Дисертації з теми "Direct surface interactions"

In this dissertation, we describe a novel method that we call Diffusing Colloidal Probe Microscopy (DCPM), which integrates Total Internal Reflection Microscopy (TIRM) and Video Microscopy (VM) methods to monitor three dimensional trajectories in colloidal ensembles levitated above macroscopic surfaces. TIRM and VM are well established optical microscopy techniques for measuring normal and lateral colloidal excursions near macroscopic planar surfaces. The interactions between particle-particle and particle-substrate in colloidal interfacial systems are interpreted by statistical analyses from distributions of colloidal particles; dynamic properties of colloidal assembly are also determined from particle trajectories. Our studies show that DCPM is able to detect many particle-surface interactions simultaneously and provides an ensemble average measurement of particle-surface interactions on a homogeneous surface to allow direct comparison of distributed and average properties. A benefit of ensemble averaging of many particles is the diminished need for time averaging, which can produce orders of magnitude faster measurement times at higher interfacial particle concentrations. The statistical analyses (Ornstein- Zernike and three dimensional Monte Carlo analyses) are used to obtain particle-particle interactions from lateral distribution functions and to understand the role of nonuniformities in interfacial colloidal systems. An inconsistent finding is the observation of an anomalous long range particle-particle attraction and recovery of the expected DLVO particle-wall interactions for all concentrations examined. The possible influence of charge heterogeneity and particle size polydispersity on measured distribution functions is discussed in regard to inconsistent particle-wall and particle-particle potentials. In the final part of this research, the ability of DCPM is demonstrated to map potential energy landscapes on patterned surfaces by monitoring interactions between diffusing colloidal probes with Au pattern features. Absolute separation is obtained from theoretical fits to measured potential energy profiles and direct measurement by sticking silica colloids to Au surfaces via electrophoretic deposition. Initial results indicate that, as colloidal probe and pattern feature dimensions become comparable, measured potential energy profiles suffer some distortion due to the increased probability of probes interacting with surfaces at the edges of adjacent pattern features. Measurements of lateral diffusion via analysis of mean square displacements also indicated lateral diffusion coefficients in excellent agreement with rigorous theoretical predictions.

Les hydroxydes doubles lamellaires (HDL) sont des composés solides constitués par un ensemble de feuilles d'hydroxydes métalliques divalents M(II) et trivalents M(III) entre lesquels s’insèrent des anions et des molécules d’eau. En raison de la flexibilité des HDL en termes de propriétés physico-chimiques, l’étude des différents mécanismes antibactériens qui leurs sont associés présente un intérêt croissant. Ce travail de thèse vise à étudier différentes hypothèses proposées pour explique l'effet antibactérien des hydroxydes doubles lamellaire (HDL) à base de zinc : (1) interactions directes entre la surface des HDL et les parois bactériennes, (2) libération d'ions métalliques divalents en solution depuis les feuillets des HDL, (3) génération d'espèces réactives de l'oxygène (ROS). Lors d’une première étude, une investigation globale a été réalisée pour déterminer les différents paramètres physico-chimiques influençant l'activité antibactérienne des HDLs M(II)Al(III) (M= Zn, Cu, Ni, Co, Mg). L'effet antimicrobien des HDLs contre les bactéries Gram-positives Staphylococcus aureus et Gram-négatives Escherichia coli a été corrélé en premier lieu à la nature du métal divalent, et en second lieu à la quantité d'ions M2+aq libérés dans le milieu de culture. Cet effet était plus prononcé pour les HDLs à base de Zn(II) possédant la plus forte activité antibactérienne et dont les propriétés antibactériennes dépendent du profil de libération des ions Zn2+aq (Mécanisme 2) contrôlée initialement par les différents paramètres physico-chimiques étudiés. De plus, rôle du contact direct (Mécanisme 2) a été validé pour les HDLs à base de Zn(II) en comparant l'activité antibactérienne d’HDLs de taille micrométrique contre S. aureus à celle des nanoparticules (NPs) d’HDL présentant un effet antibactérien supérieur. La présence d'interactions spécifiques entre les HDLs à base de Zn(II) et la paroi de S. aureus a été validée par microscopie à force atomique en mode spectroscopie de force (AFM-FS). L'amélioration des propriétés antibactériennes des NPs d’HDL à base de Zn(II) par la génération de ROS (Mécanisme 3) en présence de lumière UVA a également été évaluée. Après avoir fourni des preuves expérimentales sur les trois mécanismes suggérés, la contribution de chaque mécanisme dans l'activité antibactérienne des HDLs à base de Zn(II) a été déterminé
Layered double hydroxides (LDH) are solid compounds constituted by the stacking of divalent M(II) and trivalent M(III) metal hydroxide sheets separated by an interlayer of anions and water molecules. Due to the versatility of LDH in terms of their tunable physico-chemical properties, a growing interest arises for investigating their different antibacterial activity mechanisms. This thesis work aims at studying the different proposed hypotheses explaining the antibacterial effect of pristine zinc-based LDHs: (1) direct interactions between the surface of LDH and bacterial cell walls, (2) release of constituent divalent metal ions, (3) generation of reactive oxygen species (ROS). First a global investigation was performed to determine the different physico-chemical parameters influencing the antibacterial activity of pristine M(II)Al(III) LDHs (M= Zn, Cu, Ni, Co, Mg). The antimicrobial effect of LDHs against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria was linked in the first place to the nature of divalent metal itself, and to the amount of released M2+aq ions into the culture media in the second place. This effect was more easily identified in Zn(II)-based LDHs possessing the strongest antibacterial activity and whose antibacterial properties depended on their release profile of Zn2+aq ions (Mechanism 2) initially controlled by the different physico-chemical parameters. Moreover, the direct contact mechanism (Mechanism 1) was validated for Zn(II)-based LDHs by comparing the antibacterial activity of micron-sized LDHs against S. aureus to that of LDH nanoparticles (NPs) exhibiting a greater antibacterial effect. The presence of specific surface interactions between Zn(II)-based LDHs and the cell wall of S. aureus was further validated by atomic force microscopy-based force spectroscopy (AFM-FS). The enhancement of the antibacterial properties of Zn(II)-based LDH NPs by ROS generation (Mechanism 3) in presence of UVA light was also assessed. After providing experimental evidences about the three suggested mechanisms, the role of each mechanism contributing to the antibacterial activity of Zn(II)-based LDHs in different antibacterial tests assays was determined

