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Molecular chaperones and proteases help maintain protein homeostasis in the cell. While chaperones assist in the folding of polypeptide chains to their native state, proteases degrade misfolded or unfolded proteins and also help regulate protein levels. While mapping chaperone interaction networks, we found that tig (trigger factor chaperone gene), clpP and clpX genes co-localize next to each other on the genome of most examined bacteria. This led us to hypothesize that trigger factor (TF) chaperone and ClpXP protease might interact functionally. TF is a ribosome-associated chaperone that co-translationally folds polypeptide chains. ClpXP is a proteolytic complex that degrades a wide range of substrate proteins. We observed that TF enhanced the rate of the ClpXP degradation of the λO phage protein in vitro and in vivo. TF was also found to enhance the degradation of ribosome-stalled λO thus suggesting the existence of co-translational protein degradation in E. coli.

Factor VIII (FVIII) is an essential blood-clotting protein and mutations in the FVIII gene are the cause of hemophilia A, a severe inherited bleeding disorder. FVIII synthesis has been observed in discreet endothelial sub-populations including liver sinusoidal endothelial cells and in selected microvascular beds. The mechanistic basis for this differential expression is unknown. Differences in shear stress are believed to play an important role in determining endothelial heterogeneity. In this study, we have evaluated the effect of various shear stress conditions on FVIII expression in blood outgrowth endothelial progenitor cells (BOECs) with an in vitro flow system. Under static conditions, BOECs do not express FVIII. In contrast, after exposure to laminar shear stress for 48 hrs, a significant increase in FVIII expression was documented by qRT-PCR, regardless of the magnitude of shear stress studied (1, 5, 15 and 30 dynes/cm2). To determine the effect of prolonged shear stress, laminar flow was applied over 120 hrs and FVIII mRNA levels returned to static levels. Induction of gene expression by laminar shear stress followed by repression after longer durations is common to other pro-coagulant genes induced by non-laminar or oscillatory flow (eg. tissue factor). BOECs exposed to 15 dyne/cm2 of shear stress, oscillating every 0.5 sec for 120 hrs, had FVIII mRNA levels 4.7-fold that of cells in static conditions. This was significantly higher than FVIII expression in BOECs exposed to 15 dyne/cm2 of laminar shear stress for the same duration. Expression of KLF2, a transcription factor that suppresses endothelial pro-coagulant gene expression under laminar shear stress, was significantly reduced in BOECs exposed to oscillatory as opposed to laminar shear stress. Finally, in BOECs exposed to oscillatory shear stress, FVIII protein was synthesized and co-localizes with its carrier protein VWF in Weibel-Palade bodies. These studies show that shear stress is a significant regulator of FVIII expression in BOECs, that FVIII expression is inversely correlated with that of KLF2, and that FVIII protein co-localizes with VWF in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2013-03-04 17:00:27.994

Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.