Cell-substrate interactions influence various cellular processes such as morphology, motility, proliferation and differentiation. Actin dynamics within cells have been shown to be influenced by substrate stiffness, as NIH 3T3 fibroblasts grown on stiffer substrates tend to exhibit more prominent actin stress fiber formation. Circular dorsal ruffles (CDRs) are transient actin-rich ring-like structures within cells, induced by various growth factors, such as the platelet-derived growth factor (PDGF). CDRs grow and shrink in size after cells are stimulated with PDGF, eventually disappearing ten of minutes after stimulation. As substrate stiffness affect actin structures and cell motility, and CDRs are actin structures which have been previously linked to cell motility and macropinocytosis, the role of substrate stiffness on the properties of CDRs in NIH 3T3 fibroblasts and how they proceed to affect cell behavior is investigated. Cells were seeded on Poly-dimethylsiloxane (PDMS) substrates of various stiffnesses and stimulated with PDGF to induce CDR formation. It was found that an increase in substrate stiffness increases the lifetime of CDRs, but did not affect their size. A mathematical model of the signaling pathways involved in CDR formation is developed to provide insight into this lifetime and size dependence, and is linked to substrate stiffness via Rac-Rho antagonism. CDR formation did not affect the motility of cells seeded on 10 kPa stiff substrates, but is shown to increase localized lamellipodia formation in the cell via the diffusion of actin from the CDRs to the lamellipodia. To further probe the influence of cell-substrate interactions on cell behavior and actin dynamics, a two dimensional system which introduces a dynamically changing, reversible and localized substrate stiffness environment is constructed. Cells are seeded on top of thin PDMS nano-membranes, and are capable of feeling through the thin layer, experiencing the stiffness of the polyacrylamide substrates below the nano-membrane. The membranes are carefully re-transplanted on top of other polyacrylamide substrates with differing stiffnesses. This reversible dynamic stiffness system is a novel approach which would help in the investigation of the influence of reversible dynamic stiffness environments on cell morphology, motility, proliferation and differentiation in various cells types.

Dans les dispositifs électronique, il est prochainement attendu que la réduction de la taille des composant atteingne prochainement la limite quantique. De ce fait, manipuler l'information quantique apparait comme un nouveau challenge. Les Qubits supraconducteurs basé sur la physique du solide et les Jonctions Josephson sont des systèmes prometteurs qui profitent des avantage des technologies de la micro-électronique. Toutefois, le temps de décohérence des états quantique est encore un facteur limitant. Cette limitation est généralement attribuée à la faible qualité cristalline des matériaux utilisés (défauts cristallins, impuretés). La technique d'épitaxie par jets moléculaires a été utilisé pour la croissance de couches minces de rhénium de haute qualité cristalline sur des substrat de saphir dans un environnement Ultra Haut Vide. Le misfit existant entre les réseaux cristallins du rhénium et du saphir est suffisamment bas pour permettre une croissance épitaxiale du rhénium sur le saphir, mais également une croissance d'une barrière tunnel en oxyde d'aluminium monocristallin sur la couche de rhénium elle-même. Afin d'améliorer la qualité cristallographique de la couche de rhénium, des simulations et de nombreuses techniques de caractérisation ont été utilisées. Puis les propriétés supraconductrices des films de rhénium ont été étudié à des températures ultra basses afin de comparer ces propriétés à la qualité cristallographique de nos films. / In electronic devices, it is expected that the quantum limit will soon be reached with decreasing system size. Therefore, manipulating quantum information appears as a new challenge. Solid state Qubits based on superconducting Josephson junction are promising systems which take advantage of microelectronics technology. However, decoherence time of the quantum states is still a limiting factor. This has been generally ascribed to the poor crystallographic quality of the materials used so far (crystallographic defects, impurities). The Molecular Beam Epitaxy (MBE) technique may be used to grow rhenium (Re) films of high quality on sapphire substrates in an Ultra High Vacuum (UHV) environment. So far, the misfit between Re and sapphire is low enough to permit the growth of a single crystal aluminium oxide thin film on top of the Re layer. In order to improve the crystallographic quality of the Re film, some simulations and several characterizations techniques have been used. Then, the superconducting properties of rhenium films have been studied at Ultra Low Temperature in order to compare with their crystallographic qualities.

