During development, Dictyostelium discoideum cells assume muticellularity via their collective aggregation. Cell-cell adhesion is required for morphogenesis, cell differentiation, cell sorting and gene expression during development. TgrC1 is a heterophilic cell adhesion molecule which is indispendable for complete development. TgrC1 can be considered as the most important cell adhesion molecule for D. discoideum development because deletion of the tgrC1 gene completely arrests development at the loose aggregate stage and inhibits fruiting body formation. In order to investigate the biological role of TgrC1 during development, I have chosen to identify and charactize the extracellular heterophilic partner and the cytoplasmic binding partner(s) of TgrC1. Using different biochemical approaches, we identified TgrB1 as the heterophilic binding partner of TgrC1 and demonstrated that their association is mediated through IPT/TIG domains in the extracellular region of both proteins. Both tgrB1 and tgrC1 share the same transcriptional promoter and their spatiotemporal expression pattern is identical during development. We also examined the assembly of TgrC1-TgrB1 complexes via the split green fluorescence protein complementation assay and the fluorescence resonance energy transfer approach. Whereas TgrC1 is capable of forming cis-homodimers spontaneously, cis-homodimerization of TgrB1 depends on its trans-interaction with TgrC1. A model of the assembly process has been proposed. To investigate signalling events initiated by the interaction between TgrB1 and TgrC1, pull-down assays were employed and led to the identification of myosin heavy chain kinase C as the cytoplamic partner of TgrC1. Mutational analysis showed that the basic residues in the short cytoplasmic domain of TgrC1 are critical to the binding with MHCK-C. Disruption of the interation between MHCK-C and TgrC1 results in an alteration of cell motility at the aggregation stage and aberrant cell sorting in slugs. These studies have highlighted the role of TgrB1-TgrC1 complexes in the regulation of morphogenesis during Dictyostelium development.

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The evolution and development of multicellular organisms requires cells to differentiate, interact and "collaborate". Our understanding of the molecular mechanisms is still hazy. In this dissertation mathematical modelling is used to integrate available experimental data and to make testable predictions about such mechanisms. The thesis is split into three parts, each of which addresses one of the three challenges: differentiation, adhesion and collaboration. In the first part, a mathematical model is developed to explain how, in the absence of polarizing cues from the environment, sister cells with identical genomes can follow distinct routes of differentiation. It is shown that difference in cell size, resulting from asymmetric cell division, is sufficient to induce differential cell fate in Bacillus subtilis. The model predicts that this effect depends on the allosteric behaviour of a kinase and the low catalytic rate of the corresponding phosphatase; both properties were subsequently confirmed in experiments. During the development of multicellular organisms, differentiation can arise in response to gradients. By example of dorso-ventral patterning it is shown how a shallow maternal gradient can be converted into a sharp pattern. In the second part, a model for cell adhesion via integrins is developed, and it is shown that, for physiological parameters, binding of a ligand and of a stabilizing factor such as talin are insufficient for ligand-dependent integrin activation, and that a positive signaling feedback is required. In the final part, antibody affinity maturation is studied as an example for division of labour between collaborating cells. A novel B cell selection mechanism, based on competition for T cell help rather than for antigen, is proposed and shown to reconcile heretofore inexplicable experimental observations. Such a mechanism requires B cells to discriminate among different affinities of binding, and it is further shown that this can be achieved if B cell signaling is initiated by antigen-dependent receptor-inhibitor segregation. The predictions of the model match experimental measurements quantitatively.

To attract more customers and compete with other modes of transportation, railway transport needs to ensure safety, punctuality, high comfort, and low cost; wheel–rail adhesion, i.e., the transmitted tangential force in the longitudinal direction during driving and braking, plays an important role in all these aspects. Adhesion needs to be kept at a certain level for railway operation and maintenance. However, wheel−rail contact is an open system contact. Different contaminants can present between the wheel and rail surfaces, forming a third-body layer that affects the adhesion. Prediction of wheel–rail adhesion is important for railway operations and research into vehicle dynamics; however, this prediction is difficult because of the presence of contaminants. This thesis deals with wheel–rail adhesion from a tribological perspective. The five appended papers discuss wheel–rail adhesion in terms of dry conditions, lubricated conditions, leaf contamination, iron oxides, and environmental conditions. The research methodologies used are numerical modelling, scaled laboratory experiments, and field tests. The research objective is to understand the mechanisms of the adhesion loss phenomenon.  A numerical model was developed to predict wheel–rail adhesion based on real measured 3D surfaces. Computer simulation indicates that surface topography has a larger impact on lubricated than on dry contacts. Plastic deformation in asperities is found to be very important in the model. Ball-on-disc tests indicate that water can give an extremely low adhesion coefficient on smooth surfaces, possibly due to surface oxidation. Investigation of lubricated contacts at low speed indicates that oil reduces the adhesion coefficient by carrying a normal load, while adhesion loss due to water depends on the surface topography, water temperature, and surface oxidation. A field investigation indicates that leaves reduce the friction coefficient because of the chemical reaction between leaves and bulk materials. The thickness of the surface oxide layer was found to be an essential factor determining adhesion reduction. Pin-on-disc experiments found a transition in the friction coefficient with regard to the relative humidity, due to a trade-off between the water molecule film and the hematite on the surface. / <p>QC 20131031</p>

