Cette thèse a été consacrée à l'évolution du transcriptome de Drosophila simulans, et à son rôledans l'adaptation et la spéciation. L'étude a comporté deux parties. La première utilisant des pucesà ADN pour comparer les transcriptomes de populations de D. simulans, de son espèce jumelleD. sechellia, et de leurs hybrides. La seconde basée sur la quantification des transcrits par séquen-çage haut débit pour comparer une population de la zone d'origine (Afrique), et une populationd'une zone récemment envahie (France métropolitaine). Ces analyses ont mis en évidence plusieursgroupes ou familles de gènes montrant des variations d'expression.Un résultat majeur est l'implication prépondérante de la famille des cytochromes P450 dansl'adaptation. Cette superfamille composée de 85 gènes chez D. simulans est notamment importantepour la détoxi_cation des xénobiotiques. L'expression de plusieurs gènes de cette famille estfortement réduite chez D. sechellia, probablement à cause de la spécialisation de cette espèce surla plante Morinda citrifolia (plante toxique pour les autres drosophiles). On peut s'attendre alorsà un relâchement des contraintes de sélection sur cette famille de gènes, dû à une forte réductionde la diversité des toxines auxquelles cette espèce est exposée.Ces gènes sont également impliqués dans la différenciation entre les populations de la zoneancestrale de D. simulans et les populations dérivées. La zone ancestrale, en Afrique de l'est etdans les îles de l'Océan Indien occidental, est encore peu anthropisée. A contrario, la plupartdes populations dérivées, comme ici notre population de la vallée du Rhône, sont exposées à descontraintes chimiques sous la forme de pesticides utilisés massivement sur les grandes cultures.Ces pesticides contraignent les populations dérivées à s'adapter, ce qui peut se réaliser par uneaugmentation de l'expression. Nous avons détecter une augmentation de l'expression de treizeP450, dont un gène très bien connu pour ses fonctions de détoxifcation : Cyp6g1. Ce gène montreune augmentation d'expression corrélée à une résistance aux pesticides et à l'insertion d'élémentstransposables en 5' ; ceci a été montré en détail chez D. melanogaster, et dans une moindre mesurechez D. simulans. Nous avons mis en évidence chez cette dernière espèce un nouvel évènementd'insertion.Nos résultats montrent également que d'autres familles de gènes impliqués dans les détoxi_-cations sont concernées par ces augmentations d'expression liées à l'anthropisation des milieux,notamment les Glutathion transfèrases (GST).Nous avons également examiné la plasticité d'expression liée au changement de ressource, enélevant une partie de nos populations sur la ressource d'origine (fruits), et une autre partie sur lemilieu axénique, milieu d'élevage standard de laboratoire (stérile). Les drosophiles élevées sur unmilieu "naturel" montrent une forte activation du système immunitaire, et notamment une forteinduction des gènes effecteurs de l'immunité innée, codant les peptides anti-microbiens. Cela estprobablement dû à la présence de microorganismes sur ce milieu (ici, la banane). En conclusion,cette thèse a révélé des familles de gènes fortement impliquées dans les différenciations d'expressionentre populations, espèces, et ressources, posant les jalons d'une meilleure compréhension desmécanismes d'adaptation du transcriptome. / The topic of this thesis was the evolution of the transcriptome of Drosophila simulans, andits role in adaptation and speciation. First, we used microarrays to compare transcriptomes ofpopulations of D. simulans, its sister species D. sechellia and their hybrids. Second, we useda RNA-seq like approach to quantify gene expression of a population from the ancestral range(Africa) on the one side, and a population from a recently invaded zone (France) on the other side.These analyses revealed several gene groups or families showing gene expression variations.One main result is the major involvement of the cytochrome P450 gene family in adaptation.This superfamily is composed of 85 genes in D. simulans, and is notably important in