No description available.

Oxygenic photosynthesis in plants, algae and cyanobacteria converts sunlight into chemical energy. In this process electrons are transferred from water molecules to CO2 leading to the assembly of carbohydrates, the building blocks of life. A cluster of four manganese ions and one calcium ion, linked together by five oxygen bridges, constitutes the catalyst for water oxidation in photosystem II (Mn4CaO5 cluster). This cluster stores up to four oxidizing equivalents (S0,..,S4 states), which are then used in a concerted reaction to convert two substrate water molecules into molecular oxygen. The reaction mechanism of this four-electron four-proton reaction is not settled yet and several hypotheses have been put forward. The work presented in this thesis aims at clarifying several aspects of the water oxidation reaction by analyzing the mode of substrate water binding to the Mn4CaO5 cluster. Time-resolved membrane-inlet mass spectrometric detection of flash-induced O2 production after fast H218O labelling was employed to study the exchange rates between substrate waters bound to the Mn4CaO5 cluster and the surrounding bulk water. By employing this approach to dimeric photosystem II core complexes of the red alga Cyanidoschyzon merolae it was demonstrated that both substrate water molecules are already bound in the S2 state of the Mn4CaO5 cluster. This was confirmed with samples from the thermophilic cyanobacterium Thermosynechococcus elongatus. Addition of the water analogue ammonia, that is shown to bind to the Mn4CaO5 cluster by replacing the crystallographic water W1, did not significantly affect the exchange rates of the two substrate waters. Thus, these experiments exclude that W1 is a substrate water molecule. The mechanism of O-O bond formation was studied by characterizing the substrate exchange in the S3YZ● state. For this the half-life time of this transient state into S0 was extended from 1.1 ms to 45 ms by replacing the native cofactors Ca2+ and Cl- by Sr2+ and I-. The data show that both substrate waters exchange significantly slower in the S3YZ● state than in the S3 state. A detailed discussion of this finding lead to the conclusions that (i) the calcium ion in the Mn4CaO5 cluster is not a substrate binding site and (ii) O-O bond formation occurs via the direct coupling between two Mn-bound water-derived oxygens, which were assigned to be the terminal water/hydroxy ligand W2 and the central oxo-bridging O5. The driving force for the O2 producing S4→S0 transition was studied by comparing the effects of N2 and O2 pressures of about 20 bar on the flash-induced O2 production of photosystem II samples containing either the native cofactors Ca2+ and Cl- or the surrogates Sr2+ and Br-. While for the Ca/Cl-PSII samples no product inhibition was observed, a kinetic limitation of O2 production was found for the Sr/Br-PSII samples under O2 pressure. This was tentatively assigned to a significant slowdown of the O2 release in the Sr/Br-PSII samples. In addition, the equilibrium between the S0 state and the early intermediates of the S4 state family was studied under 18O2 atmosphere in photosystem II centers devoid of tyrosine YD. Water-exchange in the transiently formed early S4 states would have led to 16,18O2 release, but none was observed during a three day incubation time. Both experiments thus indicate that the S4→S0 transition has a large driving force. Thus, photosynthesis is not limited by the O2 partial pressure in the atmosphere.