To promote both quality and economy in the production of applications and their interactive interfaces, it is desirable to delay their mutual binding. The later the binding, the more separable the interface from its application. An ideally separated interface can factor tasks from a range of applications, can provide a level of independence from hardware I/O devices, and can be responsive to end-user requirements. Current interface systems base their separation on two different abstractions. In linguistic architectures, for example User Interface Management Systems in the Seeheim model, the dialogue or syntax of interaction is abstracted in a separate notation. In agent architectures like Toolkits, interactive devices, at various levels of complexity, are abstracted into a class or call hierarchy. This Thesis identifies an essential feature of the popular notion of direct manipulation: directness requires that the same object be used both for output and input. In practice this compromises the separation of both dialogue and devices. In addition, dialogue cannot usefully be abstracted from its application functionality, while device abstraction reduces the designer's expressive control by binding presentation style to application semantics. This Thesis proposes an alternative separation, based on the abstraction of the medium of interaction, together with a dedicated user agent which allows direct manipulation of the medium. This interactive medium is called the surface. The Thesis proposes two new models for the surface, the first of which has been implemented as Presenter, the second of which is an ideal design permitting document quality interfaces. The major contribution of the Thesis is a precise specification of an architecture (UMA), whereby a separated surface can preserve directness without binding in application semantics, and at the same time an application can express its semantics on the surface without needing to manage all the details of interaction. Thus UMA partitions interaction into Surface Interaction, and deep interaction. Surface Interaction factors a large portion of the task of maintaining a highly manipulable interface, and brings the roles of user and application designer closer.

This dissertation deals with the simulation of dispersed multiphase flow. The particle-particle and particle-fluid interactions in this class of flows play an important role on the hydrodynamics and fluid transport phenomena that govern the overall flow behaviour. Accurate computational modelling of the particle-particle and particle- fluid interactions is thus required to correctly model the flow. The aim of this study is to use a Direct Numerical Simulation approach based on a smoothed Volume Of Fluid method to model particle-particle interactions in a dispersed multiphase flow at a fundamental level, and employing a surface thin film model, to drastically reduce the computational effort required. A multiscale modelling approach is followed with the smoothed Volume Of Fluid simulation on the particle scale and the surface thin film model simulation on the thin- film scale. The resulting governing equations are the Navier-Stokes equations for an incompressible viscous multiphase Newtonian fluid undergoing laminar and isothermal three-dimensional flow, the interface advection equation and the reduced order surface thin film equation. The model equations are discretized using the Finite Volume Method and implemented into the open source software OpenFOAM®. The numerical solution is obtained by solving the resulting non-linear system of equations implicitly on a structured computational grid on parallel processors using a pressure correction algorithm to converge the pressure at each time step. The study is restricted to gas-liquid systems where particles could either be bubbles or droplets; rigid particles are not considered. The model is tested against experimental results from binary collision of hydrocarbon droplets. Good qualitative numerical results are obtained at a practical computational cost.

The contact condensation of saturated steam on liquid spray has been studied. The mathematical model of heat and mass transfer processes between dispersed liquid and steam has been usin one-parameter drop distribution function has been worked out. The rational degree of liquid dispersing and optimál irrigation scheme has been defined.

xviii, 192 p.
Gold nanoparticles in the 1-2 mn core diameter size regime have generated a great deal of interest due to their size-dependent electronic, optical, and catalytic properties. A number of proof-of-concept experiments have demonstrated that small metal nanoparticles can be integrated into single electron transistors and optical waveguides. Still, reliable incorporation of gold nanoparticles into devices requires practical methods for their assembly on surfaces. Additionally, surface modification methods must be developed in order to control interparticle interactions and nanoparticle-environment interactions for use in sensing and catalysis. In this research, nanoparticle-substrate interactions were utilized to assemble surface-bound gold nanoparticle monolayers with interesting electronic and catalytic properties. Gold nanoparticles (1.5 nm diameter) with a thiol ligand shell containing phosphonic acid terminal functionality were synthesized and assembled selectively onto hafnium-modified silicon dioxide substrates through bonding of the terminal phosphonate to Hf(IV) surface groups. By increasing the surface coverage of Hf, it was possible to assemble monolayers of gold nanoparticles dense enough to exhibit nonlinear current-voltage properties across a 5-μm electrode gap at room temperature. Moreover, by taking advantage of the selectivity of this ligand shell for ZnO over SiO 2 , small gold nanoparticles were utilized as catalysts for selective growth of patterned, vertical ZnO nanowire arrays. In addition to engineering nanoparticle-substrate interactions, new surface modification methods were introduced to manipulate the interaction of the as-deposited gold nanoparticle monolayers with the environment. For example, thiol-thiol ligand exchange reactions were carried out on the surface-bound nanoparticle monolayers by immersion in dilute thiol solutions. Contact angle and XPS measurements indicate that the upper, surface-exposed phosphonic acid ligands are replaced by incoming thiol ligands. TEM measurements indicate that nanoparticle monolayers remain surface-bound and are stable to this exchange process, as the average particle size and surface coverage are preserved. As another example, the ligand shell can be partially removed by UV/ozone treatment to expose bare gold cores to the surrounding environment. On metal oxide substrates, this approach activates the particles for room temperature oxidation of carbon monoxide to carbon dioxide. This dissertation includes both my previously published and my co-authored materials.
Adviser: James E. Hutchison

