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.

项目总结/摘要 候选人我的博士Alisa Wolberg博士（北方大学副教授）指导下的论文 查佩尔山的卡罗莱纳），需要应用细胞和分子生物学，共聚焦显微镜， 生物化学和血栓形成的小鼠模型，以确定血浆凝血因子水平升高的作用 （高凝性）在血栓性疾病的病理生理学中的作用。我在约瑟夫博士的博士后研究 Italiano的实验室（副教授，哈佛大学），增加了我的剧目的数量， 专业的细胞生物学技术，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、原代巨核细胞的细胞培养和转基因小鼠模型来研究 巨核细胞成熟和血小板产生。这些项目为我提供了必要的 专业知识，以满足我的职业目标，通过熟悉我的细胞生物学过程和信号通路 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到一个广泛的网络， 他们的意见和经验帮助指导了我的研究 这使我的分析能力、管理能力、写作能力和演讲能力得到了显著的提高。博士 Italiano还为我提供了一系列高度专业化的设备， 分子生物学技术是我的研究目标的补充，并为我提供了专门的 导师，使我成为一个有成就的巨核细胞生物学家和显微镜。的 有机会在一个以巨核细胞研究而闻名世界的研究所接受培训，使我能够 与全球专家建立有意义的合作关系。我在哈佛的联合任命 医学院和布里格姆妇女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 我是一个更好的科学家，并支持我的职业生涯走向独立调查员。 Research.我的兴趣在于研究巨核细胞分化的机制， 了解血小板是如何以及为什么产生的，并最终开发靶向治疗，以增强或 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 将导致在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 探讨细胞因子CCL 5及其受体CCR 5在造血和巨核细胞中的作用 成熟，为此提出了包括三个具体目标的研究计划。我假设在 在炎症中，细胞因子CCL 5通过其受体CCR 5发出信号。这可能有助于1）增加 造血干细胞分化成巨核细胞和/或2）增强巨核细胞 通过增加促生存信号传导来促进成熟。在目标1中，我将确定CCL 5/CCR 5轴的作用 造血干细胞的分化。这些实验将检查CCL 5对造血细胞的影响。 并确定CCL 5导致造血干细胞偏斜的机制 沿着髓系。在目的2中，我将明确CCL 5/CCR 5轴在终末巨核细胞中的作用。 成熟具体来说，我将集中在BAD磷酸化在增加巨核细胞倍性中的作用 和前血小板形成。为此目的提出的实验将确定CCL 5 通过CCR 5的信号传导导致BAD磷酸化。此外，我将定义信号通路， 将CCR 5激活与BAD磷酸化和促生存信号联系起来。在目标3中，我将研究 CCL 5/CCR 5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鼠模型来完成这一点，包括直接输注CCL 5， 炎症性肠病的模型。使用这些模型，我将确定CCL 5是否影响造血 干细胞沿着髓系分化并通过BAD促进巨核细胞成熟 体内磷酸化。通过研究CCL 5/CCR 5通路，我将深入了解 在止血和病理条件下驱动巨核细胞分化和成熟。 研究职业发展计划。所描述的目标代表了对我的主要目标的指导 他的研究重点是开发生物模拟系统，以产生人类血小板， 输液本申请中列出的具体目标与我的导师的目标不重叠，我已收到 允许我带他们去我自己的研究实验室此外，我的共同导师柏林博士，将提供 支持体外和体内造血研究，这将使我进一步偏离博士。 工作并获得额外的技能和专业知识。 在这个奖学金中，来自目标1和2的初步数据将使我能够开展一项独立的研究， 研究项目，我预计在第三年进行。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管理。我将通过成为一个成功的 他的研究重点是了解巨核细胞是如何以及为什么在 健康和疾病。通过了解驱动巨核细胞成熟的机制，我将能够 来操纵这些通路，从而开发新的、变革性的疗法。