Tissue cells exhibit varying responses according to the stiffness of their extracellular matrix (ECM). The mechanism of this stiffness sensing is not fully understood; however, it is known that cells probe stiffness by applying intracellular force to the ECM via integrin-mediated focal adhesions. The bonds between integrins and ECM have been described as “catch bonds�, and it is unclear how ECM viscoelasticity affects these bonds. We have observed the effects of ECM stiffness on the binding strength of integrins to ECM ligands by measuring the dissociation force of individual integrin-ligand bonds of 3T3 fibroblasts on collagen-coated polyacrylamide gels using atomic force microscopy. Results show that integrins exhibit higher rates of activation on stiff substrates. Furthermore, increased matrix stiffness results in the occurrence of larger, multi-bond dissociation events, which suggests that substrate stiffness may affect the cellular response by promoting integrin clustering as well as by modulating the maximum possible force between individual integrins and the ECM.

The acquisition of cellular adhesion machinery likely represented a key factor in the evolutionary transition from unicellular to multicellular organisms. Within metazoa, cellular adhesion is an integral aspect of organismal integrity through its regulation of a wide range of processes, including tissue patterning, cellular proliferation, and migration. As such, dysregulation of adhesion has been linked to diverse pathologies including cancers and neurodegenerative diseases. At the molecular level, adhesion is mediated by specific transmembrane cell adhesion molecules (CAMs) and intracellular complexes that create a dynamic link between the extracellular milieu and the intracellular cytoskeleton. At the sequence level, immunoglobulin domains act to mediate homo- and heterophilic interactions among CAMs and thus adhesion between neighboring cells. LIGs, a family of Ig-containing proteins that contain Leucine-rich repeats, represent candidates for novel CAMs with functions in axonal regeneration and synaptic pathfinding - all of which are highly dependent on cellular adhesion. In Drosophila, two LIG family members, Kekkon1 (Kek1) and Kekkon5 (Kek5) have been been implicated in EGF signaling, and Bone Morphogenetic Protein signaling as well as cellular adhesion, respectively. To investigate the putative role of Kek5 as a CAM, characterization of Kek5 activity was carried out at the cellular and molecular level. From this it was discovered that Kek5 is able to induce a dramatic upregulation of the adherens junction component Armadillo, in addition to epithelial extrusion and cell enlargement. Together, the studies presented within support a model in which Kek5 acts in a homophilic fashion to upregulate Arm and that this activity is functionally separable from other observed effects (epithelial extrusion and cell enlargement).

A semi-synthetic medium for use in determining adherent growth with Staphylococcus epidermidis and Staphylococcus saprophyticus was developed. Production of an adherent biofilm was dependent upon the presence of hematin in the growth medium. Clinical strains of Staphylococcus epidermidis were tested for production of an adherent biofilm in trypticase soy broth, the semi-synthetic medium and the hyperalimentary nutrient solution used in the neonatal hospital unit. An adherent biofilm was obtained when Staphylococcus epidermidis was cultured m hematin supplemented hyperalimentary solution. Growth in the hyperalimentary nutrient solution diluted with fetal calf serum showed the same growth rate as when the nutrient solution was diluted with water. The final growth yield was always higher in serum diluted nutrients. There was no effect of hematin on the growth rate of the organisms.

Metastatic melanoma is among the most aggressive forms of cancer for which there are no effective therapies. Emerging evidence indicates that melanomas can be sensitized to chemotherapy by increasing the function of integrin transmembrane adhesion receptors. Current integrin therapies work by targeting the extracellular domain, resulting in complete gains or losses of integrin function that lead to toxicity.An attractive alternative approach is to target proteins from inside the cell, such as vinculin, that associate with the integrin cytoplasmic domains and regulate its ligand binding properties. The work presented in this thesis describes a novel reagent, denoted vinculin activating peptide or VAP, which increases integrin activity from within the cell as measured by elevated: (1) numbers of active integrins, (2) adhesion of cells to extracellular matrix ligands, (3) numbers of cell-matrix adhesions, and (4) downstream signaling. The effects of VAP are dependent on both integrins and a key regulatory residue A50 in the vinculin head domain. I further show that VAP dramatically increases the sensitivity of melanomas to chemotherapy in clonal growth assays and in vivo mouse models of melanoma. Finally, we demonstrate that the increase in chemosensitivity results from increases in DNA damage-induced apoptosis by a mechanism that requires both p53 and β1 integrin. Collectively these findings demonstrate that integrin function can be manipulated from within the cell and validate integrins as a new therapeutic target for the treatment of chemoresistant melanomas.

