Bone tissue continuously adapts to changes in mechanical load. This process can also result in a maladaptive ectopic bone response to mechanical insult, known as heterotopic ossification. The pathological differences at the molecular and structural levels are poorly understood. In vivo models exist but can often be too complex to allow isolation of factors which may stimulate disease progression. This thesis presents the development of a biologically self-structuring bone culture system using a fibrin gel which self-organises between two calcium phosphate anchors when seeded with cells. These bioinspired wound analogues are seeded with primary femoral periosteal cells - key players in bone repair and a range of pathologies- and develop longitudinally over time, allowing to study the temporal evolution of bone mineral and microstructure in excess of a year. Raman spectroscopy and XRD revealed that the mineral was hydroxyapatite and associated with collagen, which was organized like the in vivo tissue. The initial stem cell population differentiated to the terminal osteocytic phase, was linked by longitudinal canalicular networks (demonstrated using nanoCT) and remained viable over the year of culture. This work also demonstrated that pharmacological compounds can prevent the progress of ossification, displaying promise for applications in drug screening.

Ligament injuries are commonplace, with poor native healing leaving injury sites exposed to instability and further damage. A number of surgical methods have been established for reconstruction using a range of materials, but these have a high failure rate and a number of undesirable side-effects. Much recent work has been focused on the development of tissue engineered ligament grafts. One of the major challenges for this is the formation of an effective ligament-bone interface. In native tissue a multi-phase interface enables smooth transfer of forces between the mechanically mismatched bone and ligament tissue, however this has proved hard to replicate. Previous work has developed a bone-bone ligament construct model intended to emulate the native interface through formation of a mineralised region by soluble cement anchors. Development and optimisation of the model has seen an increasing mechanical strength, but the mechanisms involved are poorly understood. This study investigates the development of the ligament construct through the use of multiple complimentary imaging techniques to provide information on the biological, chemical, and topological development of the construct as it forms from initially homogeneous and separate materials to a complex non-homogeneous system.

Sub-critical water (SCW) hydrolysis has previously been used in the extraction of antioxidant compounds from a variety of food wastes, in-particular those which are rich in protein. The brewing industry generates high volumes of waste. The most abundant component, brewers' spent grain (BSG), is high in protein content. The work presented in this thesis aimed to investigate the SCW extraction of antioxidant compounds from BSG. Whilst SCW hydrolysis has proved effective in the extraction of antioxidants from a range of compounds its mechanism of action has not been thoroughly investigated. High performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) was used to analyse peptide production from the SCW hydrolysis of proteins. Sites of cleavage were identified and a mechanism of action of SCW on proteins was postulated. The results from this analysis raised the possibility of using SCW as a proteolytic reagent during proteomics experiments. Approaches for SCW-based proteomics were further explored by investigating SCW induced amino acid side chain modifications to aid peptide identification. To assess the antioxidant capacity of mixtures generated via SCW hydrolysis oxygen radical absorbance capacity, reducing power and comet assays were used. The decomposition products responsible for antioxidant capacity were characterised using MS/MS.

This thesis describes the use of enzymes to partially deprotect or deprotect sugars. The advantages of this technique are that mild conditions may be used for the protections and that enzymatic catalysis may allow derivatives to be made which involve multi-step procedures or cannot be made using standard methods. These protected sugars can then be used to synthesise interesting derivatives. The particular aim of this project at the outset was to use biotransformations to make partially protected derivatives for the synthesis of a novel artificial sweetener, l',4,6'-trichloro-r,4,6'-trideoxy-ga/acto- sucrose, known as sucralose. The analysis of the products from a deacetylation reaction is a potential problem due to the number of possible products that may be formed. A new approach to this problem is discussed in Chapter Two. A combination of n.m.r. and mass spectrometry is used to analyse the products after they have first been perdeuterioacetylated by treatment with d6-acetic anhydride. The analysis is therefore carried out on one compound, the starting material, now containing deuteriated acetate groups in place of those that were hydrolysed during the reaction. The technique was used initially to analyse the deacetylation of sucrose octaacetate catalysed by yeast esterase. The selectivity of the enzyme for certain positions of the sugar may be determined in this way but little information can be found about the individual species that are formed. The technique can be considerably enhanced by the introduction of a chromatographic separation step. The separation of the deacetylation mixture into classes, according to the number of acetate groups, allows a much more detailed analysis of the individual components to be carried out. If the reaction shows a certain amount of selectivity then it is possible to determine the quantity of each of the individual species. This technique is used to analyse the deacetylation of glucose pentaacetate catalysed by Aspergillus niger lipase. The deacetylation of sucrose octaacetate catalysed by yeast esterase is also analysed in the same way. Chapter Three describes the conversion of N-acetyl- glucosamine to N-acetyl-galactosamine. This is of interest due to the importance of this sugar in biological systems and its high cost relative to the starting material. The synthesis involves the use of an enzyme catalysed deesterification to make a partially protected intermediate, demonstrating the practical application of biotransformations in the synthesis of sugar derivatives.