In this thesis, we are making a new approach to fabricate silicon on insulator (SOI). By replacing the buried silicon dioxide and the silicon handling wafer with silicon carbide through hydrophilic wafer bonding, we have achieved silicon on crystalline silicon carbide for the first time and silicon on polycrystalline silicon carbide substrates at 150 mm wafer size. The conditions for the wafer bonding are studied and the surface and bond interface are characterized. Stress free and interfacial defect free hybrid wafer bonding has been achieved. The thermally unfavourable interfacial oxide that originates from the hydrophilic treatment has been removed through high temperature annealing, denoted as Ox-away. Based on the experimental observations, a model to explain the dynamics of this process has been proposed. Ox-away together with spheroidization are found to be the responsible theories for the behaviour. The activation energy for this process is estimated as 6.4 eV. Wafer bonding of Si and polycrystalline SiC has been realised by an intermediate layer of amorphous Si. This layer recrystallizes to some extent during heat treatment. Electronic and thermal testing structures have been fabricated on the 150 mm silicon on polycrystalline silicon carbide hybrid substrate and on the SOI reference substrate. It is shown that our hybrid substrates have similar or improved electrical performance and 2.5 times better thermal conductivity than their SOI counterpart. 2D simulations together with the experimental measurements have been carried out to extract the thermal conductivity of polycrystalline silicon carbide as κpSiC = 2.7 WK-1cm-1. The realised Si-on-SiC hybrid wafer has been shown to be thermally and electrically superior to conventional SOI and opens up for hybrid integration of silicon and wide band gap material as SiC and GaN.

The advent of Substrate Integrated Circuit (SIC) technology, and specifically Substrate Integrated Waveguide (SIW) technology has made it feasible to design and fabricate low loss and high quality factor (Q-factor) microwave and millimeter wave structures on a compact and integrable layout and at a low cost. The SIW structure is the planar realization of the conventional rectangular waveguide (RWG). In this technology, the side walls of the waveguide are replaced with two rows of metallic vias, which are connecting two conductor sheets, located at the top and bottom of a dielectric slab. The motivation for this thesis has been to develop an analytical method to efficiently analyze SIW structures, and also design different types of passive microwave components based on this technology. As SIW structures are imitating waveguide structures in a planar format, the field distributions inside these structures are very close to those in waveguides. However, due to the very small substrate height in conventional planar technologies, and also the existence of a row of vias, instead of a solid metallic wall, there is a reduced set of modes in SIW compared to regular waveguide. This fact has given us an opportunity to deploy efficient modal analysis techniques to analyze these structures. In this thesis, we present a Mode Matching Techniques (MMT) approach for the analysis of H-plane SIW structures. One of the areas of application, which can significantly benefit from having an efficient analytical method, is designing and optimizing new circuits. Having such an analytical tool, which is faster than commercially available field solvers by an order of magnitude, new components can be designed, analyzed and optimized in a fast and inexpensive manner. Based on this technique, various types of passive microwave components including filters, diplexers, power dividers and couplers, some of which are among the first to be reported in SIW technology, are designed and analyzed in this thesis. Also based on this technique, the most accurate formula for the effective waveguide width of the SIW is presented in this thesis. In order to provide means to excite and measure SIW components, transitions between these structures and other planar topologies like microstrip and coplanar waveguide (CPW) are needed. More importantly, low-reflection transitions to microstrip are required to integrate SIW circuits with active components, and therefore it is vital to provide low-reflection transitions so that the component design is independent of the influences of the transitions. In this thesis, a new wideband microstrip-to-SIW transition, with the lowest reported reflection coefficient, is also introduced. / Graduate / 0544 / zkordi@ece.uvic.ca

The relationship between substrate oxidation (RER) and food intake (FI) is undefined. This study examined the effects of RER modified by a glucose pre-load (GL), exercise (EX) and GL with EX on, FI and energy balance (NEB) in normal-weight boys (9-12 y) and men (20-30 y). Subjects (15 boys, 15 men) were randomized with treatments of either water or GL followed by either EX or rest. Measures included RER, energy expenditure (EE)(kcal/kg), subjective appetite, FI(kcal/kg) measured at a pizza lunch and NEB (kcal/kg). FI(kcal/kg) was reduced by GL(p < 0.0001), and further decreased with GL ingested prior to EX(p = 0.0254). RER was increased with GL(p < 0.0001) and EX(p = 0.0043), and was higher in men compared to boys (p = 0.007). There was no association between RER and FI(kcal/kg). In conclusion, there was no relationship between RER and FI, suggesting that FI is not affected by substrate oxidation.