Dans cette Thèse de Doctorat, nous nous intéressons à deux problématiques: (i) le développement de stratégies de stabilisation pour des méthodes de type discontinuous Galerkin (DG) appliquées à des écoulements shallow-water fortement non-linéaires, (ii) le développement d'une stratégie de modélisation et de simulation numérique des interactions non-linéaires entre les vagues et un objet flottant en surface, partiellement immergé. Les outils développés dans le cadre du premier axe de travail sont mis à profit et valorisés au cours de la deuxième partie.Les méthodes de discrétisation de type DG d'ordre élevé présentent en général des problèmes de robustesse en présence de singularités de la solution. Ces singularités peuvent être de plusieurs natures: discontinuité de la solution, discontinuité du gradient ou encore violation de la positivité de la hauteur d'eau pour des écoulements à surface libre. Nous introduisons dans la première partie de ce manuscript deux approches de type Finite-Volume Subcells permettant d'apporter une réponse à ces problèmes de robustesse. La première approche repose sur une correction a priori du schéma DG associée à un limiteur TVB et un limiteur de positivité. La seconde approche s'appuie quant à elle sur une correction a posteriori permettant d'identifier avec une meilleure précision les cellules incriminée, ainsi que sur les propriétés de robustesse inhérentes au schéma Volumes-Finis limite d'ordre un. Cette seconde approche permet d'assurer la robustesse du schéma DG initial en présence de discontinuité, ainsi que la positivité de la hauteur d'eau, tout en préservant une excellente qualité d'approximation, bénéficiant d'une résolution de l'ordre de la sous-maille. De façon préliminaire, cette seconde approche est également étendue au cas de la dimension deux d'espace horizontal. De nombreux cas-test permettent de valider cette approche.Dans la seconde partie, nous introduisons une nouvelle stratégie numérique conçue pour la modélisation et la simulation des interactions non linéaires entre les vagues en eau peu profonde et un objet flottant partiellement immergé. Au niveau continu, l'écoulement situé dans le domaine extérieur est globalement modélisé par les équations hyperboliques non-linéaires de Saint-Venant, tandis que la description de l'écoulement sous l'objet se réduit à une équation différentielle ordinaire non linéaire. Le couplage entre l'écoulement et l'objet est formulé comme un problème au bord, associé au calcul de l'évolution temporelle de la position des points d'interface air-eau-objet. Au niveau discret, la formulation proposée s'appuie sur une approximation DG d'ordre arbitraire, stabilisée à l'aide de la méthode de correction locale des sous-cellules (a posteriori) introduite dans la première partie. L'évolution temporelle de l'interface air-eau-objet est calculée à partir d'une description Arbitrary Lagrangian-Eulerian (ALE) et d'une transformation appropriée entre la configuration initiale et celle dépendant du temps. Pour n'importe quel ordre d'approximation polynomiale, l'algorithme résultant est capable de: (1) préserver la loi de conservation géométrique discrète (DGCL), (2) garantir la préservation de la positivité de la hauteur d'eau au niveau des sous-cellules, (3) préserver la classe des états stationnaires au repos (well-balancing), éventuellement en présence d'un objet partiellement immergé.Plusieurs validations numériques sont présentées, montrant le caractère opératoire de cette approche, et mettant en évidence que le modèle numérique proposé: (1) permet effectivement de modéliser les différents types d'interactions vague / objet flottants, (2) calcul efficacement l' évolution temporelle des points de contact air-eau-objet et redéfinit en conséquence le nouveau maillage grâce à la méthode ALE, (3) gère avec précision et robustesse les possibles singularités de l'écoulement, (4) préserve la haute résolution des schémas DG au niveau des sous-cellules
In this Ph.D., we investigate two main research problems: (i) the design of stabilization patches for higher-order discontinuous-Galerkin (DG) methods applied to highly nonlinear free-surface shallow-water flows, (ii) the construction of a new numerical approximation strategy for the simulation of nonlinear interactions between waves in a free-surface shallow flow and a partly immersed floating object. The stabilization methods developed in the first research line are used in the second part of this work.High-order discontinuous-Galerkin (DG) methods generally suffer from a lack of nonlinear stability in the presence of singularities in the solution. Such singularities may be of various kinds, involving discontinuities, rapidly varying gradients or the occurence of dry areas in the particular case of free-surface flows. In the first part of this work, we introduce two new stabilization methods based on the use of Finite-Volume Subcells in order to alleviate these robustness issues. The first method relies on an a priori limitation of the DG scheme, together with the use of a TVB slope-limiter and a PL. The second one is built upon an a posteriori correction strategy, allowing to surgically detect the incriminated local subcells, together with the robustness properties of the corresponding lowest-order Finite-Volume scheme. This last strategy allows to ensure the nonlinear stability of the DG scheme in the vicinity of discontinuities, as well as the positivity of the discrete water-height, while preserving the subcell resolution of the initial scheme. This second strategy is also preliminary investigated in the two dimensional horizontal case. An extensive set of test-cases assess the validity of this approach.In the second part, we introduce a new numerical strategy designed for the modeling and simulation of nonlinear interactions between surface waves in shallow-water and a partially immersed surface piercing object. At the continuous level, the flow located in the textit{exterior} domain is globally modeled with the nonlinear hyperbolic shallow-water equations, while the description of the flow beneath the object reduces to a nonlinear ordinary differential equation. The coupling between the flow and the object is formulated as a free-boundary problem, associated with the computation of the time evolution of the spatial locations of the air-water-body interface. At the discrete level, the proposed formulation relies on an arbitrary-order discontinuous Galerkin approximation, which is stabilized with the a posteriori Local Subcell Correction method through low-order finite volume scheme introduced in the first part. The time evolution of the air-water-body interface is computed from an Arbitrary-Lagrangian-Eulerian (ALE) description and a suitable smooth mapping between the original frame and the current configuration. For any order of polynomial approximation, the resulting algorithm is shown to: (1) preserves the Discrete Geometric Conservation Law, (2) ensures the preservation of the water-height positivity at the subcell level, (3) preserves the class of motionless steady states (well-balancing), possibly with the occurrence of a partially immersed object.Several numerical computations and test-cases are presented, highlighting that the proposed numerical model(1) effectively allows to model all types of wave / object interactions, (2) efficiently provides the time-evolution of the air-water-body contact points and accordingly redefine the new mesh-grid thanks to ALE method (3) accurately handles strong flow singularities without any robustness issues, (4) retains the highly accurate subcell resolution of discontinuous Galerkin schemes

