Les acides gras à très longues chaine (VLCFA) sont essentiels dans le développement, particulièrement dans les mécanismes de trafic vésiculaires, de différenciation et division cellulaire. Cependant, le rôle de ces VLCFA dans ces différents processus chez les plantes n’est pas encore bien compris. Afin d’identifier de nouveaux acteurs associés à la biosynthèse ou la fonction des VLCFA, un crible suppresseur multicopies a été réalisé dans un mutant d’élongation des VLCFA de levure. La perte de l’activité déshydratase PHS1 chez la levure et de PASTICCINO2 chez les plantes perturbe la croissance et induit des défauts de cytokinèse. La PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) historiquement caractérisée comme une déshydratase inactive est capable de restaurer les défauts de croissance et d’élongation de phs1 mais non de pas2. PTPLA interagit avec plusieurs membres du complexe élongase dans le RE et son absence conduit à l’accumulation 3-hydroxyacyl-CoA, signature des déshydratases impliquées dans l’élongation des acides gras. Cependant, la perte de PTPLA conduit à une augmentation des VLCFA, probablement dépendante de PAS2 montrant que PTPLA serait un répresseur potentiel de l’élongation. Les deux déshydratases ont des profils d’expression divergents dans la racine. PAS2 est majoritairement exprimé dans l’endoderme tandis que PTPLA s’exprime uniquement dans les tissus vasculaires et le péricycle. La comparaison de l’expression ectopique de PAS2 et PTPLA dans leur tissus respectif confirme l’existence de deux complexe élongase indépendant associé à PAS2 ou PTPLA et interagissant de manière non cellule autonome. Les cytokinines pourraient constituer le signal entre les deux complexes élongase du fait que la biosynthèse de ces hormones est réprimée par les VLCFA. Les VLCFA répriment ainsi l'expression d'IPT3 dans les racines comme observées pour la partie apicale. Les cytokinines semblent aussi réguler la teneur en VLCFA dans la racine suggérant la présence de boucles de rétrocontrôles entre ces hormones et les VLCFA / Very long chain fatty acids (VLCFA) are involved in plant development and particularly in several cellular processes such as membrane trafficking, cell division and cell differentiation. However, the precise role of VLCFA in these different cellular processes is still poorly understood in plants. In order to identify new factors associated with the biosynthesis or function of VLCFA, a yeast multicopy suppressor screen was carried out in a yeast mutant strain defective for fatty acid elongation. Loss of function of the elongase dehydratase PHS1 in yeast and PASTICCINO2 in plants prevents growth and induces cytokinesis defects. PROTEIN TYROSIN PHOSPHATASE-LIKE (PTPLA) previously characterized as an inactive dehydratase was able to restore yeast phs1 growth and VLCFA elongation but not the plant pas2 defects. PTPLA interacted with elongase members in the ER and its absence induced the accumulation of 3-hydroxyacyl-CoA as expected from a dehydratase involved in fatty acid (FA) elongation. However, loss of PTPLA function led to increased VLCFA levels, effect that was dependent of the presence of PAS2 indicating that PTPLA activity repressed FA elongation. The two dehydratases have specific expression profiles in the root with PAS2, mostly restricted in the endodermis, while PTPLA was confined in the vascular tissue and pericycle cells. Comparative ectopic expression of PTPLA and PAS2 in their respective domains confirmed the existence of two independent elongase complexes comprising PAS2 or PTPLA that were functionally interacting in a non-cell autonomous manner. A putative regulating signal could involve cytokinins that were described to be regulated by VLCFA. VLCFA were indeed found to repress IPT3 expression in roots like in leaves. Cytokinins were also found to regulate VLCFA levels suggesting the existence of regulatory feedback loops between cytokinins and VLCFA

X–linked adrenoleukodystrophy (X–ALD) is the most common peroxisomal enzyme deficiency disorder, characterized by inborn mutations in the ABCD1 gene, an ATP–binding cassette (ABC) half–transporter. The ABCD1 gene encodes the adrenoleukodystrophy protein (ALDP), the transporter for the very–long–chain fatty acids (VLCFA; C > 22:0) from the cytosol into the peroxisomes to enter the peroxisomal B–oxidation pathway. The diagnostic disease marker is the elevated levels of VLCFAs which accumulate in different tissues and body fluids, leading to inflammatory demyelination, neuro–deterioration and adrenocortical insufficiency. At present, there is no satisfactory therapy for X–ALD available. However, another peroxisomal ABC half–transporter, ALDRP can compensate for the functional loss of ALDP and is encoded by the ABCD2 gene. This prompted