No description available.

Proteins are intimately involved in almost every cellular phenomenon, from life to death. Understanding the interactions of proteins with each other and other macromolecules and the ability to rationally redesign them to improve their activities or control their function are of considerable current interest. Split-protein methodologies provide an avenue for achieving many of these goals. Since the original discovery of conditionally activated split-ubiquitin, the field has grown exponentially to include the activities of over a dozen different proteins. The flexibility of the systems has resulted in their use across a wide spectrum, both literally and figuratively, to primarily screen, visualize and quantitate macromolecular interactions in a variety of biological systems. In another arena, there is significant interest the apoptosis-regulating proteins: the Bcl-2 family. These proteins are found in many cell types and control, through expression levels as well as other mechanisms, the apoptotic state of a protein as governed by intrinsic death signals generated from such sources as DNA damage and viral infection. The apoptotic function of these proteins are mainly governed by a single type of interaction: the helix:receptor binding of the BH3-Only helices to the anti-apoptotic receptor proteins. While this often promiscuous helix:receptor interaction has received much scrutiny, the nature of the anti-apoptotic binding pocket, especially with regard to the specific residues that govern the interaction, has been lacking. With the high sensitivity and rapid analysis platform afforded by the cell-free split-luciferase analysis methodology, we devised and carried out the first systematic and large scale alanine mutagenesis of all five major anti-apoptotic members of the Bcl-2 family, validated these results both with biophysical methods as well as correlation with previous studies. Our results help explain how different receptors can bind a wide range of helices and also uncovered details regarding binding that are not possible with structural or computational analysis alone. In a second area of research, we have utilized the interaction of BH3 helices and their receptors for designing small molecule controlled protein kinases and phosphatases. In this protein design area, BH3-Only helices were inserted using a knowledge based approach into particular loops within both a protein kinase and a protein phosphatase. The BH3-Only helix interaction with added receptors, such as Bcl-xL provided an allosteric switch for turning-off the activity of the helix-inserted enzymes. The activity of the enzymes could then be turned-on by the addition of a cell-permeable small molecule that is known to bind the receptor. This plug-and-play design was demonstrated to be successful for two very different enzyme classes and likely provides a general and tunable biological element for controlling the activity of one or more proteins and enzymes in a biochemical networks.

Mechanical conditioning has been shown to promote tissue formation in a wide variety of tissue engineering efforts. However the underlying mechanisms by which external mechanical stimuli regulate cells and tissues are not known. This is particularly relevant in the area of heart valve tissue engineering owing to the intense hemodynamic environments that surround native valves. Some studies suggest that oscillatory shear stress (OSS) caused by time-varying flow environments, play a critical role in engineered tissue formation derived from bone marrow derived stem cells (BMSCs). There is strong evidence to support this hypothesis in tissue engineering studies of bone. From observing native heart valve dynamics, OSS can be created by means of pulsatility or by cyclic specimen geometry changes. However, quantification of the individual or combined effects of these variables for the maximization of OSS environments in vitro is to date, not known. Accordingly, in this study we examined and quantified the role that i) physiologically relevant scales of pulsatility and ii) changes in geometry as a function of specimen flexure, have in creating OSS conditions for dynamic culture of tissue. A u-shaped custom made bioreactor capable of producing flow stretch and flexure was used. Computational Fluid Dynamic (CFD) simulations were performed through Ansys CFX (Ansys, Pittsburgh, PA) for both steady and pulsatile flow. We have shown that OSS can be maximized by inducing pulsatile flow over straight scaffolds. We believe that OSS promotes BMSCs tissue formation.

With over 830,000 km of operating pipeline in Canada alone, their safe and continued functioning underpins much of daily life. A key type of risk associated with pipelines is third-party damage, damage caused by actions not associated with the pipelines normal operation. The question of whether the pressurized structure like pipeline or pressure vessel would undergo “unzipping” due to the third-party impact is crucial for the safety of pipelines or pressure vessels in service needs to be answered. Thus, we endeavour to develop a methodology for assessment of design solutions effectiveness to prevent a pipeline or pressure vessel failure in an abrupt explosion-like fashion due to third-party damage. Model of crack propagation determining whether the “unzipping” rupture will occur is viewed as a key element in the safety-driven design procedure providing significant effect on the safety of operation. The crack propagation modeling is achieved through the use of nonlinear fracture mechanics technique. The method of singular integral equations is used to calculate the critical stress required for the catastrophic failure of pipeline or pressure vessel damaged due to third-party interference. The model was implemented as a FORTRAN program. Testing of the developed numerical tool was performed using experimental data available in the literature, with the results showing promising agreement. / February 2016