Transglutaminase II (TG2) is a complex protein with five different reported activities. Increases in TG2 expression and TGase activity have previously been observed during wound healing in rat studies; however, it has been unclear whether these phenomena were directly involved in the healing process or if they were simply a by-product of it. The aims of this thesis were, thus, to determine if TG2 plays a role in wound healing in vivo and to elucidate the mechanism of any effects TG2 may have at the cellular level. TG2 ablation resulted in delayed wound healing. To gain mechanistic insight into this abnormality, primary fibroblast cultures from TG2-knockout and wildtype mouse embryos were analysed. TG2-null fibroblasts displayed decreased adhesion and integrin signalling during initial stages of adhesion. Intriguingly, TG2-null cells showed faster activation of Rac1 and RhoA in response to adhesion. Long-term adhesion of TG2-null fibroblasts resulted in increased basal phosphorylation of FAK and number of paxillin-stained focal adhesions, enhanced PI3-kinase signalling, faster actin dynamics and altered activation of p44/42 MAPK. These results are indicative of futile cycling of intracellular signalling pathways resulting from reduced focal adhesion turnover in the TG2-knockout fibroblasts. Rescue experiments demonstrated that TG2-mediated effects on cell adhesion occurred in the extracellular environment and that neither GTP-binding nor TGase activity is required for these effects. Results further showed that a ???compact??? conformation of TG2 was not required for this role of TG2. Interestingly, addition of recombinant TG2 to the extracellular environment increased cell spreading of TG2-null cells to a level far greater than that seen in wildtype cells, which did not increase their spreading in response to exogenous TG2. Demonstration of faster activation of the small GTPases in the TG2-null MEFs, and the apparent inhibition of exogenous TG2???s extracellular effects on cell spreading by endogenous protein in the wildtype cells, provide tantalising evidence for a role for intracellular TG2 in regulating activation of the small GTPases to promote efficient fibroblast migration. This work identifies TG2 as a facilitator of efficient wound closure through extracellular effects on integrin-mediated signalling and intracellular effects on activation of the small GTPases.

Biodegradation of poorly water soluble hydrocarbons, such as n-alkanes and polycyclic aromatic hydrocarbons (PAHs) is often limited by the low availability of the pollutant to microbes. Adhesion of microorganisms to the oil-water interface can influence this availability. Our approach was to study a range of compounds and mechanisms to promote the adhesion of a hydrophilic PAH degrading bacterium, Pseudomonas fluorescens LP6a, to an oil-water interface and examine the effect on biodegradation of phenanthrene by the bacteria. The cationic surfactants cetylpyridinium chloride (CPC), poly-L-lysine and chlorhexidine gluconate (CHX) and the long chain alcohols 1-dodecanol, 2-dodecanol and farnesol increased the adhesion of P. fluorescens LP6a to n-hexadecane from ca. 30% to ca. 90% of suspended cells adhering. The alcohols also caused a dramatic change in the oil-water contact angle of the cell surface, increasing it from 24° to 104°, whereas the cationic compounds had little effect. In contrast, cationic compounds changed the electrophoretic mobility of the bacteria, reducing the mean zeta potential from –23 to –7 mV in 0.01M potassium phosphate buffer, but the alcohols had no effect on zeta potential. This results illustrate that alcohols acted through altering the cell surface hydrophobicity, whereas cationic surfactants changed the surface charge density. Phenanthrene was dissolved in heptamethylnonane and introduced to the aqueous growth medium, hence forming a two phase system. Introducing 1-dodecanol at concentrations of 217, 820 or 4100 mg/L resulted in comparable increases in phenanthrene biodegradation of about 30% after 120 h incubation with non-induced cultures. After 100 h of incubation with LP6a cultures induced with 2-aminobenzoate, 4.5% of the phenanthrene was mineralized by cultures versus more than 10% by the cultures containing initial 1-dodecanol or 2-dodecanol concentrations of 120 or 160 mg/L. The production and accumulation of metabolites in the aqueous phase responded similarly to the addition of 1-dodecanol. Further experiments showed that the positive influence of the alcohols could not be attributed to the changes in surface and interfacial tension or increase in biomass concentration. The results suggest that enhanced adhesion of bacterial cells to the oil-water interface was the main factor responsible for the observed increase in phenanthrene biodegradation by P. fluorescens LP6a. / Chemical Engineering

