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Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

Cell adhesion and spreading are regulated by complex interactions between the cytoskeleton, matrix adhesion receptors and extracellular matrix proteins, but the molecular determinants of these interactions in early events in cell spreading are not defined. I found that the actin-binding proteins cortactin, vinculin and filamin A were enriched in the earliest formed extensions of HEK-293 cells spreading on collagen. Knockdown of filamin A by short hairpin RNA reduced spreading and the number of cell extensions. Antibody blockade of collagen binding sites on ß1 integrin reduced (p<0.05) cell spreading and the localization of filamin A at cell extensions. Knockdown of filamin A reduced ß1 integrin occupancy by collagen as measured by 12G10 antibody, suggesting a functional co-dependence of filamin A and ß1 integrin. Based on mass spectrometry screening of potential filamin A interacting proteins I examined the interaction of filamin A with the intermediate filament protein vimentin. Filamin A and vimentin-expressing cells were well-spread on collagen and exhibited numerous cell extensions enriched with filamin A and vimentin. By contrast, knockdown of filamin A or vimentin inhibited spreading, cell adhesion, cell surface ß1 integrin expression and ß1 integrin activation. Knockdown of filamin A reduced vimentin phosphorylation and blocked recruitment of vimentin to cell extensions while knockdown of filamin A and/or vimentin inhibited the formation of cell extensions. Inhibition of cell spreading, vimentin phosphorylation and ß1 integrin surface expression and activation were all phenocopied in cells treated with the protein kinase C inhibitor bisindolylmaleimide; cell spreading was also reduced by siRNA knockdown of protein kinase Cє. By immunoprecipitation of cell lysates and by pull-down assays using purified proteins I found an association between filamin A and vimentin. Filamin A also associated with protein kinase Cє, which was enriched in cell extensions. In vitro pull-down assays using deletional mutants of purified filamin A showed that both vimentin and protein kinase Cє bound to a region of filamin A that included repeats 1-8. Reconstitution of filamin A-deficient cells with full-length filamin A or filamin A repeats 1-8 restored cell spreading, vimentin phosphorylation and the cell surface expression of ß1 integrins. I conclude that interactions of filamin A with vimentin and protein kinase Cє may be important for the trafficking and activation of ß1 integrins and cell spreading on collagen.

Adhesion of the opportunistic fungal pathogen, Candida albicans, to host surfaces is mediated through the mannoprotein-rich surface. This thesis examines the role of cell wall mannoproteins in the host-fungus interactions and the mechanism by which these proteins are tethered to the major structural polysaccharides of the wall. The mannosyl modifications of cell surface proteins are involved in host interaction. Periodate oxidation of fungal surface carbohydrates reduced adhesion to epithelial cells confirming that mannan is a component adhesion of C. albicans. Strains of C. albicans disrupted in MNT1, 2 and 3 genes encoding mannosyl transferases, were reduced in adhesion to both epithelial cells and Matrigel. Adhesion was also influenced by the Ura-status of C. albicans strains, which differs as a result of targeted gene disruption by the URA-blaster strategy. Ura-strains were less adhesive but this was not due to altered growth rate and could not be alleviated by adding excess uridine. Therefore current methods for gene deletion have to be questioned when considering adhesion as a virulence factor. Strains defective in glycosylation were altered in covalently associated cell wall proteins (CWPs) in terms of mobility during electrophoresis. The greatest alterations observed were in the CWPs of C. albicans strains with severe glycosylation defects, such as in Dochl/Doch1 and Dpmt1/Dpmt1 mutants, defective in N- and O-linked glycosylation, respectively. These strains also secreted more mannoprotein were altered in morphology or gross cell wall structure, and had elevated cell wall chitin. Therefore glycosylation is required for normal CWP incorporation and cell wall construction. For the first time C. albicans was shown to link a CWP, other than Pir-CWP, to the cell wall network via an alkali-sensitive linkage. The nature of this type of linkage remains unknown, however an O-mannan chain, whose synthesis is not initiated by Pmt1p, may be involved. Pir-CWP incorporation was increased in a Dpmt1/Dpmt1 null mutant and could be partially attributed to increased expression. Pir-CWP expression and incorporation was pH-dependent and may be regulated in response to the different pH at different host niches.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Petra Mottlová Supervisor: Ing. Petra Matoušková, Ph.D. Title of diploma thesis: Effect of valencene on adhesion of breast cancer cell line MDA-MB-231 Valencene belongs to sesquiterpenes and is constituent of essential oils of many plants. It is the main citrus flavoring component. Valencene is mainly used in the food and cosmetic industries, but recent studies have confirmed its biological activity. Its antitumor, antiinflammatory, antioxidant and antiallergic effects have been already proven. The objective of this study was to determine the cytotoxic effect and influence on the adhesion of sesquiterpene valencene on breast cancer cell line MDA-MB-231. Another objective was to study the mechanism of its effect from the perspective of the selected adhesion molecules, which have an important role in tumorigenesis and metastasis. Valencene's cytotoxic effect was tested with use of neutral red (NRU test). Valencene influence on cell adhesion was continuously monitored by means of the X-Celligence device and expression of selected adhesion molecules was studied by Western blot and qPCR methods. The results showed a slight cytotoxic effect of valencene. Cell viability was over 70% at a...

