Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The aim of this study was to explore proteomics and microbiome profiling in pigs fed high fibre diets. In the first phase, maize cobs were ensiled using whey, molasses and exogenous enzymes in the laboratory and effects on ensiling characteristics and fibre levels were evaluated. In the second phase South African Windsnyer-type Indigenous pigs (SAWIP) and Large White x Landrace crosses (LW x LR) were fed diets containing ensiled maize cobs and evaluated on; diet preferences, nutrient digestibility and colonic fermentation; growth performance, carcass traits and blood metabolite profiles; the faecal microbiome; and serum and liver proteomic profiles. Ensiling maize cobs with molasses, whey and exogenous enzymes did not improve fermentation characteristics but exogenous enzymes reduced fibre fractions and energy content of maize cob silages. Dets containing two levels of maize cobs ensiled without any additive; a low (LMC) and high (HMC) maize cob inclusion levels and a control diet which did not have any maize cobs (CON) were formulated. The SAWIP preferred the CON diet more than (P < 0.05) diets with maize cobs while the LW x LR had no feed preferences. There was no correlation between preference and diet digestibility in both breeds. The SAWIP digested nutrients better (P < 0.05) than the LW x LR in the high fibre diets. There were no differences in the diversity of the core composition of gut bacterial communities between the breeds and diets. There were differences in the ratios of Bacteroidia to Clostridia between the SAWIP and LW x LR. Verrucomicrobiae was present in SAWIP and LW x LR on HMC diet and not on the CON diet. There was a breed x diet interaction (P < 0.05) for Oscillospira. Analysis of the microbiome revealed breed differences and no dietary differences. There were differences in serum and liver proteins and in serum metabolite levels. Two specific proteins identified were Guanidinoacetate N-methyltransferase-like isoform 1 associated with creatine biosynthetic and Catalase, which is involved in cholesterol metabolic processes. At the grower stage, the SAWIP consumed more feed per metabolic body weight than the LW x LR while at the finisher stage LW x LR consumed more feed per metabolic body weight (P < 0.05) than the SAWIP. The breed of pig influenced most of the growth performance and carcass parameters more than the diet did. The SAWIP demonstrated an adaptation to high fibre diets by consuming more feed than the LW x LR per metabolic body weight at the grower stage. The inclusion of ensiled maize cobs in diets did not negatively affect selected commercial pork cuts. Analysis of faecal microbiomes revealed differences that may explain the enhanced ability of the SAWIP to digest fibrous diets better than the LW x LR breed.Proteomics can identify biomarkers that evaluate the performance of pigs consuming high fibre diets. A proof of principle to assess serum and liver protein profiles of pigs fed a a high fibre diet using a sodium dodecyl sulphate polyacrylamide gel electrophoresis matrix-assisted laser desorption ionization mass spectrometry (SDS-PAGE /MALDI MS) workflow was established. Key words: ensiling, exogenous enzymes, palatability, fermentation, fibre, metageome, biomarkers.

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Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure.

Matrix metalloproteinases (MMPs) network with other biological molecules to maintain the extracellular matrix (ECM) in normal physiology and perform different roles. Understanding and assigning specific role to each of 24 members of these endoproteinases is impeded because of lack of specific and efficient detection methods in biological samples. Moreover, MMP-based anti-cancer drug development has also been challenged because, currently, there is no robust methodology to distinguish the inactive pro-enzymes, active enzymes or those complexed with endogenous inhibitors in biological specimens. The objective of this project is to develop a chemical proteomics strategy based on Matrix assisted laser desorption ionization tandem mass spectrometry (MALDI-MS/MS) to help identify and discriminate the various MMP forms. Firstly, a triazine dye-based ligand immobilized on chromatography beads was utilized to assess whether it binds to recombinant human MMPs (rhMMPs). The results highlighted that the ligand interacts with latent forms of MMPs in agreement with the literature. Secondly, the potential of the ligand was assessed using MALDI-MS/MS based methodology in in vitro cancer models. Cell line culture supernatants were used in amounts to emulate the availability of tumour biopsies in clinical settings. The MS/MS spectral peaks specific to MMPs (MMP-2 and MMP- 14), and two endogenous inhibitors TIMP-1 and TIMP-2 were found in affinity chromatography eluates of cell culture supernatants with higher Mascot scores for the latter. While western blot detected MMP-2 in cell extracts, MALDI-MS/MS did not detect MMPs because of amounts below the limit of detection (LOD) of the instrument. Although the ligand was found to be interacting with MMPs and detergent-free salt elution buffers improved MALDI analysis, recovery of MMPs from biological samples was sub-optimal. The dye ligand was observed to bind other enzymes and despite various strategies to reduce non-specific binding of proteins or enable selective elution did not improve MMP enrichment. Further work using methodology described in this study is required after scaling up the MMP amounts in biological specimen and to resolve the issue of non-specific binding of proteins to the ligand by understanding its structure. / Shaukat Khanam Memorial Cancer Hospital and Research Centre, Pakistan and University of Bradford