This thesis presents: 1) literature review on adhesive bonding technologies in aviation industry including surface pretreatments (pre-preparation), surface quality assurance, and surface chemistry analysis methods; and 2) development and study of a novel solid-state electrochemical sensor for surface chemistry analysis of composite surfaces. The performance of an adhesive bonding is greatly determined by the adherend?s surface pretreatments which could increase surface tension, surface roughness, and change surface chemistry thereby increasing bond strength and durability of polymer composite adhesive joints. The primary goal of the surface pretreatments is to increase the surface roughness, surface energy, chemical activity, and cleanliness of the composite adherend as much as possible. Methods of surface pretreatments are reviewed in this paper, including: (1) abrasion/solvent cleaning; (2) grit blasting; (3) peel-ply; (4) tear-ply; (5) acid etching/anodizing; (6) corona discharge treatment; (7) plasma treatment; (8) flame treatment; (9) laser treatment; (10) others. One of the critical issues in aviation industry for an adhesive bonding is to analyze the prepared composite surfaces using a nondestructive inspection (NDI) or nondestructive test (NDT) method to determine whether the quality of surfaces are ready for the following bonding processes. Existing NDI methods include: (1) Near-Infrared; (2) Electrical potential; (3) Transient thermal NDT; (4) Electrical Impedance Spectroscopy (EIS); (5) Neutron radiography; and (6) X-ray Photoelectron Spectroscopy (XPS). However, up till now, these methods cannot provide definitive analysis or online and in-field analysis. Because of the non-availability of an on-line, in-field NDI method for surface chemistry analysis, excess or inadequate surface treatment and quality control processes may exist in the current aircraft manufacture processes incurring either a high cost or potentially weak adhesive bonds. Electrochemical reactions usually occur in liquid electrolyte or on conducting electrode but not on non-conducting composite. Conventional electrochemical sensors involve liquid electrolytes which will cause contamination on composite surfaces when they are used for surface chemistry analysis. In this work, we explore an all solid-sate electrochemical sensor technology. Redox pairs or mediators are combined into a solid-state electrolyte, NafionTM. The mediators can pass electrons to or from the composite surfaces causing slight reduction or oxidation of the composite surfaces. The output current in response to cyclic polarization (cyclic potential scanning) is used as the indication of the surface contamination level. The sensors included a working or sensing electrode with mediated Nafion clusters, Nafion membrane, Pt catalyzed carbon counter electrode, and Ag|AgCl reference electrode. The working electrode and counter electrode were attached to the Nafion membrane from different sides. The sensors were tested on different kinds of surfaces: original, polished, and sulfuric acid treated acrylic samples and pristine peel ply prepared, polished, and sulfuric acid treated composite laminate surface samples. The sensors showed a high sensitivity to the surface contamination. The performances and possible mechanisms related to the electrochemical sensors are discussed.

