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- 저자 : Barthelery, Miguel (검색결과 1 건)
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Nuclear proteomics of human embryonic stem cell neural differentiation
Barthelery, Miguel The Pennsylvania State University 2008 해외박사(DDOD)
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Goals of dissertation research. With the discovery of adult and embryonic stem cells, molecular medicine has expanded prospects for addressing a number of unmet medical needs. We can now imagine mending failing organs at the cellular level or replacing them altogether. However, we still face the issue of efficiently directing the differentiation of these cells. Part of the problem is a lack of knowledge of the signals and developmental timing required to commit to a certain lineage fate. Another issue is that available molecular markers of differentiation are limited. Cellular responses originate in the nucleus where gene expression is initiated. The genetic material and its expression are managed and maintained by the nuclear proteome---the collection of proteins that operate within the nuclear envelope. We were interested in understanding the fundamental transformations required to direct the differentiation of human embryonic stem cells (hESCs) into neuroprogenitors and neurons in vitro. Indeed, neural stem cells, ordinarily isolated from adult brains, can be obtained from hESCs through growth factor-mediated induction and selection. We postulated that expression proteomic technologies, applied to nuclear protein extracts, would enrich our understanding of the molecular reprogramming that occurs during in vitro neural differentiation of hESCs (Chapter 1). Therefore, we focused our efforts on investigating what changes take place in the composition of the nuclear proteome and the relative levels of individual nuclear proteins, using 2-dimensional differential in-gel electrophoresis (2-DIGE). The goal of this research encompasses the identification of these changes and the discovery of additional markers and effectors of neural lineage commitment. Specific aims: Specific aim 1. Develop a methodology for enhancing discovery proteomics for nuclear extracts (Chapter 2). The nucleus is rich in nucleic acids and salts. These species are potent inhibitors of the 2-DIGE process and need to be removed to generate optimal experimental results. Moreover, histones constitute a sizable proportion of the nuclear proteome and their selective extraction would allow better resolution of less abundant proteins. Specific Aim 2. Characterize Reptin 52 expression during neural differentiation (Chapter 3). A preliminary comparison of hESC and hESC-derived neural stem cells illuminated an increase in Reptin 52 expression during differentiation. In light of a separate study performed between hESCs and human mesenchymal stem cells (hMSCs), where the same protein showed a decreased expression in hMSCs, we used orthogonal methods to characterize this protein and its known binding partners. Specific Aim 3. Comparison of the nuclear proteomes of hESCs and hESC-derived neurospheres (Chapter 4). An extensive 2-DIGE experiment was conducted with three replicated samples to assess the differential expression of nuclear proteins during in vitro development of neural stem cells. Original breakthroughs and findings. We have developed an effective and reproducible technique to deplete nuclear protein extracts of histones and nucleic acids in a single step. This method significantly improves the efficacy and the resolution of 2-DIGE separation, enhancing the quality of image analysis for nuclear protein extracts. We also demonstrated that the expression proteomics approach can illuminate the expression of unsuspected proteins such as Reptin 52 as changed during differentiation. The use of orthogonal analytical tools confirmed this finding and suggests that this protein (and/or its binding partners) are potential regulators of neural differentiation. We have also discovered a network of nuclear proteins that are differentially expressed during in vitro differentiation and could be used as additional markers of differentiation.
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