在增强巨核细胞倍性中的作用 和前血小板的形成。为此目的提出的实验将确定 CCL5 的机制 通过 CCR5 的信号传导会导致 BAD 磷酸化。此外，我将定义信号通路 将 CCR5 激活与 BAD 磷酸化和促生存信号传导联系起来。在目标 3 中，我将检查 CCL5/CCR5轴影响造血干细胞分化和巨核细胞的机制 体内发育。我将使用多个小鼠模型来完成此任务，包括直接输注 CCL5 和 炎症性肠病模型。使用这些模型，我将确定 CCL5 是否影响造血功能 干细胞沿骨髓谱系分化并通过 BAD 增强巨核细胞成熟 体内磷酸化。通过研究 CCL5/CCR5 通路，我将深入了解 CCL5/CCR5 通路的机制 在止血和病理条件下驱动巨核细胞分化和成熟。 研究职业发展规划。所描述的目标代表了与我的初衷的指导背离 主管，其研究重点是开发仿生系统来生成人类血小板 输液。本申请中列出的具体目标与我的导师的目标不重叠，并且我已收到 允许我带他们去我自己的研究实验室。此外，我的合作导师 Berliner 博士将提供 支持体外和体内造血研究，这将使我进一步偏离意大利博士的观点 工作并获得额外的技能和专业知识。 从本次奖学金的目标 1 和 2 中得出的初步数据将使我能够启动一个独立的项目 研究计划，我预计将在第三年进行。这些数据将支持我的最终职业目标 改善血小板增多症和血小板减少症的治疗。我将通过成为一名成功人士来做到这一点 学术科学家，其研究重点是了解巨核细胞是如何以及为何产生的 健康和疾病。通过了解驱动巨核细胞成熟的机制，我将能够 操纵这些途径，从而开发新的变革性疗法。