Pseudomonas aeruginosa (PA) est l’un des principaux germes impliqués dans les maladies nosocomiales et est aussi la principale cause de mortalité et morbidité des patients atteints de la mucoviscidose malgré l’utilisation massive d’antibiotiques. Dans la lutte contre PA, une alternative aux antibiotiques est l’inhibition de ses facteurs de virulence notamment ceux impliqués dans l’adhésion et la formation du biofilm via des interactions de type sucres/protéines. Ces protéines sont appelées lectines (PA-IL, PA-IIL, FliD). L’objectif de ce travail est la recherche de molécules inhibitrices (glycoclusters) de ces lectines impliquées dans la virulence de PA. Compte tenu du grand nombre de glycoclusters à tester et des faibles quantités de matériels biologiques disponibles, un outil de criblage innovant a été développé (glycoarray) à partir d’une lame de verre microstructurée et fonctionnalisée chimiquement afin d’immobiliser de manière organisée et ordonnée les glycoclusters. La méthode d’immobilisation choisie est la méthode d’immobilisation spécifique par hybridation de l’ADN appelée DDI : DNA Directed Immobilization. Sur ces glycoarrays, 3 méthodes indépendantes (lecture de fluorescence directe, IC50 et Kd) de mesure des interactions glycoclusters/lectines ont été mises au point et validées par une étude comparative donnant un classement similaire des glycoclusters pour leur affinité vis-à-vis des lectines Il faut noter que ces mesures faites sur glycoarrays ne consomment que quelques picomoles de glycoclusters comparées aux méthodes classiques (ITC, ELLA, RMN, …) qui nécessitent des micromoles de produits. A l’aide de ces glycoarrays, un criblage d’une bibliothèque d’une centaine de glycoclusters multivalents, de différentes topologies, charges et linkers a permis d’identifier deux structures montrant une très forte affinité vis-à-vis des lectines de PA. Ces glycoclusters sont actuellement en test in vitro et in vivo. Ces études d’interactions sur DDI-glycoarray ont été étendues à d’autres agents pathogènes tels que les bactéries Burkholderia ambifaria, Viscum album ou contre le virus de la grippe. Dans le futur, pour mieux appréhender les mécanismes d’interactions sucres/protéines, il serait intéressant de pouvoir suivre en temps réel ces interactions en utilisant des systèmes de détection sans marquage tel que, par exemple, la résonance plasmonique de surface. Aussi, le dernier chapitre donne les prémices d’une adaptation de la méthode DDI sur glycoarray sur surface d’or
Pseudomonas aeruginosa (PA) is one of the predominant bacterium encountered in nosocomial infections. PA infections often lead to chronic inflammation and eventually to death despite aggressive antibiotic therapy. A promising approach is to inhibit the virulence factors of PA such as PA-IL, PA-IIL, FliD (lectins). Therefore, there is a great interest for studying carbohydrate/lectin interactions in order to design new treatments. The goal of this work is the research for inhibitory molecules (glycoclusters ) of these lectins involved in the virulence of PA. An innovative screening tool for studying carbohydrate/lectin interactions has been developed (glycoarray). Glycoarray are microstructured glass-slides, chemically functionalized in order to immobilize, organized and orderly, glycoclusters at the surface. The immobilization method is the specific immobilization method based on DNA hybridization called DDI (DNA Directed Immobilization). This miniaturized analytical biosystem allows multiplex test performed in one single microwell. Moreover, three independent methods of affinity measurement (direct fluorescence read-out, IC50 and Kd) have been developed and validated by a comparative study giving a similar ranking of glycoclusters for their affinity towards PA-IL. These measurements on glycoarrays consume only a few picomoles glycoclusters compared to conventional methods (ITC, ELLA...) that require micromoles of products. Using these glycoarrays, the screening of a library of hundreds of glycoclusters presenting different topologies, multivalencies, charges and linkers led to the identification of two structures showing a very strong affinity for PA lectins. These glycoclusters are currently in vitro assay and in vivo. These interaction studies on DDI-glycoarray were extended to other pathogens such as Burkholderia ambifaria bacteria, Viscum album or against the influenza virus. In the future, to better understand the mechanisms of sugar / protein interactions, it would be interesting to monitor in real time the interactions using label-free detection systems such as, for example, the surface plasmon resonance (SPR). Also, the last chapter gives the beginnings of an adaptation of the method of DDI glycoarray on gold surface

Multitouch technology allows the users to use both their hands and multiple fingers to manipulate digital content directly on the screen. This paper attempts to analyze the actual convenience of bimanual and multifinger manipulation on a multitouch display by conducting three observational experiments and studying how a group of volunteers use their hands and fingers when interacting with digital content on a touchscreen surface. In addition, the participants had to fill in a questionnaire where they give some additional insights on how they experienced the use of multitouch-based interface during the experiments. The results suggest that when participants were performing tasks in which they were instructed to manipulate the digital content as fast as they could, a high percentage of them resorted to the use of at least some level of bimanual manipulation of the digital content. However, when participants were told to perform the tasks calmly the big majority of participants decided to move the objects by using only one hand (unimanual). Same-hand multifinger manipulation was also used by a high percentage of participants when moving several objects simultaneously. Nevertheless, in all three experiments the most common way of moving objects across the screen was by dragging them one at a time (sequential move). Finally, in relation to the personal assessment made by the participants, a total of 70% feel that the possibility for engaging in bimanual interaction, that multitouch interface offers, is a clear benefit and advantage over traditional keyboard and mouse. However, 40% of the respondents feel that the use of mouse still is a more effective and natural form of interaction than multitouch technology.

Lors d'un hypothétique accident grave de réacteur à eau sous pression, un mélange de matériaux fondus, appelé corium, issu de la fusion du cœur peut se relocaliser dans le puits de cuve constitué par un radier en béton. Les codes d'évaluation réacteur pour simuler la phénoménologie de l'interaction corium-béton sont basés sur une description à grande échelle des échanges qui soulève de nombreuses questions, tant sur la prise en compte des phénomènes multi-échelles mis en jeu que sur la structure adoptée de la couche limite au voisinage du front d'ablation. Dans ce contexte, l'objectif principal de ce travail consiste à aborder le problème de la structure de la couche limite par simulation numérique directe. Ce travail s'inscrit dans le cadre plus général d'une description et d'une modélisation multi-échelle des échanges, c'est-à-dire de l'échelle locale associée au voisinage du front d'ablation jusqu'à l'échelle du code d'évaluation réacteur. Une telle description multi-échelle des échanges soulève le problème de la description locale de l'écoulement multiphasique multiconstituant mais aussi le problème du changement d'échelle et en particulier le passage de l'échelle locale à l'échelle de description supérieure dite macroscopique associée aux mouvements convectifs dans le bain de corium. Parmi les difficultés associées au changement d'échelle, nous nous intéressons à la problématique de la construction de conditions aux limites effectives ou lois de parois pour les modèles macroscopiques. Devant la complexité du problème multiphasique multiconstituant posé au voisinage du front, cette contribution a été abordée sur un problème modèle. Des conditions aux limites dites effectives ont été construites dans le cadre d'une méthode de décomposition de domaine puis testées pour un problème d'écoulement laminaire de convection naturelle sur parois rugueuses. Mˆeme si le problème traité reste encore éloigné des applications visées, cette contribution offre de nombreuses perspectives et constitue une première étape d'une modélisation multiéchelle des échanges pour la problématique de l'interaction corium-béton. Dans le cas plus complexe des écoulements multiphasiques multiconstituants et devant les difficultés expérimentales associées, le développement de lois de parois pour les outils existants aux échelles de description supérieures nécessite, au préalable, de disposer d'un outil de simulation numérique directe de l'écoulement au voisinage du front d'ablation. L'outil développé dans ce travail correspond à un modèle de Cahn-Hilliard/Navier-Stokes pour un mélange diphasique (liquide-gaz) compositionnel (corium-béton fondu) s'appuyant sur une description du système selon trois paramètres d'ordre associés respectivement aux fractions volumiques du gaz et aux deux espèces miscibles de la phase liquide ainsi que sur une décomposition de l'énergie libre selon une contribution diphasique et compositionnelle. Les équations de transport sont dérivées dans le cadre de la thermodynamique des processus irréversibles et résolues sur la base d'une application éléments finis de la plate-forme PELICANS. Plusieurs expériences numériques illustrent la validité et les potentialités d'application de cet outil sur des problèmes diphasiques et/ou compositionnels. Enfin, à partir de l'outil développé, nous abordons par simulation numérique directe une étude de la structure de la couche limite au voisinage du front d'ablation pour des bétons siliceux et silico-calcaire
In the late phases of some scenario of hypothetical severe accident in Pressurized Water Reactors, a molten mixture of core and vessel structures, called corium, comes to interact with the concrete basemat. The safety numerical tools are lumped parameter codes. They are based on a large averaged description of heat and mass transfers which raises some uncertainties about the multi-scale description of the exchanges but also about the adopted boundary layer structure in the vicinity of the ablation front. In this context, the aim of this work is to tackle the problem of the boundary layer structure by means of direct numerical simulation. This work joins within the more general framework of a multi-scale description and a multi-scale modeling, namely from the local scale associated with the vicinity of the ablation front to the scale associated with the lumped parameter codes. Such a multi-scale description raises not only the problem of the local description of the multiphase multicomponent flow but also the problem of the upscaling between the local- and the macro-scale which is associated with the convective structures within the pool of corium. Here, we are particularly interested in the building of effective boundary conditions or wall laws for macro-scale models. The difficulty of the multiphase multicomponent problem at the local scale leads us to consider a relatively simplified problem. Effective boundary conditions are built in the frame of a domain decomposition method and numerical experiments are performed for a natural convection problem in a stamp shaped cavity to assess the validity of the proposed wall laws. Even if the treated problem is still far from the target applications, this contribution can be viewed as a first step of a multi-scale modeling of the exchanges for the molten core concrete issue. In the more complicated case of multiphase multicomponent flows, it is necessary to have a direct numerical simulation tool of the flow at the local scale to build wall laws for macro-scale models. Here, the developed tool corresponds to a Cahn-Hilliard/Navier-Stokes model for a two-phase compositional system. It relies on a description of the system by three volume fractions and on a free energy composed by a two-phase part and a compositional part. The governing equations are derived in the frame of the thermodynamic of irreversible processes. They are solved on the basis of a finite element application of the object-oriented software component library PELICANS. Several numerical experiments illustrate the validity and the potentialities of application of this tool on two-phase compositional problems. Finally, using the developed tool, we tackle by means of direct numerical simulation the problem boundary layer structure in the vicinity of the ablation front for limestone-sand and siliceous concretes