detoxificationof xenobiotics. The expression of several genes of this family is strongly reduced in D. sechellia,likely due to the specialization of this species on Morinda citrifolia (a plant toxic for any otherdrosophila). This specialization may strongly reduce the diversity of toxines this species is exposedto, thus relaxing selective constraints on this gene family.These genes are also involved in the differentiation between populations of the ancestral range ofD. simulans and derived populations. The ancestral range, in eastern Africa and in the occidentalislands of the Indian Ocean, is not highly anthropized yet. Contrasting with this pattern, manyderived populations (here our population from the Rhône valley) are exposed to chemical pressuresdue to the massive use of pesticides on agricultural zones. These pesticides force derived populationsto adapt, which can happen via increased expression in genes such as the very famous exampleof Cyp6g1. This gene shows a strong increase in expression correlated with pesticide resistance aswell as the insertion of transposable elements upstream of the gene ; this was described in detailsin D. melanogaster, and to some extent in D. simulans. We have shown a novel insertion event inthe latter species.Our results also reveal the involvement of other gene families in detoxification linked withanthropized environment via increase of gene expression, notably the Glutathione transferases(GST).We also examined expression plasticity linked with resource change, raising half our _ies ontheir natural food resource (fruits), and the other half on axenic medium (standard sterile laboratorymedium). Drosophila raised on their natural medium show a strong activation of their immunesystem, and notably an induction of the effectors of teir innate immunity, the anti-microbial peptides.This observation can be explained by the presence of microorganisms on this medium (here,banana). To conclude, this thesis revealed gene families strongly involved in expression differentiationamong populations, species and food resources, paving the way of a better understanding ofmechanisms of adaptation of the transcriptome.

A conversão de biomassa vegetal em produtos químicos e energia é essencial a fim de sustentar o nosso modo de vida atual. O bagaço de cana-de-açúcar, matériaprima disponível em abundância no Brasil, poderá tanto ajudar a suprir a crescente demanda pelo etanol combustível como ser empregado para obtenção de produtos de valor agregado, tais como xilitol, além de trazer vantagens econômicas para o setor sucroalcooleiro. O xilitol, um poliol com poder adoçante semelhante ao da sacarose e com propriedades peculiares, como metabolismo independente de insulina, anticariogenicidade e aplicações na área clínica, no tratamento de osteoporose e de doenças respiratórias, é obtido em escala comercial por catálise química de materiais lignocelulósicos. A produção biotecnológica de xilitol como alternativa ao processo químico vem sendo pesquisada e os resultados revelam que a presença de compostos tóxicos nos hidrolisados hemicelulósicos resultantes do processo de hidrólise ácida contribui para sua baixa fermentabilidade. Isto se deve à inibição do metabolismo microbiano causada principalmente por compostos tais como ácidos orgânicos, fenólicos e íons metálicos. No presente trabalho foi avaliado o efeito de diferentes fontes de carbono (xilose, glicose e mistura de xilose e glicose) empregadas no preparo do inóculo de Candida guilliermondii FTI 20037 sobre a bioconversão de xilose em xilitol a partir de fermentações em frascos Erlenmeyer de hidrolisados hemicelulósicos submetidos a procedimentos de destoxificação. A condição de favorecimento deste bioprocesso foi empregada para a avaliação da ampliação de escala em fermentadores de 2,4L para 16L, utilizando como critério de ampliação o KLa (igual a 15h-1). De acordo com os resultados, os máximos valores dos parâmetros fermentativos como fator de conversão de xilose em xilitol e produtividade em xilitol foram alcançados com a utilização de inóculo obtido em xilose durante fermentação