Caractérisation par microscopie à force atomique des arrangements protéine/sucre impliquant la lectine PA-IL de la bactérie pseudomonas aeruginosa

La bactérie Pseudomonas aeruginosa est un pathogène opportuniste responsable de graves infections chez les personnes affaiblies immunitairement. Présentant des souches résistantes aux antibiotiques, une nouvelle approche thérapeutique est en cours de développement avec pour objectif l inhibition des facteurs de virulence de la bactérie. Lors de son processus d infection, le pathogène utilise les lectines pour reconnaître et se lier de manière spécifique aux glycoconjugués des cellules-hôtes en formant une interaction lectine/glycoconjugué. Plus particulièrement, la lectine PA-IL, spécifique du galactose, a été étudiée. A l aide de glycomimétique, il semble possible de bloquer l action de la lectine en créant une interaction lectine/glycomimétique. Pour développer cette approche, de nombreux glycocluster sont donc été élaborés et leur affinité avec la lectine PA-IL a été évaluée par plusieurs méthodes de caractérisation (SPR, HIA, ELLA, puce à sucre, ).Dans ce projet de thèse, nous avons cherché à visualiser par microscopie à force atomique (AFM) l arrangement des complexes lectine PA-IL/glycocluster formés pour trois glycoclusters différents. Nous avons ainsi pu montrer l influence du cœur du glycocluster et des bras-espaceurs sur l arrangement des complexes. Suivant le glycocluster, l arrangement prend la forme de filaments 1D,de structures dentelées avec des bras sinueux ou encore de larges structures compactes. Dans le cas des filaments, la résolution de nos images AFM nous a permis d identifier les lectines à l intérieur même de la structure filaire. Nous avons aussi démontré, en observant les lectines seules, l existence d une interaction lectine/lectine. De plus, des expériences ont été menées pour déterminer les conditions expérimentales appropriées à leur observation à l air et en milieu liquide.The bacterium P. aeruginosa is an opportunistic pathogen responsible for serious infections in immunocompromised patients. It also develops some strains resistant to antibiotics. A new approach is developed to inhibit virulence factors of the bacterium. During the process of infection, the pathogen uses lectins to recognize and bind specifically to glycoconjugates of the host cells forming alectin/glycoconjugate complex. Particularly, the lectin PA-IL, specific to galactose, was studied. Using glycomimetics, it seems possible to block the action of the lectin by creating lectin/glycomimetic interaction. To develop this approach, many glycoclusters were designed and their affinity with lectin PA-IL was evaluated by various characterization techniques (SPR, HIA, ELLA, microarrays, ).In this thesis project, we have tried to visualize by Atomic Force Microscopy (AFM) the arrangement of lectin PA-IL/glycocluster complexes with three different glycoclusters. Our results show the influence of the glycocluster core and the linker on the arrangement of complexes. Depending on glycocluster, the arrangement takes the form of 1D filaments, 2D "pinked" structures with sinuous branches or large compact structures. In the case of filaments, the resolution of AFM images allows us to identify lectins along the filament. We also demonstrated the existence of lectin/lectin interactions at high concentration of lectin. In addition, some experiments were performed to determine sample preparation techniques to observe lectins in air and in liquid.LYON-Ecole Centrale (690812301) / SudocSudocFranceF

Effect of Surface Elasticity on the Rheology of Nanometric Liquids

International audienceThe rheological properties of liquids con ned to nanometer scales are important in many physical situations. In this paper we demonstrate that the long range elastic deformation of the con ning surfaces must be taken into account when considering the rheology of nanometric liquids. In the case of a squeeze- ow geometry, we show that below a critical distance Dc the liquid is clamped by its viscosity and its intrinsic properties cannot be disentangled from the global system response. Using nanorheology experiments, we demonstrate that picometer elastic de ections of the rigid con ning surfaces dominate the overall mechanical response of nanometric liquids con ned between solid walls

Stable unidimensional arrays of coherent strained islands

We investigate the equilibrium properties of arrays of coherent strained islands in heteroepitaxial thin films of bidimensional materials. The model we use takes into account only three essential ingredients : surface energies, elastic energies of the film and of the substrate and interaction energies between islands via the substrate. Using numerical simulations for a simple Lennard-Jones solid, we can assess the validity of the analytical expressions used to describe each of these contributions. A simple analytical expression is obtained for the total energy of the system. Minimizing this energy, we show that arrays of coherent islands can exist as stable configurations. Even in this simple approach, the quantitative results turn out to be very sensitive to some details of the surface energy.Comment: 24 pages, 7 figures. to be published in Surface Scienc

Single stage electrochemical exfoliation method for the production of few-layer graphene via intercalation of tetraalkylammonium cations

We present a non-oxidative production route to few layer graphene via the electrochemical intercalation of tetraalkylammonium cations into pristine graphite. Two forms of graphite have been studied as the source material with each yielding a slightly different result. Highly orientated pyrolytic graphite (HOPG) offers greater advantages in terms of the exfoliate size but the source electrode set up introduces difficulties to the procedure and requires the use of sonication. Using a graphite rod electrode, few layer graphene flakes (2 nm thickness) are formed directly although the flake diameters from this source are typically small (ca. 100–200 nm). Significantly, for a solvent based route, the graphite rod does not require ultrasonication or any secondary physical processing of the resulting dispersion. Flakes have been characterized using Raman spectroscopy, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS)

