Ubiquitylation is a versatile post-translational modification that participates in regulation of protein stability, via proteasomal and lysosomal degradative pathways, regulation of membrane protein internalisation and other trafficking events, and regulating the biological activity of some proteins independent of degradation. The diverse functions of ubiquitylation as a post- translational protein modification allow speculation that regulation of protein ubiquitylation status may be of crucial importance during the dynamic process of development. A screen of known, and suspected, ubiquitin pathway enzymes was designed to test this hypothesis. Whole mount in situ hybridisation was conducted on early post implantation mouse embryos to determine expression patterns of the ubiquitin pathway enzyme targets. This screen was not pursued in depth due to difficulties in resolving doubts regarding the sensitivity of the method and the validity of weak ubiquitous staining patterns. The FAM deubiquitylating enzyme is a known developmentally regulated ubiquitin pathway enzyme, and although believed to antagonise the conjugation of ubiquitin to specific substrates its cell biology remains poorly characterised. In different cellular contexts FAM has been reported to localise to points of cell-cell contact or to endosomes, and circumstantial evidence suggests a role in regulating trafficking of a cell-cell adhesion complex (Murray et al., 2004; Taya et al., 1999; Taya et al., 1998). It was sought to further investigate the role of FAM in cell-cell adhesion in the well characterized polarized epithelial cell line, MDCKII, by creating clonal MDCKII cell lines that overexpress FAM. These cell lines were to be analysed for alterations in cell-cell adhesive properties and the biochemistry of proposed FAM substrates, which include the cell adhesion molecules β-catenin, E-cadherin and AF-6. MDCKII cell lines expressing exogenous V5-tagged murine FAM were successfully isolated, but failed to show changes in cell-cell adhesive properties. Generation of an antibody that reliably recognised both the canine and murine FAM protein demonstrated that the FAM-V5 expressing cell lines did not have increased total FAM protein. Other approaches taken to facilitate the study of FAM include attempts to express GFP-FAM fusion proteins and to generate an inducible FAM overexpressing cell line. Further alternative approaches are discussed. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

This research is intended to contribute toward the understanding, development, and implementation of a more fundamental design process for bituminous pavement materials, utilizing thermodynamic properties of the materials involved. The theory developed by van Oss, Chaudhury and Good forms the basis of this research. Optimization of techniques to characterize surface energy, as well as consideration and evaluation of additional factors that influence adhesion in the presence of water, are pursued. A synthesis of theories and mechanisms of bitumen-aggregate adhesion is presented, and existing and potential techniques for surface energy characterization are reviewed to establish firm background knowledge on this subject. The Wilhelmy plate technique was scrutinized and improved methodologies and analysis procedures are proposed. Inverse gas chromatography (IGC) is introduced as an alternative technique. A reasonable comparison of total surface energy values form these techniques with mechanical surface tension values were found. Results suggest that bitumen surface energies do not vary substantially. Inability of these techniques to detect the effect of a liquid additive is rationalized by the ‘potential’ surface energy concept. Suggestions for a more realistic characterization of bitumen polar surface energy components are presented. A static gravimetric sorption technique was employed to characterize aggregate surface energies. Dynamic vapor sorption was identified as a candidate alternative technique for aggregate surface energy characterization. A study on the effect of pH on surface energy components of water revealed that this effect is practically negligible. Calculation of the free energy of electrostatic interaction (DGEL) indicated that this term contributes less than 1% to the total free energy of adhesion. Despite this finding, it is shown that DGEL alone is able to distinguish moisture sensitive mixtures. The significance of electrical phenomena at the interface is elucidated through another mechanism following the work of M.E. Labib. The relationship between pH and electron donor-acceptor properties of aggregate surfaces is presented. The Labib approach potentially offers the solution to quantify the effect of pH on adhesion. In addition, it should be possible to resolve issues with the acid-base scale proposed by the founders of the current theory, by replacing it with a more absolute donor-acceptor scale.

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Ubiquitylation is a versatile post-translational modification that participates in regulation of protein stability, via proteasomal and lysosomal degradative pathways, regulation of membrane protein internalisation and other trafficking events, and regulating the biological activity of some proteins independent of degradation. The diverse functions of ubiquitylation as a post- translational protein modification allow speculation that regulation of protein ubiquitylation status may be of crucial importance during the dynamic process of development. A screen of known, and suspected, ubiquitin pathway enzymes was designed to test this hypothesis. Whole mount in situ hybridisation was conducted on early post implantation mouse embryos to determine expression patterns of the ubiquitin pathway enzyme targets. This screen was not pursued in depth due to difficulties in resolving doubts regarding the sensitivity of the method and the validity of weak ubiquitous staining patterns. The FAM deubiquitylating enzyme is a known developmentally regulated ubiquitin pathway enzyme, and although believed to antagonise the conjugation of ubiquitin to specific substrates its cell biology remains poorly characterised. In different cellular contexts FAM has been reported to localise to points of cell-cell contact or to endosomes, and circumstantial evidence suggests a role in regulating trafficking of a cell-cell adhesion complex (Murray et al., 2004; Taya et al., 1999; Taya et al., 1998). It was sought to further investigate the role of FAM in cell-cell adhesion in the well characterized polarized epithelial cell line, MDCKII, by creating clonal MDCKII cell lines that overexpress FAM. These cell lines were to be analysed for alterations in cell-cell adhesive properties and the biochemistry of proposed FAM substrates, which include the cell adhesion molecules β-catenin, E-cadherin and AF-6. MDCKII cell lines expressing exogenous V5-tagged murine FAM were successfully isolated, but failed to show changes in cell-cell adhesive properties. Generation of an antibody that reliably recognised both the canine and murine FAM protein demonstrated that the FAM-V5 expressing cell lines did not have increased total FAM protein. Other approaches taken to facilitate the study of FAM include attempts to express GFP-FAM fusion proteins and to generate an inducible FAM overexpressing cell line. Further alternative approaches are discussed. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

Nowadays, the assembly of small (<1mm) components has become an industrial reality. Many domains like MEMS, surgery, telecommunications, car industry, etc. now have large use of micro-parts. At this scale, predominant forces are different than in macroworld. The pieces often undergo adhesion problems. The adhesion forces can be splitted in different components : van der Waals, electrostatics and capillary condensation. This work focuses on capillary condensation as it often can be the major component of the adhesion force. The first part of this work details a review of literature of different fields involved in capillary condensation. A simulation tool is then implemented and theoretically validated in the second part of the work. Finally, a test bed is presented; this bed is then used to experimentally validate the simulation results. Experiments and simulation results are shown to concord. Therefore, the simulation tool can be used to model the force due to capillary condensation.

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