do hidrolisado destoxificado por resinas (YP/S = 0,81 g g-1 e QP = 0,60 g L-1 h-1, respectivamente), embora o emprego de carvão ativado tenha gerado valores de rendimento próximos para as diferentes fontes de carbono (YP/S variando de 0,78 a 0,80 g g-1). Considerando o valor de fator de conversão e que o procedimento de destoxificação com carvão ativado é o de menor custo e de mais fácil manipulação em comparação ao processo com resinas, os experimentos de ampliação de escala da produção de xilitol por C. guilliermondii foram realizados nesta condição de destoxificação e empregando-se xilose como fonte de carbono para o inóculo. Nesta etapa ficou evidente a viabilidade de ampliação de escala de produção de xilitol de fermentador de 2,4L para 16L, já que os valores dos parâmetros fermentativos avaliados foram semelhantes entre os fermentadores (valores médios: YP/S ≈ 0,68 g g-1 e QP ≈ 0,28 g L-1 h-1). No entanto, tais valores foram inferiores aos obtidos em frascos Erlenmeyer, possivelmente devido às condições de disponibilidade de oxigênio diferirem nos fermentadores de bancada, uma vez que o oxigênio é o parâmetro mais crítico neste bioprocesso. / The conversion of vegetable biomass into chemicals and energy is essential to sustain our current style of life. Sugarcane bagasse, a raw material abundantly available in Brazil, greatly contributes to the supply of the evergrowing demand for ethanol. Furthermore, biomass can be employed for obtaining value-added products, such as xylitol, as well as bring economical advantages for the sugar-ethanol sector. Xylitol, a polyol with sweetener power similar to that of saccharose and peculiar properties such as insulin-independent metabolism, anticariogenic power, and applications in the clinical area, in the treatment of osteoporosis and respiratory diseases, is obtained on a commercial scale by chemical catalysis of lignocellulosic materials. The biotechnological production of xylitol as an alternative to the chemical process has been researched and the results reveal that the presence of toxic compounds in hemicelllosics hydrolysates resulting from acid hydrolysis process contributes to its low fermentability. Such toxicity could be due to the inhibition of microbial metabolism promoted mainly by compounds such as organic acids, phenols and metallic ions. In the present work, the effect of different carbon sources (xylose, glucose and a mixture of xylose and glucose) used in the inoculum preparation of Candida guilliermondii FTI 20037 for the xylose-to-xylitol bioconversion by fermentation of hemicellulosics hydrolysates submitted to detoxification procedures in Erlenmeyer flasks was evaluated. The best condition for this bioprocess was employed to evaluate the scale up from the 2.4L to 16L fermentors, using KLa (equal to 15h-1) as scale-up criteria. According to the results the highest values of fermentative parameters such as xylitol yield and productivity were achieved with the use of inoculum cultivated on xylose during the fermentation of hydrolysate detoxified with resins (YP/S = 0.81 g g-1 and QP = 0.60 g L-1 h-1, respectively), although with the use of charcoal the yield value was similar (YP/S ranging for 0.78 to 0.80 g g-1), regardless of the carbon source employed. Considering the value of xylitol yield and that detoxification with activated charcoal is less expensive and more easily manipulated when compared to detoxification procedure with resins, the experiments for scale up xylitol production by C. guilliermondii were performed in such detoxification condition with xylose as the carbon source for the inoculum. At this stage it was evident the scale up xylitol production from a fermenter of 2.4L to 16L was feasible, since the values of fermentative parameters evaluated were similar to those of the fermentors (medium values YP/S ≈ 0.68 g g-1 e QP ≈ 0.28 g L-1 h-1). However, these values were lower than those obtained in Erlenmeyer flasks, maybe due to conditions of oxygen availability for they differ from those in fermentors, since oxygen is the most critical parameter in this bioprocess.