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2005.
Includes bibliographical references (p. 42-43).
Introduction: The ability to utilize directional, specific bonds are a fundamental property of atoms which has allowed us to predictably create molecules of consistent geometry and composition for centuries. One fundamental difference between a true atom and a nanoparticle is that to date, nanoparticles do not possess this property.
by Brian Neltner.
S.B.

L'objet de ce travail de thèse a été de mettre au point un capteur biologique base sur l'utilisation de phénomènes optiques. Un capteur biologique se compose d'une couche réceptrice d'un transducteur et d'un système de lecture. Nous avons impose un cahier des charges relatif a ce capteur en termes de sensibilité par le biais du transducteur, de détection en parallèle de plusieurs interactions biologiques, et enfin, en termes d'optimisation de la couche réceptrice. L'étude de la sensibilité a consiste a comparer théoriquement et expérimentalement deux transducteurs optiques : la résonance des plasmons de surface (RPS) et le miroir résonant. Ce dernier possède une meilleure sensibilité que l'autre en théorie, mais la comparaison expérimentale nous a fait préférer la RPS. Puis, il a fallu adapter le détecteur à la détection en parallèle. Nous avons fait l'image de la couche réceptrice sur une camera CCD, nous permettant ainsi de suivre l'évolution de la totalité de la surface du capteur. Une première série d'expériences a été réalisée pour la reconnaissance entre des antigènes et des anticorps, ces derniers étant adsorbés passivement sur le capteur. Les résultats obtenus nous amenèrent à développer des procèdes biochimiques pour immobiliser les molécules sur la couche réceptrice. C'est ainsi que nous avons essaye différentes voies d'immobilisation pour les fragments d'ADN, pour ne retenir que le polypyrrole, optimisant la densité de surface des sondes d'ADN sur le capteur. De plus l'utilisation de ce polymère conducteur était tout a fait compatible avec l'adressage de plusieurs séquences d'ADN différentes sur le capteur. Le capteur biologique pouvait alors suivre toutes les interactions relatives aux séquences immobilisées. Nous avons alors applique le capteur a un problème clinique : la détection de mutations ponctuelles au sein d'un gène. Nous avons des lors montre que notre capteur était à même de discriminer la présence d'une base mutée sur toute une séquence.

Les interactions protéine-protéine (IPPs), complexes et dynamiques, sont le cœur des réseaux protéiques cellulaires. Au niveau des synapses excitatrices, la densité post-synaptique (PSD) est un exemple typique de réseau protéique dont la structure et la composition à l’échelle nanoscopique détermine la fonction cellulaire. Ainsi, la régulation dynamique de la composition de la PSD et des mouvements des récepteurs au glutamate dans ou hors de la PSD constitue la base des théories moléculaires actuelles sur l’apprentissage et la mémoire. Dans ce contexte, durant ma thèse, j’ai étudié une classe d’IPPs faisant intervenir les domaines PDZ. En effet, durant ces dernières années, de nombreuses études ont démontré l’implication de ces interactions impliquant les domaines PDZ de la famille de PSD95 dans le ciblage synaptique et l’ancrage des récepteurs au glutamate. Cependant, en partie dû au manque d’outils adaptés, les mécanismes moléculaires sous-jacents qui contrôlent de façon dynamique leur rétention à la synapse restent mal compris. Dans le but d’étudier ces interactions impliquant des domaines PDZ, j’ai développé plusieurs stratégies de sélection par phage display basées sur l’utilisation du dixième domaine de type III de la fibronectine humaine (10Fn3) dans le but de cibler les motifs d’interaction aux domaines PDZ des récepteurs (Stargazin pour les rAMPA et GluN2A pour les rNMDA) ou les domaines PDZ eux-mêmes. En utilisant une approche multidisciplinaire, mes objectifs principaux ont été de concevoir de petits anticorps synthétiques qui nous permettront de rompre ou de stabiliser spécifiquement ces complexes protéiques, ainsi que d’observer les interactions endogènes
Complex and dynamic protein-protein interactions are the core of protein-based networks in cells. At excitatory synapses, the postsynaptic density (PSD) is a typical example of protein-based network whose nanoscale structure and composition determines the cellular function. For instance, the dynamic regulation of PSD composition and glutamate receptors movements into or out of the PSD are the base of current molecular theories of learning and memory. In this context, during my PhD, I focused on a class of protein-protein interactions mediated by PDZ domains. Indeed, over the last decade, numerous studies have shown the critical implication of PDZ domain-mediated interactions from the PSD95 scaffolding protein family in the synaptic targeting and anchoring of glutamate receptors. However, in part due to the lack of adapted tools, the molecular mechanisms that dynamically govern their respective synaptic retention remain poorly understood. In order to investigate these PDZ domain-mediated interactions, I developed several selection strategies by phage-display based on the fibronectin type III (FN3) scaffold in order to either target the PDZ domain-binding motifs of the receptors complexes (e.g., stargazin for AMPARs and GluN2A for NMDARs) or the PDZ domains themselves. Using a multidisciplinary approach, my main objectives were to engineer small synthetic antibodies that will allow us to acutely and specifically disrupt or stabilize these protein complexes, as well as monitor endogenous interactions

