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.

项目摘要/摘要 候选人。我的博士论文，由Alisa Wolberg博士(诺斯大学副教授)指导 在教堂山的卡罗莱纳)，需要应用细胞和分子生物学，共聚焦显微镜， 生化和小鼠血栓模型以确定血浆凝血因子水平升高的作用 (高凝)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 意大利的实验室(哈佛大学副教授)，为我的剧目增加了许多 专门的细胞生物学技术，包括荧光，高含量，和电子显微镜，活细胞 成像、逆转录病毒感染、原代巨核细胞培养和转基因小鼠模型研究 巨核细胞成熟和血小板生成。这些项目为我提供了必要的 通过让我熟悉细胞生物学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，可以接触到非常广泛的 有才华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 这让我大大扩展了我的分析、管理、写作和演讲技能。Dr。 意大利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 导师使我成为一名有成就的巨核细胞生物学家和显微镜专家。这个 有机会在一家以巨核细胞研究而闻名的研究所接受培训，使我能够 与世界各地的专家建立有意义的协作关系。我在哈佛的联合任命 医学院和布里格姆妇女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 我成为了一名更好的科学家，并支持了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 了解血小板是如何以及为什么制造的，并最终开发有针对性的治疗方法来增强或 抑制巨核细胞分化成熟。体内控制巨核细胞成熟的能力 将导致在血小板减少和血小板增多症中调节血小板计数的能力。我的短期目标 目的是研究细胞因子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。我假设在这个时代 炎症，细胞因子CCL5通过其受体CCR5传递信号。这可能会有效地1)增加数字 可分化为巨核细胞的造血干细胞和/或2)增强巨核细胞 通过增加有利于生存的信号来成熟。在目标1中，我将确定CCL5/CCR5轴的作用 在造血干细胞分化中的作用。这些实验将检验CCL5对造血的影响。 并确定CCL5导致造血干细胞偏斜的机制 沿着髓系血统。在目标2中，我将确定CCL5/CCR5轴在终末巨核细胞中的作用 成熟。具体地说，我将重点介绍不良的磷酸化在扩大巨核细胞倍性中的作用。 和原血小板的形成。在这一目标中提出的实验将确定CCL5 通过CCR5传递的信号会导致糟糕的磷酸化。此外，我将定义信号通路， 将CCR5的激活与不良的磷酸化和促生存信号联系起来。在目标3中，我将研究 CCL5/CCR5轴影响造血干细胞分化和巨核细胞的机制 体内发育。我将使用多种小鼠模型来实现这一点，包括直接和 炎症性肠病的模型。使用这些模型，我将确定CCL5是否会影响造血 干细胞沿髓系分化并通过BAD促进巨核细胞成熟 体内的磷酸化。通过研究CCL5/CCR5途径，我将深入了解 在止血和病理条件下促进巨核细胞分化和成熟。 研究职业发展计划。所描述的目标代表了我对初选的指导 Supervisor，他的研究重点是开发仿生系统来生成人类血小板 输液。这份申请表中列出的具体目标与我导师的目标没有重叠，我收到了 允许我把它们带到我自己的研究实验室。此外，我的合作导师伯林纳博士将提供 对体外和体内造血研究的支持，这将使我进一步偏离意大利博士的 工作并获得更多的技能和专业知识。 从这个奖学金中的目标1和目标2中获得的初步数据将使我能够启动一个独立的 这些数据将支持我的终极职业目标 加强对血小板增多症和血小板减少症的管理。我会通过成为一个成功的人来做到这一点 一位学术科学家，他的研究重点是了解巨核细胞是如何以及为什么在 无论是健康还是疾病。通过了解推动巨核细胞成熟的机制，我将能够 操纵这些通路，从而开发新的、变革性的疗法。