Les infections génitales par les virus du papillome humains (HPV) sont les infections virales sexuellement transmises, les plus communes chez les femmes en âge de procréer. Il est désormais bien établi que l’infection persistante par les HPV classés «à haut risque» est l’un des facteurs indispensables au développement de lésions précancéreuses et cancéreuses du col de l’utérus. Ces HPV semblent aussi être impliqués dans le développement d’autres cancers de la région ano-génitale et pourraient être également impliqués dans les cancers de la tête et du cou. Durant cette dernière décennie, des études croissantes tendent à établir un rôle étiologique des HPV dans les dysfonctionnements gestationnels. La détection des ADN HPV dans les placentas issus d’avortements spontanés et leur capacité exceptionnelle à se répliquer in vitro dans les cellules trophoblastiques cultivées en monocouche, ont apporté de nouvelles perspectives quant à la possibilité que le placenta pourrait constituer aussi un tropisme naturel des infections par HPV. Six jours après la fécondation et suite à l’accolement du blastocyste à l’épithélium utérin, le trophoblaste s’engage dans des processus actifs de prolifération, d’invasion et de différenciation complexe pour la construction de l’interface physiologique indispensable aux échanges essentiels entre la mère et l’enfant ; le placenta. De façon intéressante, ses propriétés sont similaires à celles de la cellule tumorale maligne. Néanmoins, ses mécanismes sont étroitement régulés dans le trophoblaste, à la fois dans l’espace et le temps, assurant un développement normal à chaque étape de la grossesse. Devant toutes ces données, nous avions émis l’hypothèse que l’expression des protéines précoces E5, E6 et E7 d’HPV de type 16 (de haut risque), pourraient modifier le développement des trophoblastes infectés. Les résultats obtenus durant ce travail de doctorat démontrent que la protéine virale E5, hautement hydrophobe, est cytotoxique et affecte la viabilité du trophoblaste. Cette cytotoxicité est neutralisée, et la viabilité est améliorée, lorsque les oncoprotéines majeures E6 et E7 sont exprimées en présence de la protéine E5. Lorsque toutes les protéines précoces sont exprimées sous le contrôle de leur propre promoteur (LCR), la viabilité est favorisée. Ces observations ont été confirmées dans les cellules cervicales également. Il a été précédemment rapporté que les oncoprotéines E6 et E7 affectaient l’adhésion du trophoblaste aux cellules endométriales. Dans le présent travail, il a été retrouvé que la protéine E5 diminuait elle aussi l’adhésion, non seulement aux cellules endométriales, mais aussi au support de culture cellulaire. Les capacités de migration et d’invasion de la matrice extracellulaire sont augmentées par l’expression de E5 et dans une plus large proportion par l’expression de E6 et E7. Des résultats similaires ont été obtenus lorsque toutes les protéines de la région précoces sont exprimées sous le contrôle de leur propre promoteur (LCR). La diminution de l’expression de la E-cadhérine est considérée comme un marqueur de malignité et de mauvais pronostic pour les cancers. Nous avons démontré que l’expression de E5, E6 ou de E7, inhibait l’expression de la E-cadhérine, reflétant l’impact des oncoprotéines du virus HPV-16 sur la diminution de l’adhésion et l’augmentation du pouvoir invasif des cellules trophoblastiques. L’investigation d’autres marqueurs de malignité et de tolérance immunitaire, l’étude de l’impact du virus HPV-6 (de bas risque) sur la migration et l’invasion des cellules trophoblastiques, et l’étude de la capacité des protéines précoces d’HPV-16 à influencer l’entrée des particules virales, ont fait l’objet de résultats préliminaires, ouvrant de larges perspectives. Genital Human Papillomavirus (HPV) infections are the most common sexually transmitted infections amongst women on the age of reproduction. It is well established that persistent infection with high-risk HPVs is the necessary factor in the causation of precancerous and cancerous cervical lesions. High-risk HPVs have also been reported to be involved in the causation of head and neck cancers and other anogenital cancers. On this last decade, growing data are attempting to study the potential etiological association of HPV with gestational dysfunctions. The detection of HPV DNA in placentas resulting from spontaneous abortions and the unique ability of multiple HPV types to replicate in vitro in trophoblastic cells cultured in a monolayer system, rise new questions over the HPV tropism. Six days following fertilization and once the apposition of the blastocyst on the uterine wall takes place, the trophoblast, in a very active and complex process, starts to proliferate, invade and to differentiate in order to build a physiological interface; the placenta, from where multiple mother/foetus exchanges occur. Interestingly, the way that the trophoblast behaves is very similar to malignant tumoural cells. However, the trophoblast obeys to strict spatial-temporal regulatory confines, insuring a proper development all along the pregnancy. In regard to these data, we hypothesised that the expression of the high-risk HPV type 16 oncoproteins E5, E6 and E7, might modify the development of the infected trophoblast. During my Ph.D study, I demonstrated that the highly hydrophobic protein E5 is localized in many interne membranes compartments of the transfected trophoblast. E5 affects the viability of transiently and stably transfected trophoblastic cells. E6 and E7, favouring cell growth, neutralised the E5 cytotoxic effect. All HPV-16 early proteins, when expressed under the control of their endogenous promoter (LCR), favoured trophoblastic growth. These observations were also observed in cervical cell lines. In addition, E5 decreased the adhesiveness of trophoblastic cells to the tissue culture plastic and to endometrial cells similarly as previously described for E6 and E7. Cells expressing E6, E7 and in less extend E5 favoured chemotaxic migration and matrigel invasion compared to the cells expressing the LacZ control. These effects were also observed when early proteins were expressed under the control of their own viral promoter (LCR). Interestingly, the E-cadherin was down regulated in trophoblastic cells expressing E5, E6 and E7. In conclusion, HPV-16 early proteins enhanced trophoblastic growth and intensify the malignant phenotype by impairing cell adhesion leading to increased cellular motile and invasive properties. HPV-16 E5 participated, with E6 and E7, in these changes by impairing E-cadherin expression, a hallmark of malignant progression. Additional preliminary results consisting on the investigation of other markers of malignancy and immune tolerance, on studying the impact of the low-risk HPV type 6 early proteins on the migratory and invasive properties of trophoblastic cells and on the study of the ability of HPV-16 to influence the entry of virus particules, allowed to open wide perspectives.