In the Xenopus embryo, cell rearrangements during early development require the dynamic modulation of adhesion. Cells primarily use the integrin family of transmembrane receptors for attachment to and interpretation of the extracellular environment. While acting as adhesion receptors, integrins also have bidirectional signalling properties essential for driving cellular movements. The regulation of integrin activity is thought to stem from cytoplasmic assemblies of constitutively expressed molecules. PINCH (Particularly Interesting New cysteine-histidine rich protein), an adapter protein, is part of an IPP complex that has emerged as a key signalling scaffold indispensable for integrin function in vitro. As such, I tested the hypothesis that PINCH regulates integrin function in the Xenopus embryo. Xenopus PINCH was successfully cloned using RT-PCR. The predicted amino acid sequence of PINCH shares a 98% similarity with mammalian orthologs, and comprises of five highly conserved LIM domains. PINCH mRNA and protein are ubiquitously expressed throughout embryogenesis. In situ hybridization indicates that PINCH mRNA is expressed in the blastocoel roof and the pre-involution mesoderm. The localization and temporal expression of PINCH suggests a role in mediating cell adhesive events during gastrulation. A functional approach was used to examine the role of PINCH during gastrulation. I used site-directed mutagenesis to generate non-functional LIM1 (LIM1mut) and LIM4 (LIM4mut) domains that have been proposed to bind ILK and Grb4 respectively. Over-expression of PINCH leads to a delay in blastopore closures, while the expression of both LIM1mut and LIM4mut relieve this inhibition at lower concentrations. Further analysis indicates that PINCH, LIM1mut, and LIM4mut inhibit FN matrix assembly independent of integrin adhesion. Contradictory to in vitro studies, co-immunoprecipitation analysis indicates that endogenous PINCH does not bind ILK, confirming an integrin-independent role during gastrulation. Furthermore, in the embryo PINCH is found at cell boundaries but does not appear to directly modulate cadherin adhesion. As such this thesis provides evidence that PINCH regulates cell intercalation movements independent of integrin and cadherin receptors and raises the possibility that the LIM4 domain is involved in PINCH regulation of cell adhesion during early development.

Cement - within - Cement (C-C) revision techniques have been demonstrated to reduce the complications associated with removal of secure cement from the femoral canal during revision hip joint arthroplasty. Material failure at the interface between new and old cement mantles represents a theoretical limitation of this technique. The objectives of this thesis are to describe the variability in material properties of uniform and bilaminar polymethylmethacrylate (PMMA) cement mantles in shear with respect to duration of post-cure and the influence of commercial inclusion of antibiotics on bilaminar cement mantle interfacial shear strength. Uniform mantles of Surgical Simplex P and Antibiotic Simplex PMMA cements demonstrated variability in ultimate shear stress to failure with respect to duration of post-cure (p < 0.001), however the variations were quantitatively small and unlikely to be of clinical relevance. Bilaminar cement mantles were 15 - 20 percent weaker than uniform mantles (p < 0.001) and demonstrated similar time dependant material property variations in shear (p < 0.001). Bilaminar PMMA test specimens manufactured using Antibiotic Simplex cement demonstrated equivalent ultimate shear stress to failure as bilaminar specimens manufactured from Surgical Simplex (p=0.52). High C-C interfacial strengths are demonstrated as early as one hour after cement application. Interfacial adhesion by mechanisms other than mechanical interlock significantly influence the bond formed between layered PMMA cements, with an important contribution by diffusion based molecular interdigitation. In the presence of a secure cement-bone interface, C-C femoral revision can be recommended as a viable technique on the basis of the strong interfacial bond formed between new and old cement mantles. The use of Antibiotic Simplex in C-C revision is recommended as detrimental effects on the interfacial shear properties have not been demonstrated with the commercial addition of Tobramycin.

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.

