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Postdoctoral Fellow: Mechanisms of Blood Stem Cell Differentiation
Posted by:
Northwestern University
Posted date:
03-Nov-09
Mechanisms of Nicotinamide-Mediated Megakaryocytic Polyploidization
Platelets are transfused for many thrombotic deficiencies and each transfusion requires pooling of platelet harvests from multiple donors. Production of autologous or matched platelets by megakaryocytic cells (Mks) derived from cultured hematopoietic stem and progenitor cells (HSPCs or CD34+ cells) would greatly decrease the risks from bacterial contamination, blood-borne pathogens, and alloimmunization. However, generating 500 billion platelets for a single transfusion using current technology would require 25 umbilical cord blood harvests or a complete apheresis of HSPCs from donors treated (or mobilized) with cytokines. Thus, major advances will be required. Human bone marrow Mks have a modal (predominant) ploidy (DNA content) of 16N (diploid cells are 2N), which is twice as great as the highest reported modal ploidy for human Mks in culture. We have shown that the vitamin nicotinamide (NIC) greatly increases Mk ploidy under a wide range of Mk-promoting culture conditions (Br. J. Haematol. 135:554, 2006; Exp. Hematol. 37:1340, 2009). NIC is known to have multiple effects on cells including inhibition of sirtuins and PARP (poly(ADP-ribose) polymerase) and greatly increased NAD(H) content. Elucidation of the particular mechanism that is responsible for increased ploidy would facilitate the use of more selective inhibitors or activators that could increase Mk ploidy to levels higher than those obtained with NIC. Based on our preliminary results, we are using RNA interference-mediated knockdown to test the hypothesis that NIC increases Mk ploidy via inhibition of the SIRT1 and SIRT2 Class III histone/protein deacetylases. The sirtuins SIRT1 and SIRT2 are known to act on a wide range of proteins; we will examine changes in acetylation to investigate which target proteins directly modulate Mk polyploidization. Finally, we will directly modulate the activity of the most promising SIRT target proteins via over-expression or knockdown.
Successful applicants will have a PhD in chemical engineering, cell biology, immunology, molecular biology, biomedical engineering or a related field. Prior experience with several of the following skills is highly desirable: multiparametric flow cytometry, intracellular flow cytometry, culture of primary human or animal cells, hematopoietic cell culture and colony assays, transfection of human or animal cells for over-expression or knockdown, Western blots, immunoprecipitation, and immunofluorescence microscopy.
Initial appointment will be for a period of one year with the intention to renew for a second year. Interested candidates should send a single pdf file email attachment containing their CV, a statement of career objectives and availability, and the names and contact information for two references to Professor Bill Miller at wmmiller northwestern.edu.
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