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Understanding bacterial adhesion and biofilm formations on abiotic surfaces are important biological processes that affect the growth of bacteria, with its far-spreading impacts on in everyday life, either as a benefactor or as an inhibitor. To study these bacterial interactions, tools to probe these interfaces are also important to provide further means for discovery of the adhesion mechanisms. In this thesis, a customized imaging platform was developed, utilizing brightfield microscopy to study E. coli K12 on silica surfaces over the stages of bacteria growth. Results observed bacteria adhering onto silica surfaces in a preferential pattern to already existing bacteria-adhered colonies. This suggest that bacteria, once adhered to the surface, enhance attraction of other planktonic bacteria. The platform was designed to enable concurrent Raman measurements, with further optimization required in order to enhance the Raman signals from individual cells. Results from this study provide strong evidence to link changes in interfacial water structure with previous surface vibrational spectroscopy experiments, where the surface coverage of bacteria was found to reach a maximum earlier in the stages of growth compared to the surface water response, indicating that adhesion alone is not the primary contributing factor to modification of the surface microenvironment. / Graduate

Serratia marcescens is an opportunistic pathogen involved in predominantly nosocomial infections, however, it is also implicated as a common cause of microbial keratitis. Since many S. marcecens strains are also resistant to multiple antibiotics, this organism represents a growing public health problem. S. marcescens MG1 utilises a regulatory system for regulation of swarming motility and exo-enzyme secretion that relies on the production of a diffusible signal identified as N-butanoyl-L-homoserine lactone (C4-HSL). The aim of this study was to determine the role of C4-HSL in surface colonisation (adhesion and biofilm formation). In this thesis, the development of a novel biofilm in S. marcescens MG1 is described. The biofilm comprises of an intricate and complex structure consisting of long filamentous cells, cell aggregates and cell chains. Two C4-HSL controlled genes (bsmA and bsmB) are shown to be crucial for biofilm formation. It is proposed that C4-HSL regulated bsmA and bsmB gene products are engaged in fine tuning aggregation at a specific time point in late biofilm development. Since adhesion is the first stage of colonisation, the role of C4-HSL in adhesion to a hydrophilic abiotic surface (HAS) and a human corneal epithelial (HCE) cell line was assessed. While adhesion to the HAS was found to be C4-HSL controlled, this was not the case for adhesion to the HCE cells. In adhesion to the HAS, mutations in the following C4-HSL regulated genes resulted in reduced adhesion; a sensor kinase gene (rssA), a type I transporter gene (lipB), bsmA and bsmB. These four genes were found to effect the expression of type I fimbriae which is proposed to be the adhesin affecting C4-HSL regulated adhesion. While C4-HSL is not involved in adhesion to the HCE cell line, the genes bsmA and bsmB are important. It is proposed that bsmA and bsmB dependent HCE adhesion is due to the requirement of these genes for type I fimbriae production. Furthermore, C4-HSL was found to regulate capsule polysaccharide and OmpX production and repress cytotoxic activity against HCE cells and erythrocytes. It is proposed that cytotoxicity is mediated by ShlA haemolysin.

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