<p> Bcl-2 proteins play a critical role in the regulation of apoptosis, a form of programmed cell death. Apoptosis is important during development to facilitate the elimination of supernumerary, damaged or harmful cells in multicellular organisms. Altered regulation of apoptosis is associated with many diseases such as several forms of cancer as well as autoimmune and degenerative disorders. The way in which Bcl-2 proteins regulate apoptosis is unknown and much research is focused on elucidating the molecular mechanism of their function. Bcl-2, an anti-apoptotic member of this family, is localized to the mitochondria, endoplasmic reticulum and nuclear envelope. In healthy cells, Bcl-2 adopts a typical tail-anchored topology in which the carboxyl-terminal helix (a9) is inserted into the membrane, anchoring the protein, leaving the majority of the protein in the cytosol. Previous results from our lab have shown that after the induction of apoptosis, Bcl-2 undergoes a conformational change in which the endogenous cysteine residue, C158, in the a5 helix becomes protected from a membrane impermeant cysteine specific labelling reagent, IASD (4-acetamido-4' ((iodoacetyl)amino)-stilbene-2,2'disulfonate). Modification of cysteine residues results in a change in migration ofBcl-2 in an isoelectric focusing, IEF, gel system. To investigate the nature of this conformational change, cysteine scanning mutagenesis was used to determine the topology of Bcl-2 in the late stages of apoptosis. The results from the current study showed that in rat 1 myc ERTM fibroblasts, a discontinuous sequence of residues in the a5 and a6 helices of Bcl-2 become protected from IASD labelling after the induction of apoptosis by etoposide or serum starvation. The data support a model topology in which, during apoptosis, Bcl-2 undergoes a functionally significant conformational change, going from a single spanning transmembrane protein to a polytopic membrane protein in which three helices span the membrane, a5, a6 and a9. </p> / Thesis / Master of Science (MSc)

Automated and continuous processes are the future trends in downstream protein purification. A functionalized nanometer-scale membrane electrode system, mimicking the function of cell wall transporters, can selectively capture genetically modified proteins and subsequently pump them through the system under programmed voltage pulses. Numerical study of the two-step pulse pumping cycles coupled with experimental His-GFP releasing study reveals the optimal 14s/1s pumping/repel pulse pumping condition at 10 mM bulk imidazole concentration in the permeate side. A separation factor for GFP: BSA of 9.7 was achieved with observed GFP electrophoretic mobility of 3.1×10-6 cm2 s-1 V-1 at 10 mM bulk imidazole concentration and 14 s/1 s pumping/repel duration. The purification of His6-OleD Loki variant directly from crude E. coli extracts expression broth was demonstrated using the pulse pumping process, simplifying the separation process as well as reducing biopharmaceutical production costs. The enzymatic reactions showed that His6-OleD Loki was still active after purification. A nanoporous membrane/electrode system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzymes-complex system was demonstrated. The substrates residence time on the immobilized enzyme can be precisely controlled by changing the pumping rate and thereby prevent a secondary hydrolysis reaction. Immobilized enzyme showed long term storage longevity with activity half-life of 50 days at 4℃ and the ability to be regenerated. One-step immobilization and purification of His-tagged OleD Loki variant directly from expression broth, yielded 98% Uridine Diphosphate glycosylation and 80% 4-methylumbelliferone glycosylation conversion efficiency for the sequential reaction. A flow-through electroporation system, based on a novel membrane/electrode design, for the delivery of membrane-impermeant molecules into Model Leukocyte cells was demonstrated. The ability to apply low voltage between two short distance electrodes contributes to high cell viability. The flow-through system can be easily scaled-up by varying the micro-fluidic channel geometry and/or the applied voltage pulse frequency. More importantly, the system allows the electrophoretical pumping of molecules from the reservoir across the membrane/electrode system to the micro-fluidic channel for transfection, which reduces large amount of reagents used.

Dielectric elastomers (DEs) are a relatively new electroactive polymer (EAP) transducer technology. They are capable of over 100% strain when actuated, and can be used as sensors to measure large strains. In actuation mode, the DE is subject to an electric field; in sensing mode, the capacitance of the dielectric elastomer is measured. In this work, a dielectric elastomer configured as a circular membrane clamped around its outer edge over a sealed chamber and inflated by a bias pressure is studied in order to characterize its static and dynamic electromechanical behavior. In both cases, the experiments were conducted with prestretched dielectric elastomer actuators fabricated from 0.5 mm or 1 mm thick polyacrylate films and unless stated otherwise carbon grease electrodes were used. The static tests investigate the effect of flexible electrodes and passive layers on the electromechanical response of dielectric elastomer membrane actuators and sensors. To study the effect of the flexible electrodes, four compliant electrodes were tested: carbon grease, silver grease, graphite spray, and graphite powder. The electrode experiments show that carbon grease is the most effective electrode of those tested. To protect the flexible electrodes from environmental hazards, the effect of adding passive elastic layers to the transducers was investigated. A series of tests were conducted whereby the position of the added layers relative to the transducer was varied: (i) top passive layer, (ii) bottom passive layer, and (iii) passive layers on both the bottom and top of the transducer. For the passive layer tests, the results show that adding elastic layers made of the same material as the DE dramatically changes both the mechanical and electrical response of the actuator. The ability to use capacitance measurements to determine the membrane's maximum stretch was also investigated. The experiments demonstrate that the capacitance response can be used to sense large mechanical strains in the membrane ï ³ 25%. In addition, a numerical model was developed which correlates very well with the experimental results especially for strains up to 41%. The dynamic experiments investigate the dynamic response of a dielectric elastomer membrane due to (i) a time-varying pressure input and (ii) a time-varying voltage input. For the time-varying pressure experiments, the prestretched membrane was inflated and deflated mechanically while a constant voltage was applied. The membrane was cycled between various predetermined inflation states, the largest of which was nearly hemispherical, which with an applied constant voltage of 3 kV corresponded to a maximum strain at the pole (center of membrane) of 28%. These experiments show that for higher voltages, the volume displaced by the membrane increases and the pressure inside the chamber decreases. For the time varying voltage experiments, the membrane was passively inflated to various predetermined states, and then actuated. Various experiments were conducted to see how varying certain system parameters changed the membrane's dynamic response. These included changing the chamber volume and voltage signal offset, as well as measuring the displacement of multiple points along the membrane's radius in order to capture its entire motion. The chamber volume experiments reveal that increasing the size of the chamber onto which the membrane is clamped will cause the resonance peaks to shift and change in number. For these experiments, the pole strains incurred during the inflation were as high as 26 %, corresponding to slightly less than a hemispherical state. Upon actuation using a voltage signal with an amplitude of 1.5 kV, the membrane would inflate further, causing a maximum additional strain of 12.1%. The voltage signal offset experiments show that adding offset to the input signal causes the membrane to oscillate at two distinct frequencies rather than one. Lastly, experiments to capture the entire motion of the membrane revealed the different mode shapes the membrane's motion resembles. / Master of Science