A l’arrivée d’un potentiel d’action au niveau d’une synapse neuronale, des ions calcium (Ca2+) pénètrent dans le neurone, permettant aux protéines SNAREs (N-ethylmaleimide-sensitive factor activating protein receptor) de s’assembler entièrement, engendrant la fusion des vésicules synaptiques contenant les neurotransmetteurs avec la membrane plasmique du neurone. Des protéines régulatrices telles que la Complexine et la Synaptotagmine sont étroitement couplées aux SNAREs et permettent une fusion rapide et synchrone. La Synaptotagmin-1 (Syt1), une protéine transmembranaire localisée sur les vésicules synaptiques, est le senseur calcique de la neurotransmission. Syt1 possède deux domaines de liaison au Ca2+, C2A et C2B, un domaine flexible reliant la région membranaire au C2A, ainsi qu’un court lien entre C2A et C2B. Il a été montré qu’une région polybasique dans le C2B se liait aux lipides anioniques tels que phosphatidylserine (PS) et phosphatidylinositol-4,5-bisphosphate (PIP2) en l’absence de Ca2+. A l’entrée du Ca2+, les ions Ca2+ se lient au C2A et au C2B. La liaison de Syt1 aux ions Ca2+ permet aux résidus non polaires à proximité des sites de liaison au Ca2+ de s’insérer dans la membrane. Si ces mécanismes sont relativement bien acceptés, les mécanismes biochimiques et biophysiques précis du déclenchement de la fusion induit par la liaison de Syt1 au Ca2+ restent flous. Dans ce travail, nous mesurons directement les interactions de Syt1 liée à une membrane avec des membranes anioniques comprenant des lipides PS et PIP2 par un appareil à force de surface (SFA), afin d’imiter la membrane d’une vésicule synaptique contenant Syt1 interagissant avec la membrane plasmique anionique. Nous réalisons une mutagénèse dirigée sur les sites de liaison au Ca2+ de C2A et C2B, ainsi que sur le site polybasique de C2B, pour entièrement cartographier les énergies de liaison à la membrane relatives à ces sites, à la fois en présence et en l’absence d’ions divalents. Nous trouvons que Syt1 se lie avec une énergie de ~6 kBT dans l’EGTA, ~10 kBT dans le Mg2+, et ~18 kBT dans le Ca2+. Des réarrangements moléculaires mesurés pendant le confinement de Syt1 entre les membranes prévalent dans le Ca2+ et dans le Mg2+, et suggèrent que Syt1 se lie initialement via le C2B puis réoriente ses domaines C2 dans la conformation de liaison privilégiée. La neutralisation des sites de liaison au Ca2+ de C2B engendre une réduction radicale de l’énergie de liaison de Syt1 dans le Ca2+, alors que la même mutation dans le C2A a un effet plus nuancé. Ces résultats éclairent sur la coopérativité de C2A et C2B dans leur liaison à la membrane, et montrent un rôle apparent prédominant de C2B
Upon arrival of an action potential at the neuronal synapse, calcium ions (Ca2+) enter the neuron, allowing soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins to fully zipper, leading to the fusion of pre-docked synaptic vesicles containing neurotransmitters with the plasma membrane of the neurone. Regulatory proteins such as Complexin and Synaptotagmin are closely coupled to SNAREs during synaptic vesicle fusion and lead to synchronous, fast fusion. Synaptotagmin-1 (Syt1) is a transmembrane protein found in synaptic vesicles and is the Ca2+ sensor for synaptic transmission. Syt1 has two Ca2+ binding domains, C2A and C2B, with a flexible linker domain from the membrane region to C2A, and a short linker between C2A and C2B. A polybasic patch in C2B has been shown to bind to anionic lipids such as phophidylserine (PS) and phosphisotinol (PIP2) in the absence of Ca2+. Upon Ca2+ influx, Ca2+ ions bind in C2A and C2B. Ca2+ binding to Syt1 allows non-polar residues nearby the Ca2+ binding sites to insert into the membrane. While these mechanisms are relatively well-accepted, the precise biochemical and biophysical mechanisms for the Syt1 Ca2+ trigger remain unclear. In this work, we directly measure the interactions of Syt1-coated membranes with anionic membranes including PS and PIP2 lipids by the surface forces apparatus (SFA) technique, in order to mimic a Syt1-coated synaptic vesicle membrane interacting with the anionic plasma membrane. We perform site directed mutagenesis of the Ca2+ binding sites of C2A and C2B, along with the polybasic patch in C2B, to fully map the site-binding energetics of Syt1 with membranes, both in the absence and presence of divalent ions. We find that Syt1 binds with ~6 kBT in EGTA, ~10 kBT in Mg2+, and ~18 kBT in Ca2+. Molecular rearrangements measured during confinement of Syt1 between membranes are more prevalent in Ca2+ and Mg2+ and suggest that Syt1 initially binds through C2B, then reorients the C2 domains into the preferred binding configuration. Neutralization of C2B Ca2+ binding site leads to a drastic decrease of Syt1 binding energy in Ca2+, while the same mutation in C2A has a milder effect. These results illuminate that C2A and C2B cooperate in membrane binding, with an apparent predominant role of C2B

