L’objectif de mon travail de thèse a été de mettre au point une nouvelle technique de nanoscopie de fluorescence par excitation non radiative pour l’étude des interactions membrane/substrat. Cette technique repose sur la modification d’une lamelle de verre par une monocouche de boîtes quantiques (QDs). Les QDs joueront alors le rôle de donneurs lors du transfert d’énergie non radiatif. Afin d’obtenir un transfert d’énergie entre cette surface et une membrane (vésicule géante unilamellaire ou cellule vivante), cette dernière est marquée par un fluorophore amphiphile jouant le rôle d’accepteur. Notre étude s’est principalement portée sur l’étude de l’adhésion des vésicules (système modèle de cellule) sur une surface de QDs recouverte de poly-L-lysine. Une attraction électrostatique forte est alors induite, conduisant à l’adhésion des vésicules sur la surface En ajoutant un sel en solution, nous avons pu contrôler finement la force de l’interaction et donc modifier la distance d’équilibre entre la surface et la membrane. A partir de mesure quantitative du quenching des QDs et de la fluorescence émise par le transfert non radiatif, nous avons pu calculer les distances d’équilibre et obtenir une cartographie de ces distances avec une résolution optique nanométrique. Nous avons également utilisé cette technique pour étudier l’adhésion membranaire des cellules U87MG sur différentes surfaces afin d’observer leur points focaux / The objective of my thesis work was to develop a new technique of Non-radiative Excitation Fluorescence Microscopy to study the interactions membrane/substrate.This technique is achieved by coating the substrate with donor species, such as quantum dots (QDs). Thus the dyes are not excited directly by the laser source, as in common fluorescence microscopy, but through a non-radiative energy transfer.To prevent dewetting of the donor film, we have implemented a silanization process to covalently bond the QDs on the substrate. A monolayer of QDs was then deposited on only one side of the coverslips. We highlight the potential of our method through the study of Giant Unilamellar Vesicles (GUVs) labeled with DiD as acceptor, in interaction with surface functionalized with poly-L-lysine. In the presence of GUVs, we observed together a quenching of QDs emission and emission of DiD located in the membrane, which clearly indicated that non-radiative energy transfer from QDs to DiD occurs. By changing salt concentration in the solution, we have been able to finely control the force of the interaction and thus modify the equilibrium distance between the surface and the membrane. From quantitative measurements of quenching of QDs and fluorescence emitted by non-radiative transfer, we calculate the equilibrium distances and obtain a mapping of these distances with a nanometric optical resolution. Based on this study, our functionalization technique is also used to observe the adhesion of living cells, U87MG on different surfaces in order to observe their focal points

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Polysialic acid is aunique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acidis an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma,strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST(ST8SiaIV)and STX(ST8SiaII),withSTX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferasecould lead to control of tumour dissemination and metastasis.The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferaseinhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells(SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acidsurface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acidsurface expression and invasion. Of the potential polysialyltransferase inhibitorsevaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p<0.05)reduction in cell-surface polysialic acidre-expression and invasion in polysialic acid expressing cells.Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over othermembers of the wider sialyltransferase family (α-2,3-and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitrocell based assays to discovery more potent derivatives.

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Esta tesis aborda la actividad biológica de la fibronectina (FN) como proteína de interfase en la interacción célula-material. La tesis investiga la respuesta de la proteína, en términos de cantidad adsorbida y conformación, ante diferentes propiedades físico-químicas del material. Además, se correlaciona la respuesta celular temprana y la funcionalidad celular con el estado de la proteína adsorbida sobre el material. Para ello se prepararon diferentes series de materiales con propiedades físico-químicas controladas. La distribución de FN sobre las diferentes superficies se caracterizó mediante el uso de la microscopía de fuerza atómica (AFM) y la densidad superficial adsorbida fue cuantificada mediante técnicas de marcado radioactivo y western blot. La respuesta celular se evaluó en términos de la adhesión inicial a las superficies, así como los procesos posteriores de diferenciación, proliferación, reorganización y producción de matriz extracelular. Se investigó el efecto de la nanotopografía en la adsorción de la FN y el comportamiento celular sobre una serie de topografías controladas en la escala nanométrica, obtenidas mediante el spin casting de soluciones de ácido poli(L-láctico)/poliestireno (PLLA/PS) de distintas concentraciones. La migración del PLLA hacia la superficie del film durante el proceso de spin coating proporciona superficies de PLLA con nanopicos de diferentes tamaños (14, 29 y 45 nm). El tamaño de la nanoestrutura afecta a la densidad de FN adsorbida, siendo mayor en la superficie de menor nanotopografía. En cuanto a la respuesta celular inicial, se observan adhesiones focales más desarrolladas y mejor reorganización celular de la capa de FN adsorbida en las superficies de mayor topografía (29 and 45 nm), lo que resulta en una mayor producción y organización de nueva matriz. Por otra parte se empleó una familia de materiales con sutiles variaciones en la composición química: polímeros acrílicos (polimetil, etil y butil acrilato -PMA, PEA y P / González García, C. (2012). BIOLOGICAL ACTIVITY OF FIBRONECTIN AT THE CELL-MATERIAL INTERFACE [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17701

No / Intensive research in bio-engineering has been conducted in the search for flexible biomaterials that could support cell growth and cells attachment. Flexible synthetic materials that support cell growth without the aid of synthetic extracellular matrix proteins are still rare. Cholesteryl liquid crystal containing cholesteryl moieties may have suitable biological affinity. Human keratinocytes (HaCat) were cultured with a nematic liquid crystal and three cholesteryl liquid crystals of different formulation. Subsequently, the trypan blue dye exclusion assay was used to determine cell viability in the liquid crystals. The two classes of liquid crystal were characterized by Differential Scanning Calorimeter (DSC) and polarizing microscope (POM) to understand the nature of the interface material. The cell viability study in medium containing liquid crystals verified that liquid crystals had no effects on cell viability. However, only the surface of cholesteryl liquid crystal has shown affinity to HaCat cells. In addition, cells continued to proliferate in the presence of liquid crystals without a change of medium for eight days. No sign of exothermic and endothermic activities at 370C were observed from the DSC test results for the three samples. Biological and mechanical test result of the cholesteryl liquid crystals has shown that cholesteryl liquid crystals are non toxic and support cell attachment without extracellular matrix protein at very low elasticity.