Os processos de separação por membrana (PSM) vêm ganhando destaque em aplicações industriais por conta de suas vantagens, principalmente o baixo custo de implementação e o baixo consumo de energia para sua operação. A utilização de subprodutos agrícolas na obtenção de materiais é uma tendência crescente, sendo os seus maiores atrativos a grande disponibilidade desses subprodutos e por serem uma matéria-prima barata. O presente trabalho teve como principais objetivos a produção de membranas sua utilização na detoxificação do hidrolisado hemicelulósico originado do tratamento ácido do bagaço de cana-de-açúcar. Para tanto foram produzidos dois tipos de membranas a partir de três polímeros diferentes, o acetato de celulose obtido a partir do bagaço de cana, o acetato de celulose comercial e a poliamida 66. Na produção de acetato a partir do bagaço foi realizado um estudo exploratório para extrair a celulose, matéria-prima do acetato, de uma maneira que se obtivesse um material com alto grau de pureza e que as perdas de celulose durante o processo fossem minimizadas. Para a produção das membranas foi utilizada a técnica de inversão de fases. No caso das membranas de acetato de celulose, foi realizada uma variação dos parâmetros utilizados no processo de confecção das membranas (tempo de evaporação do solvente, temperatura do banho de coagulação e tratamento térmico), com o intuito de se estabelecer as melhores variáveis do processo, enquanto que para a poliamida 66, foram utilizadas condições previamente determinadas por outros estudos. Depois de prontas, as membranas foram caracterizadas fisicamente e pelas suas propriedades de fluxo de água pura, fluxo de vapor de água, rejeição de sais, rejeição de açúcares e rejeição de compostos tóxicos. Finalmente, as membranas foram aplicadas no processo de detoxificação do hidrolisado hemicelulósico para testar sua capacidade de remoção de furfural, hidroximetilfurfural (HMF), ácido acético e compostos fenólicos. No estudo de extração da celulose do bagaço, as melhores condições produziram uma celulose com pureza de 84,01%. O acetato produzido apresentou um grau de substituição de 2,52, podendo ser classificado como um triacetato de celulose. Em comparação, o acetato comercial apresentou um grau de substituição de 2,85. Fisicamente, todas as membranas apresentaram uma morfologia que intercalava a presença de poros com regiões nodulares. As membranas de bagaço de cana apresentaram uma considerável fragilidade, por isso nos testes de permeação sob pressão, elas foram suportadas por uma membrana de polissulfona comercial. Todas as membranas de acetato de bagaço e membrana de poliamida apresentaram fluxo de água pura, enquanto que apenas algumas membranas de acetato comercial conseguiram permear água pura. As membranas apresentaram diferentes resultados nos experimentos de rejeição de compostos, resultado das diferenças estruturais entre elas. No ensaio de detoxificação, a membrana que alcançou o melhor desempenho foi a membrana obtida a partir do acetato comercial. Essa membrana conseguiu remover 89,92% de HMF, 91,99% de furfural, 51,52% de ácido acético e 8,35% de compostos fenólicos. As membranas produzidas a partir do bagaço de cana alcançaram uma remoção de 71,66 de HMF, 60,87% de furfural, 91,79% de ácido acético e 10,86% de fenólicos. / Membrane separation processes (MSP) have been highlighted at industrial processes because of their advantages, mainly the low cost of implementation and the low energy consumption during their operation. The utilization of agriculture co-products for the obtainment of material is a increasing trend, wherein the main attractive are the high availability and the low cost of these co-products. The aims of this work were to produce membranes and to investigate their utilization in the detoxification of the hemicellulosic hydrolisate originated from the acid treatment of sugarcane bagasse. For that, two types of membranes were produced from three different types of polymers, cellulose acetate obtained from sugarcane bagasse, commercial cellulose acetate and polyamide 66. For the production of the sugarcane bagasse cellulose acetate it was conducted an exploratory study in order to extract cellulose, raw-material of the acetate, in a manner that the final material possessed high purity degree and the losses of cellulose during the process were minimized. The technique of phase inversion was utilized to produce the membranes. In the case of cellulose acetate membranes, the variation of the membrane production parameters (time of solvent evaporation, temperature of coagulation bath and thermical treatment) was performed for the purpose of establishing the best process parameters, whereas it was utilized previously established conditions found in the literature for the polyamide membrane production. The membranes were characterized physically and for their properties of pure water flux, vapor water flux, salt rejection, sugar rejection and toxic compound rejection. Finally, the membranes were applied in the process of hemicellulosic hydrolysate detoxification for testing their capacity of furfural, hydroxymethylfurfural (HMF), acetic acid and phenolic compound removal. The best conditions of cellulose extraction from sugarcane bagasse were able to produce cellulose with 84.01% of purity. The sugarcane cellulose acetate presented a substitution degree of 2.52, being classified as cellulose triacetate. In comparison, commercial cellulose acetate presented a substitution degree of 2,85. Physically, all membranes possessed a morphology that interspersed the presence of porous and nodular regions. Due to their fragility, sugarcane bagasse membranes were supported by a polysulfone commercial membrane in the tests of permeation under pressure. All sugarcane bagasse membranes and polyamide membrane achieved pure water flux. Nevertheless, just some commercial cellulose acetate membranes could permeate pure water. In the assays of compound rejection, the membranes reached different results, on behalf of their structural differences. The membrane that obtained the best performance in the detoxification process was the membrane produced form commercial cellulose acetate. This membrane was able to remove 89.92% of HMF, 91.99% of furfural, 51.52% of acetic acid and 8.35% of phenolic compounds. The membranes produced from sugarcane bagasse reached a removal of 71.66 of HMF, 60.87% of furfural, 91.79% of acetic acid and 10,86% of phenolics.