La réalisation de nouveaux matériaux bidimensionnels est un domaine en plein essor de la matière condensée, à la fois pour les aspects fondamentaux, avec les propriétés exotiques émergeant de la dimensionnalité réduite, et pour les applications technologiques potentielles, avec des promesses telles que des courants sans dissipation et des hétérostructures 2D plus performantes que la technologie actuelle à base de silicium à une fraction de la taille. Dans ce travail, nous avons adopté une approche expérimentale pour la réalisation et la caractérisation de matériaux prédits pour accueillir des lignes nodales de Dirac (DNL), qui malgré de nombreuses prédictions théoriques ont vu peu de réalisations expérimentales rapportées jusqu'à présent. Ces matériaux appartiennent à la classe récemment mise en évidence des semi-métaux topologiques, dont la spécificité est un croisement de bandes protégé par symétrie entre les bandes de valence et de conduction le long d'une ligne dans l'espace réciproque, avec une dispersion linéaire. Dans un premier temps, nous nous sommes concentrés sur Cu2Si, le premier matériau 2D dans lequel des DNL ont été mis en évidence lorsqu'il est préparé sur un substrat Cu(111). Après avoir reproduit avec succès les résultats existants, nous avons montré à l'aide de l'ARPES et du XPS que, contrairement aux attentes, la structure électronique et les DNL étaient préservées après le dépôt de Pb sur la surface. Nous avons ensuite étudié Cu2Si/Si(111), et constaté que malgré une structure atomique fortement liée, le substrat Si(111) interagit assez fortement avec les orbitales hors plan de la couche Cu2Si pour empêcher l'existence des lignes nodales. Nous nous sommes ensuite penchés sur le système 2D Cu2Ge, prédit pour accueillir la DNL, et avons tenté de le synthétiser en déposant du Ge sur Cu(111). En combinant nos résultats LEED, XPS et ARPES, nous avons constaté que toutes les mesures correspondaient étroitement à ce que l'on attendait d'une monocouche de Cu2Ge libre, ce qui montre l'absence presque totale d'interactions entre le substrat Cu(111) et la couche de Cu2Ge superficielle formée sur celui-ci. Il s'agit de la première réalisation expérimentale rapportée de Cu2Ge. Dans une étude miroir, nous avons déposé Cu sur Ge(111) et observé une structure de bande dissemblable. À l'aide du STM, nous avons expliqué ces différences par une structure atomique différente, résultant d'un substrat à forte interaction. Nous soulignons par ce travail l'influence du substrat, qu'il soit métallique ou semi-conducteur, sur les propriétés électroniques des systèmes 2D à DNL
The realization of new two-dimensional materials is a booming field of condensed matter, at once for the fundamental aspects, with the exotic properties emerging from the reduced dimensionality, and for the potential technological applications, with promises such as dissipationless currents and 2D heterostructures outperforming the current silicon-based technology at a fraction of the size. In this work, we took an experimental approach to the realization and characterization of materials predicted to host Dirac nodal lines (DNLs), which despite many theoretical predictions have seen few experimental realizations reported so far. These materials belong to the recently evidenced class of topological semimetals, whose specificity is a symmetry-protected band crossing of the valence and conduction bands along a line in momentum space, with linear dispersion. As a first step, we focused on Cu2Si, the first 2D material in which DNLs have been evidenced when prepared on a Cu(111) substrate. After successfully reproducing existing results, we showed using ARPES and XPS that contrary to expectations, the DNLs were preserved after deposition of Pb on the surface without any gap, and that a band splitting occurred. We followed by the investigation of Cu2Si/Si(111), and found that despite a strongly related atomic structure, the Si(111) substrate interacts strongly enough with the out-of-plane orbitals of the Cu2Si layer to prevent the existence of the nodal lines. We then looked at the 2D Cu2Ge system, predicted to host DNL, and attempted to synthesize it by depositing Ge on Cu(111). By combining our LEED, XPS and ARPES results we found that all measurements matched closely what was expected from a free-standing Cu2Ge monolayer, showing the almost complete absence of interactions between the Cu(111) substrate and the surface Cu2Ge layer grown on it. This is the first reported experimental realization of the two-dimensional Dirac nodal line semimetal Cu2Ge. In a mirroring study, we deposited Cu on Ge(111) and observed a dissimilar band structure. Helped by STM, we explained those differences by a different atomic structure, and by a strongly interacting substrate. We highlight through this work the influence of the substrate, whether metallic or semiconductor, on the electronic properties of 2D DNL systems

Block copolymers demonstrate potential in next-generation lithography as a solution for overcoming the limitations of conventional lithographic techniques. Ideal block copolymer materials for this application can be synthesized on a commercial scale, have high [chi]-parameters promoting self-assembly into sub-20 nm pitch domains, have controllable alignment and orientation, and have high etch contrast between the domains for facilitating pattern transfer into the underlying substrate. Block copolymers that contain silicon in one domain are attractive for nanopatterning since they often fulfill at least three of these requirements. However, silicon-containing materials are notoriously difficult to orient in thin films due to the low surface energy of the silicon-containing block, which typically wets the free surface interface. In this work, the methodology behind material choice and the synthesis of new silicon-containing block copolymers by a variety of polymerization techniques will be described. Thin film self-assembly of the block copolymers with domains oriented perpendicular to the plane of the substrate is achieved using different solvent annealing and neutral surface treatments with thermal annealing conditions. Block copolymer patterns are transferred to the underlying substrate by reactive ion etching and directed self-assembly of the polymers is demonstrated using chemical contrast patterns. Interesting thermodynamics governing the self-assembly of block copolymers with solvent annealing will also be discussed. Finally, new amphiphilic block copolymers will be described that were created with lithographic applications in mind but that are most useful for biological applications in drug delivery.
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The following dissertation presents a comprehensive study of the interaction between Dirac fermion quasiparticles (DFQs) and surface phonons on the surfaces of the topological insulators Bi2Se3 and Bi2Te3. Inelastic helium atom surface scattering (HASS) spectroscopy and time of flight (TOF) techniques were used to measure the surface phonon dispersion of these materials along the two high-symmetry directions of the surface Brillouin zone (SBZ). Two anomalies common to both materials are exhibited in the experimental data. First, there is an absence of Rayleigh acoustic waves on the surface of these materials, pointing to weak coupling between the surface charge density and the surface acoustic phonon modes and potential applications for soundproofing technologies. Secondly, both materials exhibit an out-of-plane polarized optical phonon mode beginning at the SBZ center and dispersing to lower energy with increasing wave vector along both high-symmetry directions of the SBZ. This trend terminates in a V-shaped minimum at a wave vector corresponding to 2kF for each material, after which the dispersion resumes its upward trend. This phenomenon constitutes a strong Kohn anomaly and can be attributed to the interaction between the surface phonons and DFQs. To quantify the coupling between the optical phonons experiencing strong renormalization and the DFQs at the surface, a phenomenological model was constructed based within the random phase approximation. Fitting the theoretical model to the experimental data allowed for the extraction of the matrix elements of the coupling Hamiltonian and the modifications to the surface phonon propagator encoded in the phonon self energy. This allowed, for the first time, calculation of phonon mode-specific quasiparticle-phonon coupling λⱱ(q) from experimental data. Additionally, an averaged coupling parameter was determined for both materials yielding ¯λ^Te ≈ 2 and ¯λ^Se ≈ 0.7. These values are significantly higher than those of typical metals, underscoring the strong coupling between optical surface phonons and DFQs in topological insulators. In an effort to connect experimental results obtained from phonon and photoemission spectroscopies, a computational process for taking coupling information from the phonon perspective and translating it to the DFQ perspective was derived. The procedure involves using information obtained from HASS measurements (namely the coupling matrix elements and optical phonon dispersion) as input to a Matsubara Green function formalism, from which one can obtain the real and imaginary parts of the DFQ self energy. With these at hand it is possible to calculate the DFQ spectral function and density of states, allowing for comparison with photoemission and scanning tunneling spectroscopies. The results set the necessary energy resolution and extraction methodology for calculating ¯λ from the DFQ perspective. Additionally, determining ¯λ from the calculated spectral functions yields results identical to those obtained from HASS, proving the self-consistency of the approach.

