TY - GEN
T1 - Proteome-wide analyses og human stem cell differentiation using quantitative mass spectrometry and bioinformatics
AU - Barrio-Hernandez, Inigo
PY - 2016/3
Y1 - 2016/3
N2 - Evolution from unicellular to more complex multicellular organisms brought the advantage of having cells fully committed to one particular function. This specialization comes with loss of cell division capabilities making necessary the existence the existence of a pool of cells less specialized, called stem cells. Cell differentiation is the process of going from the second type to the first and it has become of great interest in several fields such as regenerative medicine. Stem cell commitment involves great changes in protein expression and furthermore in cell signalling events triggered by protein phosphorylation, what makes it a perfect area to apply mass spectrometry based quantitative proteomics and phosphoproteomics. However, identifying and quantifying thousands of features in large scale experiments need to be coupled with computational approaches.The work presented in this thesis shows the power of mass spectrometry based studies combined with bioinformatics analysis to unravel molecular mechanisms in cell differentiation, at embryonic and adult organism level. In Paper 1 we focused in the first day of osteoblastic differentiation. SILAC based quantitation of phosphosites and proteins followed by bioinformatics analysis at different levels allowed us to study the signalling events orchestrating this process. We were able to predict and prove PRKD1 implication in RunX2 regulation as well as other important pathways controlling osteoblastic lineage. In Paper 2 we set up a SILAC based quantitative approach to study human primary myoblast differentiation. We applied c-fuzzy clustering to group the regulated proteins following the different dynamic on muscle differentiation: cell cycle arrest, cell adhesion, migration and myofibril formation. Not only in silico transcription factor analysis complemented this information but also it was possible to track candidate proteins implicated in myofibril development and muscular diseases; demonstrating the strength of our model. In Paper 3, we studied the role of MDM2 in the first steps of adipocyte differentiation. Here, we used two pre-adipocyte mutant cell lines differing in Mdm2 expression and set up a SILAC based assay to see the difference in protein expression. Transcription factor analysis showed STATs implication and, subsequent follow ups the mediation of CRTC transcription factors in controlling adipocytic fate. In Paper 4 we studied Pim2 kinase role in hESC. SILAC based quantitative phosphoproteomic studies coupled with Pim2 inhibitors and random differentiation induction allowed to determine a pool of direct and indirect targets that further confirm Pim2 involvement in transition between self-renewal and differentiation.
AB - Evolution from unicellular to more complex multicellular organisms brought the advantage of having cells fully committed to one particular function. This specialization comes with loss of cell division capabilities making necessary the existence the existence of a pool of cells less specialized, called stem cells. Cell differentiation is the process of going from the second type to the first and it has become of great interest in several fields such as regenerative medicine. Stem cell commitment involves great changes in protein expression and furthermore in cell signalling events triggered by protein phosphorylation, what makes it a perfect area to apply mass spectrometry based quantitative proteomics and phosphoproteomics. However, identifying and quantifying thousands of features in large scale experiments need to be coupled with computational approaches.The work presented in this thesis shows the power of mass spectrometry based studies combined with bioinformatics analysis to unravel molecular mechanisms in cell differentiation, at embryonic and adult organism level. In Paper 1 we focused in the first day of osteoblastic differentiation. SILAC based quantitation of phosphosites and proteins followed by bioinformatics analysis at different levels allowed us to study the signalling events orchestrating this process. We were able to predict and prove PRKD1 implication in RunX2 regulation as well as other important pathways controlling osteoblastic lineage. In Paper 2 we set up a SILAC based quantitative approach to study human primary myoblast differentiation. We applied c-fuzzy clustering to group the regulated proteins following the different dynamic on muscle differentiation: cell cycle arrest, cell adhesion, migration and myofibril formation. Not only in silico transcription factor analysis complemented this information but also it was possible to track candidate proteins implicated in myofibril development and muscular diseases; demonstrating the strength of our model. In Paper 3, we studied the role of MDM2 in the first steps of adipocyte differentiation. Here, we used two pre-adipocyte mutant cell lines differing in Mdm2 expression and set up a SILAC based assay to see the difference in protein expression. Transcription factor analysis showed STATs implication and, subsequent follow ups the mediation of CRTC transcription factors in controlling adipocytic fate. In Paper 4 we studied Pim2 kinase role in hESC. SILAC based quantitative phosphoproteomic studies coupled with Pim2 inhibitors and random differentiation induction allowed to determine a pool of direct and indirect targets that further confirm Pim2 involvement in transition between self-renewal and differentiation.
M3 - Ph.D. thesis
PB - Syddansk Universitet. Det Naturvidenskabelige Fakultet
CY - Odense
ER -