The principal aim of this thesis was to investigate the ability of surface topography in inducing bone cell differentiation for biomedical purposes. In orthopedic research, regeneration of bone defects can be performed in vitro using biomaterials. Third generation biomaterials aim not only to support tissue (first generation) and not only to be ‘bioactive’ (second generation), but to stimulate specific, known and desirable responses at the molecular level. Nanoscale topography offers a possible route to the development of third generation biomaterials. Two-dimensional fluorescence difference gel electrophoresis (2D DIGE) is a new method for assessing protein expression strategies and here, a micro-grooved topography was used as a model for protocol optimization. The protocol was successfully developed and proved that 2D DIGE can be used as a powerful tool in the evaluation of biomaterial can direct cell behavior and cell fate. Next, the refined protocol was applied to the evaluation of the novel nanotopographic features; near-square nanopits (120 nm diameter, 100 nm depth with the pitch between the pits was set to an average of 300 nm with a ± 50 nm error). Protein expression profiles indicated that ERK1/2 might play part in cell proliferation and cell differentiation. However, to make a clear conclusion about molecular signalling, the study of sub-cellular proteome is needed in the future work. Additionally, the use of another comparative proteomic technique; dimethyl labelling, implicated the possibility of sub-population differentiation, i.e. the formation of multiple cell types that could be advantageous in tissue engineering of complex organs. Furthermore, the application of fluid-flow bioreactors was shown to enhance the growth rate and possibly increased differentiation of cells cultured on nanotopographical features.

Trypanosoma brucei evades host acquired immunity by antigenic variation, involving periodic switches in the variant surface glycoprotein (VSG) coat. DNA recombination is critical in this process and a key component of homologous recombination, RAD51, also plays a role in VSG switching. T. brucei encodes four proteins distantly related to RAD51; termed RAD51 paralogues, named RAD51-3, RAD51-4, RAD51-5 and RAD51-6. Two of these RAD51 paralogues, RAD51-3 and RAD51-5, have been shown to function in homologous recombination, DNA repair and RAD51 re-localization into foci following DNA damage. Surprisingly, however, only RAD51-3 appears to act in VSG switching. To examine the functions of all the RAD51 paralogues in T. brucei, reverse genetics has been used to generate mutants of the two remaining unstudied paralogues, RAD51-4 and RAD51-6. Phenotypic analysis of both mutant cell lines indicates that these factors also play critical roles in RAD51-directed recombination and repair, and both influence VSG switching in the parasite. As homozygous mutant cell lines of RAD51 and the RAD51 paralogues were available, it was possible to comprehensively compare the phenotypes of rad51 -/- with rad51-3 -/-, rad51-4 -/-, rad51-5 -/-, and rad51-6 -/-. From these results it was observed that the phenotypes of the rad51 paralogue mutants are broadly equivalent, with two exceptions. Firstly, as mentioned above, RAD51-5 does not function in VSG switching, and RAD51-4 and RAD51-6 may not have direct roles in VSG switching. Secondly, rad51-4 -/- mutants are less sensitive to the DNA damaging agent phleomycin and a higher percentage of rad51-4 -/- cells form DNA damage-induced RAD51 foci compared with the other homozygous mutant cell lines. These results may imply that RAD51-4 and RAD51-5 have a less central role in RAD51-directed DNA repair or that their functions can be performed by other factors. In addition, the physical interactions of all the RAD51 paralogues were examined. It was found by yeast two-hybrid and co-immunoprecipitation that they form at least two complexes, and probably function in sub-complexes in a similar manner to the Rad51 paralogues of higher eukaryotes. These analyses shed light on the evolution and role of eukaryotic RAD51 paralogues in DNA recombination and repair in general, as well as the contribution that recombination makes to antigenic variation in T. brucei.