Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.

In recent years the application of single-molecule techniques to probe biomolecules and intermolecular interactions at single-molecule resolution has expanded rapidly. Here, I investigate a series of peptides and proteins in an attempt to gain a better understanding of nanopore sensing as a single-molecule technique. The analysis of retro, inversed, and retro-inversed isomers of glucagon and α-helical Fmoc-D2A10K2 peptide showed that nanopore sensing utilizing a wild-type α-hemolysin pore can distinguish between all four isomers while circular dichroism can only distinguish between chiral isomers, but not between directional isomers. The investigation of a series of proteins of different chemical and physical properties revealed important information about nanopore analysis of proteins. Contrary to some reports in the literature, all proteins analysed here induced large blockade events. The frequency of total events and the proportion of large blockade events were significantly reduced in tris(hydroxymethyl)aminomethane or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffers and were only restored by the addition of ethylenediaminetetraacetic acid or the use of phosphate buffer, both of which can sequester metal ions. Furthermore, the results obtained with the proteins in the presence of ligands demonstrated that transient or partial unfolding of proteins can be detected by nanopore analysis confirming the usefulness of this technique for conformational studies or for protein/ligand interactions. Interestingly, while the blockade current histograms were different for each protein there was no obvious correlation between the properties of the proteins and the blockade current histograms. In an attempt to identify whether the large blockade events were translocation or intercalation, both an indirect and a direct approach were taken. The indirect approach which relies on the effect of voltage on the interaction of the molecule with the pore provided no conclusive answer to the question of protein translocation through the α-hemolysin pore. In contrast, the direct approach in which ribonuclease A is added to the cis side of the pore and then the trans side is tested for enzyme activity showed that ribonuclease A doesn't translocate through the α-hemolysin pore.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Les objectifs principaux des travaux de recherche réalisés sont de caractériser, quantifier et corréler les changements induits lors du recyclage sur les fibres, la pâte et le papier. Pour ce faire, des techniques de caractérisation spécifique ont été utilisées telles que la chromatographie inverse d'exclusion stérique (ISEC), l'analyse mécanique dynamique (DMA), la microscopie électronique à balayage environnementale (ESEM), la microscopie à force atomique (AFM) et la tomographie à rayons X. Le racornissement des fibres à elle seule ne peut pas expliquer totalement la perte de résistance des fibres. Lors du recyclage la largeur des fibres, l'épaisseur des parois fibreuses, la courbure, le nombre de coudes et d'irrégularité diminuent. Les points faibles à l'intérieur de la paroi augmentent dans les premiers et les derniers cycles de recyclage. Les fibres deviennent plus dures et cassantes à l'état sec. Les forces capillaires et la friction de surface augmentent à l'état humide. La surface de liaison entre fibres dans le réseau fibreux diminue initialement à cause de la perte de la flexibilité des fibres à l'état humide et des éléments fins alors que l'augmentation qui suit peut être reliée au collapse du lumen. Comme la solidité des fibres de diminue pas, la baisse des caractéristiques mécaniques du papier pourrait être attribuée à la dégradation de la qualité des liaisons et plus particulièrement à une délamination partielle de la couche P/S1.