Adhesion of bacteria is a key event in biofilm formation and is mediated by bacterial adhesins recognising host or bacterial partner receptors. In oral biofilm formation, primary Actinomyces and Streptococcus colonizers adhere to salivary pellicle proteins such as proline-rich proteins (PRPs) as well as to mucosal surfaces. Subsequently, Actinomyces and Streptococcus strains and other bacteria, such as Veillonella, Fusobacterium and Porphyromonas, adhere to each other. The nature of this community is highly important for the health or disease status, although specific pathogenic species may also have been implicated. The aim of this thesis was to study key players in early oral colonisation, Actinomyces and Streptococcus species, and more specifically the nature of their adhesins and ligands. A further aim was to study the function of the salivary PRP proteins and an innate peptide derived thereof on bacterial adhesion, proliferation and regulation of pH, i.e. key factors in biofilm formation. In paper I and II, adhesion, proliferation and pH affecting features of the RGRPQ (arginine-glycine-arginine-proline-glutamine) peptide, derived from PRP-1, were demonstrated. By use of an alanine-scan (I), motifs for adhesion inhibition and desorption of Actinomyces naeslundii, and proliferation stimulation, ammonia production and inhibition of sucrose induced pH drop by Streptococcus gordonii were indicated. The RGRPQ peptide also stimulated S. gordonii colonisation in vivo. In paper II, a more sophisticated quantitative structure-activity relationship (QSAR) study, using statistical molecular design (SMD) and multivariate modelling (partial least squares projections to latent structures, PLS), further narrowed down the RGRPQ peptide motifs. The R and Q amino acids were crucial for activity. For proliferation a hydrophobic and large size third position amino acid was crucial, while adhesion inhibition and desorption needed a small hydrophilic second position amino acid. All functions depended on a low polarity hydrophobic fourth position. Accordingly, activities could be optimized separately, with decreased function in the others. In paper III and IV, focus was on the bacterial adhesins and their binding epitopes. The genes for FimA major subunit proteins of type-2 fimbriae were sequenced from A. naeslundii genospecies 1 and 2 and Actinomyces odontolyticus, each with unique carbohydrate binding specificities (III). Three major subtypes of FimA proteins were found that correlated with binding specificity, including a novel fimA gene in A. odontolyticus. All subtypes contained a pilin, LPXTG and E box motif. In paper IV, multiple PRP binding patterns for Actinomyces and Streptococcus strains were mapped using a hybrid peptide construct. The two most deviating binding groups deviated in type-1 fimbriae mediated binding to milk and saliva protein ligands. In conclusion, differences in bacterial adhesins and their ability to utilise salivary proteins may render bacteria tropism for different niches. Peptides derived from protein receptors, such as RGRPQ, may be important modulators of biofilm formation, giving commensal bacteria a competitive edge in the bacterial community.

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mso-font-signature:-1610611985 1107304683 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman","serif"; mso-fareast-font-family:"Times New Roman";} .MsoChpDefault {mso-style-type:export-only; mso-default-props:yes; font-size:10.0pt; mso-ansi-font-size:10.0pt; mso-bidi-font-size:10.0pt;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 70.85pt 70.85pt 70.85pt; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> <!--[if gte mso 10]><mce:style><! /* Style Definitions */ table.MsoNormalTable{mso-style-name:"Normal tabell";mso-tstyle-rowband-size:0;mso-tstyle-colband-size:0;mso-style-noshow:yes;mso-style-priority:99;mso-style-qformat:yes;mso-style-parent:"";mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-para-margin:0cm;mso-para-margin-bottom:.0001pt;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Calibri","sans-serif";mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-fareast-font-family:"Times New Roman";mso-fareast-theme-font:minor-fareast;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;} --><!--[endif]--> The aim of the project was to develop a method to investigate differences in adhesion of microorganisms to materials that contains lignocellulose. The method was tested on a gram-positive (Micrococcus lutea) and one gram-negative (E-coliJM109) bacteria. The study was begun by cultivation of the two microorganisms. The cultivation was done to calculate the generation times of the bacteria and to obtain growth curves. Cells from these cultivations were also frozen (-70ºC) and later used for inoculation. At STFI-Packforsk AB the total charge on the mass was measured and later a conductivity titration on the mass was executed as well, all to find out more about the different properties of the mass. Properties that in a later part of this study could possibly be linked to the adhesion of cells to the pulp. The adhesion experiments that were executed gave poor results. The adhesion experiment with M. lutea was the only experiment that gave a reproducible result. In this experiment M. lutea was contacted with bleached leaf. A reduction of cells was observed in all of the dilutions where M. lutea had been in contact with the mass. The number of colony forming units of the culture was 1,2×107 before the adhesion and 2×106 subsequently.