Siderophores are a series of important iron chelators secreted by many bacteria that normally have high affinity of iron ions and contain hydroxamate, catecholate and carboxylate and other ligand groups. These organic ligands play significant roles in bacterial metabolism: solubilising iron (III) from environments, enabling iron uptake and acting as a cell-signalling molecule to control gene expression. Recent observations of initial stages of Pseudomonas aeriginosa biofilm formation on metal oxides surfaces indicate that siderophores may also facilitate bacterial adhesion to metals. However, details of how siderophores interact with metal surfaces and the relationship of their chemical nature with bacterial adhesion were not fully understood. To test the generality of bacteria attachment to metals via siderophores, the adsorption behaviour of siderophores and their functional ligands groups on particle films of metal oxides were investigated with in-situ ATR-IR spectroscopy. In this study, nanoparticle films of titanium oxide, boehmite, iron oxide and chromium oxyhydroxide were prepared as substrates to simulate titanium, alumnium, stainless steel surface and more detailed work were carried out on titanium dioxide. Monohydroxamic acids (acetohydroxamic acid, N-methylformohydroxamic acid, N-methyl-acetohydroxamic acid and 1-hydroxy, 2-piperidone) and catechol and catechol-like (L-dopa and esculetin) ligands were selected as modelling compounds for the most frequently occured functional groups in natural siderophores. IR spectra of these ligands in aqueous solution and adsorbed on TiO₂ were obtained with a flowing cell system fit to a horizontal accessory by in situ ATR-IR spectroscopy and interpreted based on vibrational mode analysis with density function theory. Results show that all these ligands can form surface complexes with metal surfaces and possible adsorption modes of these molecules were discussed. The pH dependence of absorbance of IR absorption of these adsorbed ligands showed that most of the ligands exhibited maximium adsorption to TiO₂ at about pH 8, only N-methylformohydroxamic acid, 1-hydroxy, 2-piperidone groups having maximum absorption at pH~3 and pH~6. Infrared spectroscopic studies of adsorbed siderophores (desferroximme B, enterobactin and pyoverdine) were also carried out in variation of concentration and pH. Possible adsorption modes of these iron scavenger ligands on TiO₂ and other metal oxides were discussed. Results showed that all these ligands adsorbed on metal oxide surfaces and form surface complex via hydroxamate or catecholate lignd groups which confirm the generality of siderphore-metal bond formation. Siderophore based-bacterial adhesion to metals of was conducted with Pesudomonas aeruginosa and E. coli strains on titanium oxide and iron oxide films. These observations support that siderophore play a significant role in bacterial adhesion to metals. Further work needs to be carried out on the wider involvement of siderophores in bacterial adhesion initiation to metals and siderophore-mediated iron transport.

The ongoing interest in bacterial interactions with various surfaces, followed by attachment and subsequent biofilm formation, has been driven by the importance of bacterial activities in number of medical, industrial and technological applications. However, bacterial adhesion to surfaces has not been completely understood due to the complexity of parameters involved. The study presented herein investigates the attachment pattern of nine medically and environmentally significant bacteria belonging to different taxonomic lineages: Firmicutes - Bacillus, Gammaproteobacteria, Alphaproteobacteria and Bacteriodetes. Physicochemical assessment techniques such as contact angle and surface charge measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), confocal microscopy (CLSM), as well as X-ray photoelectron spectroscopy (XPS), X-ray fluorescence spectroscopy (XRF) and time-of-flight secondary ion mass spectroscopy (ToF-SIMS) analysis were all employed in order to attain better insight into the factors that influence bacterial interactions with surfaces. Bacterial surface characteristics such as surface wettability and charge in addition to substratum surface wettability, tension, charge and chemistry were also considered. However due to the recent interest in designing micro-textured surfaces with antibacterial and/or antifouling effects the prime was given to the influence of micro- and nano-meter scale surface textures on bacterial adhesion. The interactions between selected bacteria and glass, polymer and optical fibre surfaces were studied. Carefully designed methods for surface modification allowed alteration of the topography of glass, polymer and optical fibre surfaces while maintaining other surface parameters near constant. This allowed isolated assessment of only the effects of surface roughness on bacterial adhesion. Obtained results indicated consistent cellular inclination towards the smoother surfaces for all of the tested species. Enhanced bacterial presence on the smoother surfaces was also accompanied by changes in the bacterial metabolic activity as indicated by the elevated levels of secreted extracellular polymeric materials (EPS) and modifications in the cells morphology. The results indicate that nano-scale surface roughness exert greater influence on bacterial adhesion than previously believed and should therefore be considered as a parameter of primary interest alongside other wellrecognized factors that control initial bacterial attachment.

Lactoferrin (Lf) is an iron binding protein produced in mammals. It has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonize mammalian hosts have adapted to living in high Lf environments. Helicobacter pylori, which inhabits the human gut, was chosen as a model organism to investigate how bacteria may adapt to Lf. H. pylori was able to use iron from fully saturated human Lf (hLf) in various low iron media, achieving growth levels similar to the ironreplete control. Partially saturated hLf decreased growth, yet both partially saturated bovine Lf (bLf) and hLf were able to increase internalization of bacteria into mammalian tissue culture cells. A substantially larger increase in internalization was seen when bacteria were supplemented with hLf in low iron conditions, possibly mediated by iron-regulated cellular receptors or bacterial lactoferrin binding proteins. In eukaryotes, Lf is known to bind and facilitate internalization of DNA into cells and sometimes the nucleus, and upregulate gene expression. Here, one hundred bacterial genomes were surveyed for known Lf binding sites as an indication that Lf had similar functions using bacterial DNA. While the frequency and location of Lf binding sites suggest they occur at random, their presence in all genomes suggests that Lf may be able to act as a vector for bacterial DNA, and facilitate the movement of genes between species. Lf is being widely considered for commercial and therapeutic uses, with significant interest in producing it in genetically modified organisms (GMO). Widespread production and use of Lf could increase the number of bacteria that are adapted to it. How Lf interacts with bacteria adapted to it, and the ability of it to act as a DNA vector, may have relevance for GMO risk assessment.