.La densification du névé en glace est un processus essentiel à comprendre pour interpréter les enregistrements climatiques. Une bonne connaissance des mécanismes permet une datation précise de l'air capturé dans la glace lors de la fermeture des pores. Celle-ci est plus vieille que l'air capturé à cause du transport des gaz dans la colonne de névé plus rapide que la densification de celui-ci. Cette différence d'âge entre la glace et le gaz est généralement appelé le Δage. La densification de la neige consiste en un processus complexe de réarrangement de grains, de frittage et de déformation viscoplastique. Bien que le comportement viscoplastique du cristal de glace soit fortement anisotrope, les modèles de densification actuels ne tiennent pas compte de cette anisotropie. De plus, le caractère granulaire du névé affecte aussi sa densification. La relation entre la fermeture des pores et les mécanismes microstructuraux sous-jacents est encore méconnue. Le but de cette thèse est d'incorporer l'aspect granulaire ainsi que l'anisotropie du cristal de glace dans une approche de modélisation innovante de la densification. Des expériences sur l'indentation viscoplastique de cylindres monocristallins de glace ont été réalisées pour proposer une loi de contact basée sur la théorie de l'indentation, et prenant en compte la déformation préférentielle du cristal de glace sur les plans basaux. Cette loi de contact a été implémentée dans un code utilisant la méthode des éléments discrets pour prédire la densification du névé.La micro-tomographie aux rayons X a été utilisée pour caractériser ex situ le névé polaire en trois dimensions à différentes étapes de la densification (ρ= 0.55-0.88 g/cm3), i.e. pour différentes profondeurs (~23 à 130m). Une étude fine de la fermeture des pores et de différentes caractéristiques morphologiques et physiques a été réalisée pour les sites polaires Dome C et Lock In. Des essais mécaniques ont aussi été réalisés in situ sur du névé extrait de Dome C dans le but de modéliser la densification du névé. Les observations microstructurales des expériences ex situ et in situ révèlent d'importantes différences dues aux vitesses relativement importantes utilisées lors des essais mécaniques. Ces vitesses rapides permettent de découpler la contribution des cinétiques de diffusion de la contribution viscoplastique de la déformation. Les effets de ces contributions sur la morphologie des pores et leurs fermetures sont discutés. Pour caractériser la fermeture des pores, cette thèse propose un indice de connectivité définit par le ratio entre le volume du plus gros pore sur la porosité totale. En effet, cet indice est plus approprié lors de l'utilisation de la tomographie aux rayons X que le ratio de pores fermés pour prédire la densité au close-off. / Densification from firn to ice is an essential phenomenon to understand for the interpretation of the climate record. A good knowledge of this mechanism enables the precise dating of the air embedded in the ice. The step at which the air becomes entrapped is the pore closure (or close-off). Because of gas flow in the firn column, the ice is older than the entrapped air. The difference between ice and gas is generally defined as Δage.Snow densification consists of grain rearrangements, sintering and viscoplastic deformation. Although the viscoplastic behaviour of the ice crystal is strongly anisotropic, densification models do not take into account this anisotropy. Firn also bears some granular characteristics that may affect its densification. The interactions between pore closure and microstructural mechanisms in the firn are still misunderstood.The aim of this PhD thesis is to incorporate both the granular aspect of firn and its anisotropy into an innovating approach of firn densification modelling. The mutual indentation of viscoplastic monocrystalline ice cylinders was experimentally carried out to propose a contact law that is based on indentation theory and that takes into account the preferential viscoplastic deformation on the basal plane. We have integrated this contact law into a DEM (Discrete Element Method) code for the prediction of firn densification.3D X-ray micro-tomography was performed on polar firn at different stages of the densification (ρ= 0.55-0.88 g/cm3) and depths (~23 to 130m). A thorough investigation of pore closure and of different morphological and physical parameters was achieved for the Dome C and the newly drilled Lock In polar sites. In addition to these ex situ analyses, in situ X-ray micro-mechanical experiments were carried out on firn extracted from Dome C in order to model its densification. Ex situ and in situ microstructural observations indicate significant differences that can be explained by the relatively large strain-rates imposed to the firn during in situ tests. These large strain rates allow for a decoupling of the effects of diffusion kinetics and of viscoplastic deformation. Their relative weights on the morphology of pores and on their closure are discussed. To measure pore closure, we propose a connectivity index, which is the ratio of the largest pore volume over the total pore volume. We show that this index is better suited for X-ray tomography analysis than the classic closed porosity ratio to predict the close-off density

Une pile à membrane échangeuse de protons (PEMFC) est un dispositif convertissant l’hydrogène en électricité grâce à une réaction électrochimique appelé électrolyse inverse. Comme chaque pile à combustible ou batterie, les PEMFC sont composées d’une série de couches. Nous nous intéressons à la couche de diffusion (GDL) du côté de la cathode. La GDL est constituée de fibres de carbone traitées pour être hydrophobes. Elle peut être vue comme un milieu poreux mince avec une taille moyenne de pores de quelques dizaines de microns. Une question clé dans ce système est la gestion de l'eau produite par la réaction. Dans ce contexte, le principal objectif de la thèse est le développement d'un outil numérique visant à simuler la formation de l'eau liquide dans la GDL. Une approche réseau de pores est utilisée. Nous nous concentrons sur un scénario où l’eau liquide se forme par condensation dans la GDL. Les comparaisons entre simulations et expériences effectuées grâce à un dispositif microfluidique bidimensionnel, sont d'abord présentées pour différentes conditions de mouillabilité, de distributions de température et d'humidité relative à l’entrée, afin de valider le modèle. Une étude de sensibilité est alors effectuée afin de mieux caractériser les paramètres contrôlant l'invasion de l'eau. Enfin, les simulations sont comparées à des distributions d’eau obtenues in-situ par micro-tomographie à rayons X, ainsi que des distributions expérimentales de la littérature obtenues par imagerie neutronique. / A Proton Exchange Membrane Fuel Cell (PEMFC) is a device converting hydrogen into electricity thanks to an electrochemical reaction called reverse electrolysis. Like every fuel cell or battery, PEMFCs are made of a series of layers. We are interested in the gas diffusion layer (GDL) on the cathode side. The GDL is made of carbon fibers treated hydrophobic. It can be seen as a thin porous medium with a mean pore size of few tens of microns. A key question in this system is the management of the water produced by the reaction. In this context, the main objective of the thesis is the development of a numerical tool aiming at simulating the liquid water formation within the GDL. A pore network approach is used. We concentrate on a scenario where liquid water forms in the GDL by condensation. Comparisons between simulations and experiments performed with a two-dimensional microfluidic device are first presented for different wettability conditions, temperature distributions and inlet relative humidity in order to validate the model. A sensitivity study is then performed to better characterize the parameters controlling the water invasion. Finally, simulations are compared with in situ experimental water distributions obtained by X-ray micro-tomography as well as with experimental distributions from the literature obtained by neutron imaging.