博士
中國醫藥大學
藥物化學研究所博士班
94
(一) A non-radioactive, sensitive, rapid and specific method for the determination of methionine adenosyltransferase activity has been established. In this method, the methyl group of S-adenosyl-L-methionine was enzymatically transferred to esculetin with the aid of catechol-O-methyltransferase and then the resulting scopoletin was extracted with n-hexane:ethyl acetate (7:3, v/v) and measured by high-performance liquid chromatography with Si 60 column and fluorometric detection with excitation and emission wavelengths at 347 and 415 nm, respectively. The detection limit for scopoletin was about 100 fmol. Using this method to determine MAT activity in HL-60 cells only required about 2.5 mg of protein and the incubation time needed for enzymatic reaction is less than 30 min. The HPLC analysis procedure took only 5 min per sample. The kinetic study showed that MAT in HL-60 cells exhibited negative cooperativity with a Hill coefficient of 0.5. The values of Km and Vmax were 6.1 ± 0.3 mM and 135.4 ± 1.5 nmol AdoMet formed/mg protein/h, respectively. (二) Molecular docking is an increasingly important method for computer-aided drug design, and scoring functions play an essential role in docking strategy. However, high false-positive rates remain a common issue in structure-based virtual screening. In this study, we developed a pharmacophore-based, knowledge-based and empirical-based scoring function (HotLig) to predict protein-ligand interactions. Connolly surface of binding pocket was constructed by PscanMS first and was used to estimate the hydrophobic effect between protein and ligand. The distance-dependant potential for polar interactions (including H-bond, ionic bond and metal bond) and hydrophobic effect were derived from 600 complexes in protein data bank (PDB) according to the statistic frequency of each molecular interaction. Then the energy parameter of each interaction was determined by 214 complexes of training set. The training set was classified into three subsets: basic set (101 complexes), ionic set (56 complexes) and metal set (57 complexes). The Wang 100 dataset and GOLD 100 dataset were used for evaluation of prediction of binding modes. The success rate (RMSD £ 2 Å) for Wang 100 dataset and GOLD 100 dataset were 91% and 87%, respectively. The Wang 100 dataset was also used for evaluation of prediction of binding affinities. The Spearman correlation coefficient (Rs) was 0.6091. In the evaluation of virtual screening, thymidine kinase (PDB code: 1kim) was used for docking. The ligand database contained 1000 decoys and 10 actives. All submicromolar actives were found in the top 25 ligands of ranked database. Thus, HotLig predicts the interactions between protein and ligand with high accuracy, and provides a novel choice of scoring function in molecular docking studies.

Dissertação de mestrado em Genética Molecular
Xylan is the main carbohydrate of the hemicellulosic fraction of plant tissues and accounts for one third of all renewable organic carbon available. Xylanases (EC 3.2.1.8), act in nature by catalysing the hydrolysis of xylan molecules into oligomeric and monomeric pentosan units that bacteria and fungi can use as a carbon source. Xylanase producers have been isolated from all ecological niches where plant material accumulates. pXyl, a highly active psychrophilic xylanase, has previously been isolated from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a. This cold-adapted xylanase, retains 90 % and 60 % of its activity at 20 ºC and 5 ºC, respectively. It is fully stable (at 25 ºC) between pH 6 and 9 and has maximum activity (>80 % at 25 ºC) between pH 5 and 7.8. At lower pHs irreversible inactivation, protein precipitation and eventually protein unfolding occurs. To study the influence of surface hydrophobic interactions on low pH adaptation and protein precipitation, site-directed mutagenesis and a comparative characterisation of the pH dependency of activity, stability and precipitation of mutant and wild-type enzymes was carried out. In particular, surface hydrophobic residues in an exposed patch as well as neighbouring negative residues were mutated to the polar amino acids serine and/or lysine. Of the 10 mutants prepared, Y43S, V58S and E94S revealed improvements in acid pH stability as compared to the wild-type enzyme, with reduced irreversible inactivation, a maintenance of tertiary structure as monitored by tryptophan fluorescence and increases in the solubility at pH 5 and 5.5 being observed. Y43S and E94S also increased their melting temperature at pH 5 by 2.0 and 0.9 ºC, respectively, whereas for V58S this was decreased 2.2 ºC. It is suggested that specific exposed surface hydrophobes as well as negative residues which display an increased hydrophobicity on protonation at low pHs lead to the increased precipitation of pXyl at acidic pHs. Here we show that substitution of these with the highly polar amino acid serine reduces precipitation and irreversible inactivation.
Xilano é o principal hidrato de carbono da fração hemicelulósica dos tecidos das plantas e corresponde a um terço de todo o carbono orgânico renovável disponível. As xilanases (EC 3.2.1.8), atuam na natureza ao catalisar a hidrólise das moléculas de xilano em unidades oligoméricas e monoméricas da xilose que as bactérias e fungos podem usar como uma fonte de carbono. Vários produtores de xilanase foram isolados de todos os nichos ecológicos onde material vegetal se acumula. pXyl, uma xilanase psicrófila altamente ativa foi isolada na Antártida da bactéria Pseudoalteromonas haloplanktis TAH3a. Esta xilanase adaptada ao frio, retém 90 % e 60 % da sua atividade a 20 °C e 5 °C, respetivamente. É totalmente estável (a 25 ºC) entre pH 6 e 9 e possui uma atividade máxima (> 80 %, a 25 C) entre pH 5 e 7,8. A pHs inferiores a inativação irreversível, precipitação de proteína e desnaturação da mesma ocorrem. Para estudar a influência das interações hidrofóbicas na superfície à adaptação a baixo pH, técnicas de mutagénese dirigida, caracterização comparativa da dependência de atividade, estabilidade térmica, espectroscopia de fluorescência e a precipitação das enzimas mutantes e do tipo selvagem, foram utilizadas. Especificamente resíduos expostos em manchas hidrofóbicas bem como os resíduos negativos vizinhos foram mutados para aminoácidos polares serina e/ou lisina. Cada um dos resíduos investigados foram selecionados a partir de um conglomerado de manchas hidrofóbicas com a presença de resíduos negativos. Dos 10 mutantes preparados, Y43S, V58S e E94S revelaram melhorias na estabilidade a pH ácido, com reduzida inativação irreversível, uma manutenção da estrutura terciária monitorizado por fluorescência do triptofano e aumento na solubilidade a pH 5 e 5,5. Y43S E94S também aumentaram a sua temperatura de fusão em 2,0 e 0,9 °C a pH 5 respetivamente, enquanto que a para V58S esta foi reduzida de 2,2 °C. Sugere-se que resíduos hidrofóbicos expostos na superfície, bem como resíduos negativos que exibem uma hidrofobicidade aumentada aquando da protonação a pH baixos levam ao aumento da precipitação de pXyl a pHs acídicos. Aqui, mostramos que a substituição destes pela altamente polar serina reduz a precipitação e a inativação irreversível.