Os processos de separação por membrana (PSM) vêm ganhando destaque em aplicações industriais por conta de suas vantagens, principalmente o baixo custo de implementação e o baixo consumo de energia para sua operação. A utilização de subprodutos agrícolas na obtenção de materiais é uma tendência crescente, sendo os seus maiores atrativos a grande disponibilidade desses subprodutos e por serem uma matéria-prima barata. O presente trabalho teve como principais objetivos a produção de membranas sua utilização na detoxificação do hidrolisado hemicelulósico originado do tratamento ácido do bagaço de cana-de-açúcar. Para tanto foram produzidos dois tipos de membranas a partir de três polímeros diferentes, o acetato de celulose obtido a partir do bagaço de cana, o acetato de celulose comercial e a poliamida 66. Na produção de acetato a partir do bagaço foi realizado um estudo exploratório para extrair a celulose, matéria-prima do acetato, de uma maneira que se obtivesse um material com alto grau de pureza e que as perdas de celulose durante o processo fossem minimizadas. Para a produção das membranas foi utilizada a técnica de inversão de fases. No caso das membranas de acetato de celulose, foi realizada uma variação dos parâmetros utilizados no processo de confecção das membranas (tempo de evaporação do solvente, temperatura do banho de coagulação e tratamento térmico), com o intuito de se estabelecer as melhores variáveis do processo, enquanto que para a poliamida 66, foram utilizadas condições previamente determinadas por outros estudos. Depois de prontas, as membranas foram caracterizadas fisicamente e pelas suas propriedades de fluxo de água pura, fluxo de vapor de água, rejeição de sais, rejeição de açúcares e rejeição de compostos tóxicos. Finalmente, as membranas foram aplicadas no processo de detoxificação do hidrolisado hemicelulósico para testar sua capacidade de remoção de furfural, hidroximetilfurfural (HMF), ácido acético e compostos fenólicos. No estudo de extração da celulose do bagaço, as melhores condições produziram uma celulose com pureza de 84,01%. O acetato produzido apresentou um grau de substituição de 2,52, podendo ser classificado como um triacetato de celulose. Em comparação, o acetato comercial apresentou um grau de substituição de 2,85. Fisicamente, todas as membranas apresentaram uma morfologia que intercalava a presença de poros com regiões nodulares. As membranas de bagaço de cana apresentaram uma considerável fragilidade, por isso nos testes de permeação sob pressão, elas foram suportadas por uma membrana de polissulfona comercial. Todas as membranas de acetato de bagaço e membrana de poliamida apresentaram fluxo de água pura, enquanto que apenas algumas membranas de acetato comercial conseguiram permear água pura. As membranas apresentaram diferentes resultados nos experimentos de rejeição de compostos, resultado das diferenças estruturais entre elas. No ensaio de detoxificação, a membrana que alcançou o melhor desempenho foi a membrana obtida a partir do acetato comercial. Essa membrana conseguiu remover 89,92% de HMF, 91,99% de furfural, 51,52% de ácido acético e 8,35% de compostos fenólicos. As membranas produzidas a partir do bagaço de cana alcançaram uma remoção de 71,66 de HMF, 60,87% de furfural, 91,79% de ácido acético e 10,86% de fenólicos. / Membrane separation processes (MSP) have been highlighted at industrial processes because of their advantages, mainly the low cost of implementation and the low energy consumption during their operation. The utilization of agriculture co-products for the obtainment of material is a increasing trend, wherein the main attractive are the high availability and the low cost of these co-products. The aims of this work were to produce membranes and to investigate their utilization in the detoxification of the hemicellulosic hydrolisate originated from the acid treatment of sugarcane bagasse. For that, two types of membranes were produced from three different types of polymers, cellulose acetate obtained from sugarcane bagasse, commercial cellulose acetate and polyamide 66. For the production of the sugarcane bagasse cellulose acetate it was conducted an exploratory study in order to extract cellulose, raw-material of the acetate, in a manner that the final material possessed high purity degree and the losses of cellulose during the process were minimized. The technique of phase inversion was utilized to produce the membranes. In the case of cellulose acetate membranes, the variation of the membrane production parameters (time of solvent evaporation, temperature of coagulation bath and thermical treatment) was performed for