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.

No description available.

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

La giardiose, une maladie causée par le parasite protozoaire Giardia, est l'une des infections gastro-intestinales les plus détectées dans le monde. Son traitement repose principalement sur l'utilisation d'antibiotiques ou d'antiparasitaires, puisqu'il n'existe pas de vaccin disponible pour prévenir l'infection. Cependant, depuis 60 ans, la même molécule, le métronidazole (MTZ), est utilisée à cette fin comme première ligne de défense, chez les humains et les animaux, ce qui a entraîné une émergence de souches résistantes du parasite. De même, un autre médicament, le fenbendazole (FBZ), principalement utilisé pour traiter les infections helminthiques chez les animaux, a été découvert pour être efficace contre ce protozoaire au début les années 2000. De même, l'apparition de parasites résistants au FBZ est déjà en augmentation. À cette fin, nous avons réalisé une nouvelle approche basée sur le profilage protéomique thermique (TPP) pour caractériser davantage le mode d'action de ces médicaments anti-Giardia, ainsi que pour approfondir nos connaissances des mécanismes de résistance utilisés par Giardia. En interagissant avec un ligand, la dénaturation des protéines induite par la chaleur peut varier. En utilisant la protéomique quantitative multiplexée basée sur la spectrométrie de masse, nous avons observé le comportement de dénaturation de multiples protéines solubles exprimées dans le protéome de Giardia, à la fois en présence et en l'absence de ces médicaments. Des analyses bioinformatiques ont été effectuées, y compris la normalisation, l'ajustement du profil de dénaturation et l'identification des protéines qui ont démontré un changement de température supérieure ou égale à 2 (-2  ΔTm  2) causé par l'interaction avec les médicaments. Grâce à cette nouvelle approche, nous avons pu valider les cibles précédemment signalées dans la littérature de ces médicaments, telles que la thiorédoxine réductase pour le MTZ et la tubuline pour FBZ, en plus d’identifier de nouvelles cibles possibles dans le protéome du parasite, telles que la protéine 14.3.3 et les giardins. En outre, plusieurs protéines associées à la résistance aux médicaments ont révélé une interaction avec ces médicaments, ce qui permet de mieux comprendre les mécanismes de résistance déployés par le parasite. Nos résultats préparent le terrain pour la future création de nouveaux médicaments ciblant des mécanismes spécifiques au sein du parasite. / Giardiasis, a disease caused by the protozoan parasite Giardia, is one of the most detected pathogens of the gastrointestinal tract worldwide. Its treatment relies primarily on the use of antibiotic or antiparasitic agents since there is no vaccine available to prevent infection. However, since the past 60 years, the same molecule, metronidazole (MTZ), has been used for this purpose as first line of defense, in humans and animals, causing an increase of emerging resistant strains of the parasite. Likewise, another drug, fenbendazole (FBZ), primarily used to treat helminths infection in animals, was shown to be effective against this protozoan since the years 2000. In the same way, apparition of resistant parasite is already on the rise. To this end, we have carried out a novel approach based on thermal proteomic profiling (TPP) to further characterize the mode of action of these antigiardial drugs, as well as to give insight in the mechanisms of resistance employed by Giardia. By interacting with a ligand, proteins heat-induced denaturation can vary. Using multiplexed quantitative mass spectrometry-based proteomics, we observed the melting behavior of multiple expressed soluble proteins within Giardia’s proteome, both in the presence and absence of these drugs. Bioinformatics analyses were executed, including normalization, melting profile fitting and identification of proteins that showed a change of temperature higher or equal to 2 (-2  ΔTm  2) caused by the interaction with the drugs. With this novel approach, we were able to validate previously reported target of these drugs, such as the thioredoxin reductase for MTZ and tubulin for FBZ. Furthermore, we identify new possible targets in the parasite proteome, such as protein 14.3.3 and giardins. Moreover, several proteins associated with drug resistance were shown to interact with these drugs, giving possible insight in mechanisms of resistance deployed by the parasite. Our findings lay the groundwork for the future creation of new treatment drugs targeting specific mechanisms within the parasite.

The choanoflagellate Salpingoeca rosetta is an important model system to study the evolution of multicellularity. In this study we developed a new, modular, and scalable synthesis of sulfonolipid IOR-1A (six steps, 27% overall yield), which acts as bacterial inhibitor of rosette formation in S. rosetta. The synthesis features a decarboxylative cross-coupling reaction of a sulfonic acid-containing tartaric acid derivative with alkyl zinc reagents. Synthesis of 15 modified IOR-1A derivatives, including fluorescent and photoaffinity-based probes, allowed quantification of IOR-1A, localization studies within S. rosetta cells, and evaluation of structure-activity relations. In a proof of concept study, an inhibitory bifunctional probe was employed in proteomic profiling studies, which allowed to deduce binding partners in bacteria and S. rosetta. These results showcase the power of synthetic chemistry to decipher the biochemical basis of cell differentiation processes within S. rosetta.