Bioactive peptides can be released, during gastrointestinal digestion or food processing, from many plant and animal proteins and represent an important source of potential health_enhancing components. Therefore, the aim of tIus study is to investigate antioxidant, anticancer and antihypertensive activities of proteins and peptides extracted from egg yolk. Defatted egg yolk (EY) was hydrolysed using pepsin and pancreatin and sequentially fractionated by ultrafiltration, followed by gel filtration (EYGF), resulting in tlu'ee different fractions of which two (EYGF-23 and EYGF-33) effectively inhibited the oxidation of linoleic acid in a dose-dependent malmer. Both fractions (80 mglml) exhibited antioxidant activity comparable to trolox and butylated hydroxytoluene (0.2 mg/ml). The antioxidant mechanism involved superoxide anion and hydroxyl radicals scavenging, fenous chelating and reducing ability. The protective effect of one of the fractions (EYGF-23) was also tested in Caco-2 colon cancer epithelial cells, exposed to 3 mM of tert-butyl hydroperoxide (t-BHP) to induce oxidative stress. Induced cell toxicity by t-BHP was significantly inhibited when Caco-2 cells were treated with 1.0 mglml of the fraction for 24 hours. This fiaction protected the cells by inhibiting intracellular ROS as well as lipid peroxidation products by 60.0% (p <0.001) and 24.15%, (p <0.05). In addition, total glutathione level and superoxide dismutase activity were significantly elevated to 0.29 IlM (p <0.001) and 55.10% (p <0.01), respectively, in comparison to cells treated with t-BHP alone. Amino acids lysine, proline, tyrosine and tryptophan in this fraction may be responsible for antioxidant activity.

This thesis describes the structural and functional analysis of two related proteins, PI31 and Fbxo7, both of which act within the ubiquitin-proteasome system. This system is involved in a wide variety of cellular processes. Proteins modified with ubiquitin are often targeted for degradation by the proteasome, an ATP-dependent multi-subunit complex. Specificity for ubiquitin transfer is controlled by the E3 ubiquitin ligases. The multi-subunit SCF E3 ubiquitin ligases use their F-box domain protein to engage substrates. Analysis of possible binding partners of the novel F-box protein Fbxo7 using an affinity pull-down in Jurkat cell lysates identified PI31, a putative proteasome inhibitor. Fbxo7 was also shown to bind Skpl, Cullinl and Rbxl inferring that it forms part of a functional E3 ubiquitin ligase. Crystallisation of the Fbxo7-Skpl complex yielded poorly diffracting crystals with a maximum resolution of 8A. The PI31 N-terminal domain (NTD) shows significant sequence similarity to a globular domain of Fbxo7. The PI31-NTD structure was obtained at 2.64A by MAD phasing after introducing an additional methionine to engineer a non-centrosymmetric selenium substructure. The structure reveals a homodimer of novel a/p topology, which we define as the FP (Fbxo7 - PI31) domain since its distribution is limited to Fbxo7 and PI31 proteins in higher eukaryotes. Biophysical analysis of structure-based mutations identified the relevant residues in the homodimer interface by confirming the production of a monomeric form of PI31. Further mutations also revealed the binding surface between Fbxo7 and PI31 by using isothermal calorimetry. Fbxo7 and PI31 were shown to interact in vivo by co-immunoprecipitation and yeast two-hybrid screens using either PI31 or Fbxo7 as bait. Fbxo7 was shown to ubiquitinate FLAG-tagged PI31 in vivo. Abolishing the interaction between Fbxo7 and PI31 by mutations in PI31 prevents this ubiquitination. A model for the interaction between PI31 and Fbxo7 is presented.

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The human plasma proteome is potentially rich in novel biomarkers for the diagnosis, prognosis of disease and drug development. In the quest for low abundant biomarkers, the problems associated with the complexity of sample and vast dynamic range in concentration have to be overcome. Such strategies include the depletion of the most abundant proteins from plasma, the employment of prefractionation techniques and multidimensional separations. Mass spectrometry can be used to identify and quantify these biomarkers. The most abundant proteins can be depleted from plasma using antibody or dye based affinity columns. Several abundant protein depletion kits were investigated with the aim of selecting the most effective kit for subsequent analyses.

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