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During the skin ageing which is accelerated by environmental factors, the generated reactive oxygen species make unstable the "proliferation/differentiation/apoptosis" coupling of epidermal cells. A biochemical study, previously realized on human keratinocyte cultures treated by a pesticide, glyphosate, allowed the validation of an in vitro model of skin ageing. In this work, we propose to study the loss of HaCaT cell integrity appearing after this chemical induced-oxidative stress. An original approach, combining a micro- to nanoscale cell characterization and analysis of fluctuating protein expression, is proposed: a) the exploration of cell growth and morphology through fluorescence and AFM imaging, b) a functional investigation using force spectroscopy for detection of oxidative stress biomarkers in membranes and c) the establishment of proteomic comparative profiles between safe and glyphosate-treated cells. Our results revealed up to now that a glyphosate treatment induced cell impairment in an acute dose-dependency. Indeed, we observed a breakage of plasma membrane and a release of cytosol, microtubules disorganization and fragmentation, and finally aggregation of chromatin. These observations characterized apoptotic key events. Our first force spectroscopy results revealed that a glyphosate coated tip interacted specifically with keratinocyte membrane structures. This nanobiotechnological tool will allow cell mapping establishment and screening original anti-oxidant molecules. The proteomic profiling would highlight cell oxidative stress cascade and identify corresponding major biomarkers in our skin ageing model. Original molecules with cytoprotective potentials will be tested and compared to well-known antioxidants, in order to correct pesticide induced cutaneous disorders. These data all together will contribute to better encircle the mechanisms of chemical and environmental hazards acting on the human skin.

National audience; Vous attendez une nouvelle révolution ? Vous souhaitez élaborer des méthodes d’investigation sur les cellules de vigne, les levures, les bactéries, des procédures d’identification de pesticides, des biocapteurs qui pourront détecter des molécules spécifiques comme le resvératrol, des nanorobots qui pourront pénétrer à l'intérieur des bouteilles pour évaluer la qualité d’un vin... Les nanotechnologies vous permettent de refaire le monde atome par atome, molécule par molécule, d’assembler des nano-objets en vue d’applications particulières : modification des propriétés du conditionnement pour garder l’oxygène à distance, le nettoyage ionique... Les nanotechnologies peuvent s’impliquer dans le développement de laboratoire sur puce capable de détecter la présence de résidus de pesticides toxiques dans le vin et utilisable par tous les acteurs de l’élaboration du vin. La modification des caractères organoleptiques du vin, le développement de bactéries, l’apparition de nanomatériaux en suspension, la contamination par des substances chimiques provenant des surfaces en contact avec le vin sont autant de problèmes que les nanotechnologies via la miniaturisation et les possibilités de diagnostic et de détection peuvent résoudre. L’industrie agroalimentaire utilise déjà les revêtements « nanotechnologiques » à base de dioxyde de titane connus pour prévenir le développement de germes (bactéries, virus) sans mutation génétique. A l’université de Dijon, la technique de Microscopie à Force Atomique (AFM) nous a permis de caractériser les renforcements pariétaux (modification de la morphologie et des propriétés élastiques) sur des cellules de vigne (Vitis vinifera cv Gamay) après stimulation des mécanismes de défense par les ultra-violets et les oligogalacturonates. L’AFM est un outil qui permet « d’imager » en trois dimensions, avec une grande résolution (grossissement x1.000.000), la surface et les propriétés visco-élastiques des échantillons. Son grand intérêt en biologie réside dans le fait qu’elle peut être utilisée directement sur des échantillons biologiques tels que des cellules en suspension et de visualiser en temps réel les effets de traitements réalisés in situ.

The approach lies on the development of an original DNA biosensor. This self-assembled DNA biochip is constituted of a tethered lipid membrane presenting « protein/DNA » complexes. This unique assembly, using a highly flexible bifunctional molecular spacer and a fluidic matrix, allows the control of oligonucleotides surface density and their mobility thus favouring hybridization of the DNA probes in liquid environment and under weak constraints. Recent Surface Plasmon Resonance experiments lead to building of protein/DNA dimers presenting two special DNA sequences recognized by regulation proteins. We demonstrated the capacity and relevance of this biosensor in the case of an estrogen receptor/DNA specific recognition, opening the possibility of specific drugs screening. We present here preliminary characterizations of these thin biomolecular films through Atomic Force Microscopy in liquid. Homogeneity of the lipid film was controlled, the surface coverage in protein was evaluated and specificity of binding is under examination at the present time. Indeed, the visualization of the self-assembled « edifice » at a molecular scale and in a bio-mimetic environment is a precious and complementary way to improve the understanding of the protein/DNA interaction mechanisms.

International audience; In situ chemical surface analyses using X-ray photoelectron spectroscopy (XPS), completed by ex situ atomic force microscopy (AFM) analyses, were performed in order to compare the initial stages of MOCVD growth of TiO2 thin films on two different surface types. The first type was a silicon native oxide free hydrogen terminated surface and the second one was a silicon dioxide surface corresponding to a thin layer of 3.5 nm thick in situ thermally grown on silicon substrate. Si(100) was used as substrate, and the growths of TiO2 thin films were achieved with titanium tetraisopropoxide (TTIP) as precursor under a temperature of 675 -C, a pressure of 0.3 Pa and a deposition time of 1 h. Whatever the surface is, the deposited titanium amount was globally the same in the two cases. On the contrary, the deposit morphology was different: a covering layer composed of a SiO2 and TiO2 phases mixture on the hydrogen terminated surface, and small TiO2 clusters homogeneously spread on the SiO2 surface.

The properties of gramicidin, a linear antibiotic polypeptide of 15 amino acids, have been studied at the air-water interface. Analysis of the pressure-area isotherm is not able to conclude about the conformational behavior of gramicidin in the monolayer. Langmuir-Blodgett deposition of gramicidin layers onto a mica substrate has been developed for atomic force microscopy (AFM) observations. At high pressure of deposition, the gramicidin monolayer is composed of dimers perpendicular to the surface. The possibility of removing the half upper part of this dimer monolayer with the AFM tip is more in favor of a structure of single-stranded helical dimers.

The cytoplasmic face of ventral cell membranes of Madin-Darby canine kidney (MDCK) cells grown on glass coverslips was imaged by atomic force microscopy (AFM) in air and under aqueous medium, in "contact" mode. Micrometer range scans on air-dried samples revealed a heterogeneous structure with some filaments, likely corresponding to actin filaments that abut the inner leaflet of the membrane, and a few semi-organized lattice structures that might correspond to clathrin lattices. Experiments in phosphate-buffered saline confirmed the heterogeneity of the inner membrane surface with the presence of large (> 100 nm) globular structures emerging from the surface. Using sub-micrometer scan ranges, protruding particles, that occupy most of the membrane surface, were imaged in liquid medium and in air. These particles, 8 to 40 nm x-y size, were still present following ethanol dehydration which extracts a large fraction of membrane lipids, indicating their proteic nature. Due, at least partly, to the presence of some peripheral proteins, high magnification images of the inner membrane surface were heterogeneous with regard to particle distribution. These data compare with those previously reported for the external membrane leaflet at the surface of living MDCK cells. They show that details of the cytosolic membrane surface can be resolved by AFM. Finally, the images support the view of a plasma membrane organization where proteins come into close proximity.