In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with ‘‘known’’ function are mis-annotated. Several global approaches are being employed to predict function, including sequence similarity searches, analysis of gene expression, protein interaction, and protein structure. Enzymes comprise a group of target proteins that require experimental characterization for accurate functional annotations. Here I applied enzyme genomics to identify new enzymes by screening individually purified proteins for enzymatic activity under relaxed reaction conditions, which allowed me to identify the subclass or sub-subclasses of enzymes to which the unknown protein belongs. Further biochemical characterization of proteins was facilitated by the application of secondary screens with natural substrates (substrate profiling). Application of general enzymatic screens and substrate profiling greatly sped up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches. As a test case, I used this approach to characterize the members of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which consists mainly of uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Low sequence similarity between the members of the HAD superfamily precludes the computational prediction of their substrates and functions. Using a representative set of 80 phosphorylated substrates I characterized the phosphatase activities of 21 soluble HADs from Escherichia coli and seven soluble HADs from Saccharomyces cerevisiae. E. coli HADs show broad and overlapping substrate specificity against a wide range of phosphorylated metabolites. The yeast enzymes were more specific, and one protein also showed protein phosphatase activity. Comparison of HAD substrate profiles from two model organisms showed several “functional niches” that are occupied by HADs, which include hydrolysis of nucleotides, phosphoglycolate, phosphoserine, and pyridoxal phosphate. I proposed the cellular function for a number of HADs from both organisms based on substrate specificities. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, E. coli YniC.

The relationship between substrate oxidation (RER) and food intake (FI) is undefined. This study examined the effects of RER modified by a glucose pre-load (GL), exercise (EX) and GL with EX on, FI and energy balance (NEB) in normal-weight boys (9-12 y) and men (20-30 y). Subjects (15 boys, 15 men) were randomized with treatments of either water or GL followed by either EX or rest. Measures included RER, energy expenditure (EE)(kcal/kg), subjective appetite, FI(kcal/kg) measured at a pizza lunch and NEB (kcal/kg). FI(kcal/kg) was reduced by GL(p < 0.0001), and further decreased with GL ingested prior to EX(p = 0.0254). RER was increased with GL(p < 0.0001) and EX(p = 0.0043), and was higher in men compared to boys (p = 0.007). There was no association between RER and FI(kcal/kg). In conclusion, there was no relationship between RER and FI, suggesting that FI is not affected by substrate oxidation.

The human protein kinase superfamily consists of over 500 members that individually control specific aspects of cell behavior and collectively control the complete range of cellular processes. That such a large group of proteins is able to uniquely diversify and establish individual identities while retaining common enzymatic function and significant sequence/structural conservation is remarkable. The means by which this is achieved is poorly understood, and we have begun to examine the issue by performing a comparative analysis of the catalytic domain of protein kinases. A novel approach for protein structural alignment has revealed a high degree of similarity found across the kinase superfamily, with variability confined largely to a single region thought to be involved in substrate binding. The similarity detected is not limited to amino acids, but includes a group of conserved water molecules that play important structural roles in stabilizing critical residues and the fold of the kinase domain. The development of a novel technique for identifying kinase substrates on a large scale directly from cell lysate has revealed that substrate specificity is not what discriminates the closely related p38α and β mitogen-activated protein kinases. Instead cellular localization appears to be their distinguishing characteristic, at least during myoblast differentiation. Together these results highlight the extent of conservation, as well as the minimal variability, that is found in the catalytic domain of all protein kinase superfamily members, and that while distantly related kinases may be distinguished by substrate specificity, closely related kinases are likely to be distinguished by other factors. Although these results focus on representative members of the kinase superfamily, they give insight as to how all protein kinases likely diversified and established unique non-redundant identities. In addition, the novel techniques developed and presented here for structural alignment and substrate discovery offer new tools for studying molecular biology and cell signaling.