the purpose of establishing the best process parameters, whereas it was utilized previously established conditions found in the literature for the polyamide membrane production. The membranes were characterized physically and for their properties of pure water flux, vapor water flux, salt rejection, sugar rejection and toxic compound rejection. Finally, the membranes were applied in the process of hemicellulosic hydrolysate detoxification for testing their capacity of furfural, hydroxymethylfurfural (HMF), acetic acid and phenolic compound removal. The best conditions of cellulose extraction from sugarcane bagasse were able to produce cellulose with 84.01% of purity. The sugarcane cellulose acetate presented a substitution degree of 2.52, being classified as cellulose triacetate. In comparison, commercial cellulose acetate presented a substitution degree of 2,85. Physically, all membranes possessed a morphology that interspersed the presence of porous and nodular regions. Due to their fragility, sugarcane bagasse membranes were supported by a polysulfone commercial membrane in the tests of permeation under pressure. All sugarcane bagasse membranes and polyamide membrane achieved pure water flux. Nevertheless, just some commercial cellulose acetate membranes could permeate pure water. In the assays of compound rejection, the membranes reached different results, on behalf of their structural differences. The membrane that obtained the best performance in the detoxification process was the membrane produced form commercial cellulose acetate. This membrane was able to remove 89.92% of HMF, 91.99% of furfural, 51.52% of acetic acid and 8.35% of phenolic compounds. The membranes produced from sugarcane bagasse reached a removal of 71.66 of HMF, 60.87% of furfural, 91.79% of acetic acid and 10,86% of phenolics.

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds. However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT) catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how, genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues, systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

Awareness of detoxification, nowadays known as biotransformation, has become an integral part of our daily lives. It is a modern buzz word that is used to promote anything from health food to enhancement of performance in sports. Another lesser known application for detoxification is as a therapy for alleviating symptoms of inborn errors of metabolism. Detoxification is the process where endogenous and xenobiotic metabolites are transformed to less harmful products, in the liver and kidneys, in two phases. Phase 1 detoxification includes oxidation, hydroxylation, dehydrogenation metabolic reduction and hydrolysis. Phase 2 detoxification uses conjugation reactions to increase hydrophillicty of metabolites for excretion in bile and urine. Glycine N-acyltransferse (GLYAT; EC 2.3.1.13) is one of the amino acid conjugation enzymes. There are two variants of human GLYAT. I focused on the full-length mRNA human GLYAT isoform a, with a long term view of using it as a viable therapeutic enzyme for enhanced detoxification of harmful metabolites. I investigated if it is possible to clone and express a biologically active GLYAT. To achieve this goal I used three expression systems: traditional bacterial expression using the pET system; second generation cold shock bacterial expression using the pCOLDTF expression vector to improve solubility of the recombinant protein; and baculovirus expression in insect cells since therein some form of post translation glycosylation of the recombinant protein can occur which might improve solubility and ensure biological activity. The recombinant GLYAT expressed well in all three expression systems but was aggregated and no enzyme activity could be detected. A denature and renature system was also used to collect aggregated recombinant GLYAT and used to try to refold the recombinant protein in appropriate refolding buffers to improve solubility and obtain biological activity. The solubility of the recombinant GLYAT was improved but it remained biologically inactive. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