Enseignement des nanotechnologies en école d'ingénieur et en master international

International audienc

Microscopie à Force Atomique (AFM) & Microscopie Electronique en Transmission (TEM)

4h de Cours dans Ecole Thématique Surf' Ing' Chem' 2015, 8-12 Juin 2015 La Londe Les Maures , Franc

Caractérisation par microscopie à force atomique d'une puce ADN, de l'élaboration à l'hybridation

Les puces à ADN sont des systèmes de reconnaissance génétique, élaborées à partir d'un support solide sur lequel sont greffées des monobrins-sondes de séquences connues. Elles permettent d'identifier dans une solution, les brins d'ADN-cibles possédant la séquence complémentaire. Dans le contexte d'une miniaturisation des puces, une analyse par microscopie à force atomique (AFM) de chaque étape de fabrication des puces a été entreprise, de l'immobilisation des sondes jusqu'à la lecture de l'hybridation. Il a été montré que la nature des sondes, séquences et marqueurs fluorescents, influençaient leur organisation en surface et que chaque sonde de 25 bases pouvait être identifiée individuellement. Après hybridation de faibles quantités de cibles, les images AFM ont révelé la présence de brins couchés et dispersés, sur lesquels la zone d'hybridation a été localisée.DNA chips rely on the specific recognition of immobilized singles strands DNA (ssDNA), called probes, and unknown free ssDNA targets in solution. Integration has created a great deals in studying chips under micron scale. Atomic Force Microscopy (AFM) was used to follow each step preparation of the chip, from probes immobilization to the ssDNA recognition by hybridization process. It was shown that probes sequences and fluorescent groups influence greatly on the surface organization and that the molecular resolution could be achieved on 25 bases probes. After hybridization of low quantity targets, AFM images have revealed scarcely distributed targets lying on the complementary probes area. The hybridized zone was also identified along the targets.LYON-Ecole Centrale (690812301) / SudocSudocFranceF

Soft oxidation of graphite studied by XPS and STM

The modification of the graphite surface after weak oxidation using a nitric acid solution has been studied by X-ray photoelectron spectroscopy (XPS) and scanning tunneling microscopy (STM). A new corrugation at the atomic scale has been revealed by STM on the basal planes. It is attributed to the presence, in other respects demonstrated by XPS, of some oxygenated groups created by oxidation and regularly distributed on the surface. This soft oxidation conserves the graphite basal planes and is interpreted by an oxygen chemisorption on graphite through a bond involving π electrons of the aromatic cell. Organic groups which need dangling σ bonds to fix on the surface should preferencially attach on ridges of large defects.La modification de la surface de graphite après une faible oxydation dans une solution d'acide nitrique a été étudiée par spectroscopie de photoélectrons (XPS) et par microscopie tunnel (STM). Une nouvelle corrugation a été mise en évidence à l'échelle atomique par STM sur les plans de base du graphite. Elle est attribuée à la présence d'autre part démontrée par XPS, de groupements organiques créés par l'oxydation et distribués de façon régulière sur la surface. Cette oxydation douce n'altère pas les plans de base du graphite et est interprétée par la chimisorption d'atomes d'oxygène sur le graphite, les liaisons s'effectuant à partir d'électrons π de la cellule aromatique. Les groupements organiques nécessitant des liaisons pendantes σ pour se fixer sur la surface semblent se positionner préférentiellement sur les arètes de gros défauts physiques

Curious Binding Energy Increase between the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein and Angiotensin-Converting Enzyme 2 Adsorbed on a Silane Monolayer from Molecular Dynamics Simulations

International audienceIn the context of the COVID-19 outbreak since December 2019, antigenic tests are widely used, for diagnosis purposes, to detect the SARS-CoV-2 spike protein in nasopharyngeal fluid through its interactions with specific antibodies. However, the SARS-CoV-2 spike protein is subject to rapid mutations yielding more and more variants that might lose their affinity toward the currently used antibodies. The virus entry into the host cell involves interactions between the angiotensin-converting enzyme 2 (ACE2) and the SARS-CoV-2 spike protein receptor-binding domain. Consequently, ACE2 could be a target with limited mutation escaping possibilities. However, as the enzyme has not evolved to recognize the virus, its affinity with the spike protein receptor-binding domain is lower than that with specific antibodies. The present molecular dynamics simulations study suggests that the adsorption of the ACE2 on specific silane monolayers could increase its affinity toward the spike protein receptor-binding domain. Indeed, silane monolayers, combining silane molecules with short alkyl chains and positively charged head groups and silane molecules without charged head groups, could adsorb the ACE2 while maintaining its bioactivity (orientation compatible with the spike protein trapping, low conformational changes) and increasing its interactions with the spike protein receptor-binding domain (number of hydrogen bonds and electrostatic interactions) to lead to an increase by 20% both in the binding free energy and in the enzyme/receptor-binding domain rupture force. This work could help develop biosensing tools efficient toward any variants of the SARS-CoV-2 spike protein