Molecular mechanisms of megakaryocyte differentiation and maturation during inflammation

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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.