No description available.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

BACKGROUND: Netarsudil, a Rho kinase and norepinephrine transport (NET) inhibitor, is a new FDA approved drug used for decreasing raised intraocular pressure (IOP) in ocular hypertensive and primary open-angle glaucoma (POAG) patients. Previous studies reported that netarsudil increased outflow facility and lowered IOP by increasing active outflow areas around the circumference of the eye and dilating the episcleral veins (ESV; Kiel and Kopczynski, 2015; Ren et al., 2016). However, the mechanisms by which netarsudil increases outflow facility have not yet been fully elucidated. Moreover, the effects of netarsudil on the inner wall (IW) endothelium I-pores and B-pores of the Schlemm’s canal (SC) have also not been investigated yet. AIM: The goal was to determine if netarsudil-treatment increased the effective filtration areas (EFA) by increasing pore density in both high- and non-flow type areas, compared to untreated control eyes. METHODS: In this study, the effects of netarsudil on the pore densities on IW of SC were investigated by serial block-face scanning electron microscopy (SBF-SEM). Two pairs of eyes were perfused with green fluorescent tracers in order to determine the outflow pattern prior to treatment. Then, one eye of each pair was perfused with netarsudil, while the fellow eye of each pair was perfused with vehicle solution. All eyes were then perfused with red fluorescent tracers in order to determine the outflow pattern once they were treated with netarsudil. Both pairs of eyes were perfused and fixed at 15 mmHg. Global imaging was performed for all eyes to visualize high- and non- flow areas in the trabecular meshwork (TM) and ESV’s. A SBF-SEM was used to image eight wedges of tissue including the IW of SC and TM (high- and non-flow areas from four eyes) for a total of 16,378 images. The study analyzed the percentage of pore-types (GV-associated I-pores, Non-GV associated I-pores, B-pores), the median pore spans, the GV-associated I-pore locations, and the pore densities (per IW nuclei and IW area) between the equivalent control and netarsudil-treated flow areas. RESULTS: In global images, an increase in high-flow areas were observed in netarsudil-treated eyes due to recruitment from low-flow and non-flow areas. A greater percentage of GV-associated I-pores, B-pores, and total pores were found in high-flow in contrast to non-flow areas in both control and netarsudil-treated eyes (all P ≤ 0.05). However, the percentage of GV-associated I-pores in non-flow areas were significantly greater in treated compared to control eyes (P ≤ 0.05). Qualitative observations from two pairs of eyes showed a trend of greater I-pore, B-pore, and total pore density/per IW nucleus and density/per IW surface area in high-flow in contrast to non-flow areas for both treated and control eyes. No difference in I-pore, B-pore, and total pore density/per IW nucleus and density /per IW surface area were observed in equivalent flow-type areas when comparing control and netarsudil-treated eyes. In addition, there was a significant greater percentage of I-pores located on the side of GVs than the top of GVs in all cases (P ≤ 0.05). CONCLUSIONS: Netarsudil increased high-flow areas. A greater pore density was found in high-flow in contrast to non-flow areas. Netarsudil also significantly increased the proportion of GV-associated I-pores in non-flow areas when compared to control eyes. Our results suggests that one mechanism of netarsudil increasing outflow facility is acting through recruiting the high-flow areas around the circumference of the eye, which is associated with higher pore density and increasing the proportion of GV-associated I-pores in non-flow areas.

No description available.