The concept of accumulating xenobiotics within the human body as a health risk is well known. However, these compounds can also be endogenous, as in the case of inborn errors of metabolism. Biotransformation of both exogenous and endogenous toxic compounds is an important function of the liver, and the critical balance between these systems is of fundamental importance for cellular health. Fatty acid ?-oxidation deficiencies are associated with characteristic clinical symptoms as a consequence of the accumulation of specific metabolites. For these accumulated metabolites various nutrients are indispensable for optimal biotransformation and continuous accumulation of metabolites can ultimately result in the depletion of biotransformation substrates and cofactors. In this study, a novel model (the unbalanced biotransformation metabolism model) is proposed that describes the critical balance between Phase I and Phase II biotransformation and how a disturbance in this balance will increase the oxidative stress status. The significance of this model lies within the treatment possibilities, as the assessment of biotransformation metabolism and oxidative stress status can lead to the development of nutritional treatment strategies to correct imbalances. The value of this model is illustrated by its application to a clinical case investigated. In addition to the use of nutritional supplementation in treatment, biotransformation substrates and cofactors were also used to develop a ?substrate loading cocktail?. This cocktail ensured sufficient availability of biotransformation substrates and precursors to stimulate coenzyme A biosynthesis. The application of this ?substrate loading cocktail? in subjects with both induced and inborn errors in fatty acid oxidation demonstrated that such a novel approach is a useful tool to give new insight into these kinds of deficiencies and open the possibility for the identification of new deficiencies. Interesting observations made in subjects originally referred for biotransformation and oxidative stress status profiles led to the first in vivo evidence of an inhibitory effect of acetylsalicylic acid on short-chain fatty acid metabolism possibly at the level of isobutyryl-CoA dehydrogenase. Since not all individuals were affected to the same degree, this observation can potentially be used to detect individuals with rate-limiting polymorphisms or mutations in the isobutyryl-CoA dehydrogenase enzyme. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

The concept of accumulating xenobiotics within the human body as a health risk is well known. However, these compounds can also be endogenous, as in the case of inborn errors of metabolism. Biotransformation of both exogenous and endogenous toxic compounds is an important function of the liver, and the critical balance between these systems is of fundamental importance for cellular health. Fatty acid ?-oxidation deficiencies are associated with characteristic clinical symptoms as a consequence of the accumulation of specific metabolites. For these accumulated metabolites various nutrients are indispensable for optimal biotransformation and continuous accumulation of metabolites can ultimately result in the depletion of biotransformation substrates and cofactors. In this study, a novel model (the unbalanced biotransformation metabolism model) is proposed that describes the critical balance between Phase I and Phase II biotransformation and how a disturbance in this balance will increase the oxidative stress status. The significance of this model lies within the treatment possibilities, as the assessment of biotransformation metabolism and oxidative stress status can lead to the development of nutritional treatment strategies to correct imbalances. The value of this model is illustrated by its application to a clinical case investigated. In addition to the use of nutritional supplementation in treatment, biotransformation substrates and cofactors were also used to develop a ?substrate loading cocktail?. This cocktail ensured sufficient availability of biotransformation substrates and precursors to stimulate coenzyme A biosynthesis. The application of this ?substrate loading cocktail? in subjects with both induced and inborn errors in fatty acid oxidation demonstrated that such a novel approach is a useful tool to give new insight into these kinds of deficiencies and open the possibility for the identification of new deficiencies. Interesting observations made in subjects originally referred for biotransformation and oxidative stress status profiles led to the first in vivo evidence of an inhibitory effect of acetylsalicylic acid on short-chain fatty acid metabolism possibly at the level of isobutyryl-CoA dehydrogenase. Since not all individuals were affected to the same degree, this observation can potentially be used to detect individuals with rate-limiting polymorphisms or mutations in the isobutyryl-CoA dehydrogenase enzyme. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Dans cette thèse, le processus d'assemblage ainsi que la maturation du cytochrome c oxydase de type cbb3 (cbb3-Cox) ont été étudiés dans la proteobactérie phototrophique pourpe non soufrée Rhodobacter capsulatus. R. capsulatus contient une chaîne de transfert d'électrons très ramifiée et represente un modèle d’organisme très utilisé dans l'étude des processus respiratoires et photosynthétiques.Les cbb3-Coxs spécifiques des bactéries représentent la deuxième catégorie la plus abondante des cytochromes c oxydases après le type Cox-aa3, mais n'ont jusqu'à présent pas été étudiées en détail. Récemment, la première structure cristalline cbb3-Cox de P. stutzeri a été obtenue, fournissant ainsi une avancée majeure invitant à des etudes plus détaillées sur le mécanisme catalytique et le processus d'assemblage. Les études sur les procédés d'assemblage et de maturation sont d'une très grande importance en raison du fait que de nombreux agents pathogènes humains tels que Helicobacter pylori, Neisseria meningitidis ou Campylobacter jejuni utilisent ce type de Cox, ce qui par conséquent pourrait amener a développer une interessante cible thérapeutique.Cbb3-Cox dans R. capsulatus est encodé par le gène opéron ccoNOQP codant quatre protéines membranaires constitutives de cbb3-Cox. CcoP et CcoO sont des cytochromes de type c, contenant des motifs périplasmiques fixés à l’hème. CcoN est la sous-unité centrale catalytique contenant deux molécules d’hèmes de type b et un ion cuivre. L’étude de la distribution de l’ion Cu à la sous-unité CcoN et l'assemblage des quatre sous-unités dans le complexe actif cbb3-Cox complexe sont les thèmes principaux de ce travail.Ici, le rôle du facteur d'assemblage putatif CcoH, sa structure et son interaction avec cbb3-Cox ont été étudiés en détail. CcoH est une petite protéine membranaire codé dans le groupe de gènes ccoGHIS situé à proximité des gènes codant cbb3-Cox. L'analyse in vivo de la formation de cbb3-Cox dans une souche ne contenant pas le facteur CcoH a montré une absence totale de cbb3-Cox. De même, la stabilité du facteur CcoH a été considérablement altérée dans une souche avec deletion du gene ccoNOQP. La dépendance mutuelle des deux protéines suggère leur interaction directe, ce qui a été confirmé par la photoréticulation directe de CcoH à la sous-unité CcoN, l’immunodétection de CcoH dans les cbb3-Cox complexes sur gels Blue Native, la co-purification par marquage CcoH-cbb3-Cox et le marquage radioactive in vitro des complexes cbb3-Cox avec CcoH.[...] / In this thesis, the assembly and maturation process of the cbb3-type cytochrome c oxidase (cbb3-Cox) was studied in the purple-non-sulphur phototrophic α-proteobacterium Rhodobacter capsulatus. R. capsulatus contains a highly branched electron-transfer chain and is a well studied model organism for investigating respiratory and photosynthetic processes.The bacteria-specific cbb3-Coxs represent the second most abundant class of cytochrome c oxidases after the aa3-type Cox, but have so far not been investigated in much detail. Recently, the first crystal structure of cbb3-Cox from P. stutzeri was obtained, providing a major breakthrough and inviting detailed studies on the catalytic mechanism and the assembly process. Studies on the assembly and maturation processes are of wide significance due to the fact that many human pathogens like Helicobacter pylori, Neisseria meningitides or Campylobacter jejuni use this type of Cox and it therefore might develop into an attractive drug-target. cbb3-Cox in R. capsulatus is encoded by the ccoNOQP gene operon which codes for four membrane proteins constituting cbb3-Cox. CcoP and CcoO are c-type cytochromes, containing periplasmic heme-binding motifs. CcoN is the central catalytic subunit which contains two b-type hemes and a copper ion. Investigating the delivery of Cu to the CcoN subunit and the assembly of all four subunits into the active cbb3-Cox complex were the main topics of this work. Here the role of the putative assembly factor CcoH, its structure and interaction with cbb3-Cox was investigated in detail. CcoH is a small membrane protein encoded in the ccoGHIS gene cluster located adjacent to the genes coding for cbb3-Cox. In vivo analysis of cbb3-Cox formation in a strain lacking ccoH showed the total absence of cbb3-Cox. Likewise, the stability of CcoH was drastically impaired in a ccoNOQP deletion strain. The mutual dependency of both proteins suggested their direct interaction, which was confirmed by site-directed photocrosslinking of CcoH to the CcoN subunit, by immunodetection of CcoH in cbb3-Cox complexes on Blue Native gels, by CcoH-cbb3-Cox co-purification and by in vitro labelling of cbb3-Cox complexes with radioactively labelled CcoH.[...]