a new approach to treatment strategies. Phenylbutyric acid (PBA) over–expresses the ABCD2 gene, leading to an increased expression of ALDRP and PBA decreases VLCFA levels by increasing peroxisomal B–oxidation. This study had a dual aim: to determine the antioxidant capacity of PBA and to verify known and identify new metabolites of PBA. In vitro, HeLa cells were cultivated and treated with 0.5 mM, 1 mM, 2 mM and 5 mM PBA for 48 hours. The ROS, lipid peroxidation, apoptosis and cell viability were determined using fluorescein–based flow cytometry. Images were taken to visualize the peroxisome proliferation. In vivo, a vervet monkey was given a single dose of 130 mg/kg PBA. Blood was collected before treatment and 15 minutes, 30 minutes, 1, 2 and 3 hours after treatment. ROS, apoptosis and lipid peroxidation were determined by fluorescein–based flow cytometry. Urine was collected before treatment and 15 minutes, 30 minutes, 1, 2, 3, 7 and 24 hours after PBA treatment. A standardised method, employing gas chromatography–mass spectrometry (GC/MS), was used to analyse the organic acids in the urine and fatty acids in the blood. In vitro results showed decreased levels of ROS and lipid peroxidation with increased concentrations of PBA. PBA showed a protective effect towards the HeLa cells with reduced apoptosis and a high number of viable cells. In vivo levels of ROS en lipid peroxidation decreased over time of treatment with PBA. The fluorescence microscope images confirmed an increased number of peroxisomes after PBA treatment. The short term effect of PBA showed an initial, but small decrease in the levels of the fatty acids, suggesting induction over a longer period rather than activation of peroxisomal B–oxidation. New metabolites of phenylbutyrate were identified in the urine of a vervet monkey. These new metabolites originated from monooxygenase, N–phenylacetyl–glutamine synthases and B–oxidation byproducts. Recently discovered metabolites in humans and rats were also verified and confirmed in the vervet monkey. We therefore propose that treatment with PBA, on account of its beneficial effects of restoring VLCFA levels and reducing oxidative stress, could be considered a novel approach for the treatment of X–ALD. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.

X–linked adrenoleukodystrophy (X–ALD) is the most common peroxisomal enzyme deficiency disorder, characterized by inborn mutations in the ABCD1 gene, an ATP–binding cassette (ABC) half–transporter. The ABCD1 gene encodes the adrenoleukodystrophy protein (ALDP), the transporter for the very–long–chain fatty acids (VLCFA; C > 22:0) from the cytosol into the peroxisomes to enter the peroxisomal B–oxidation pathway. The diagnostic disease marker is the elevated levels of VLCFAs which accumulate in different tissues and body fluids, leading to inflammatory demyelination, neuro–deterioration and adrenocortical insufficiency. At present, there is no satisfactory therapy for X–ALD available. However, another peroxisomal ABC half–transporter, ALDRP can compensate for the functional loss of ALDP and is encoded by the ABCD2 gene. This prompted a new approach to treatment strategies. Phenylbutyric acid (PBA) over–expresses the ABCD2 gene, leading to an increased expression of ALDRP and PBA decreases VLCFA levels by increasing peroxisomal B–oxidation. This study had a dual aim: to determine the antioxidant capacity of PBA and to verify known and identify new metabolites of PBA. In vitro, HeLa cells were cultivated and treated with 0.5 mM, 1 mM, 2 mM and 5 mM PBA for 48 hours. The ROS, lipid peroxidation, apoptosis and cell viability were determined using fluorescein–based flow cytometry. Images were taken to visualize the peroxisome proliferation. In vivo, a vervet monkey was given a single dose of 130 mg/kg PBA. Blood was collected before treatment and 15 minutes, 30 minutes, 1, 2 and 3 hours after treatment. ROS, apoptosis and lipid peroxidation were determined by fluorescein–based flow cytometry. Urine was collected before treatment and 15 minutes, 30 minutes, 1, 2, 3, 7 and 24 hours after PBA treatment. A standardised method, employing gas chromatography–mass spectrometry (GC/MS), was used to analyse the organic acids in the urine and fatty acids in the blood. In vitro results showed decreased levels of ROS and lipid peroxidation with increased concentrations of PBA. PBA showed a protective effect towards the HeLa cells with reduced apoptosis and a high number of viable cells. In vivo levels of ROS en lipid peroxidation decreased over time of treatment with PBA. The fluorescence microscope images confirmed an increased number of peroxisomes after PBA treatment. The short term effect of PBA showed an initial, but small decrease in the levels of the fatty acids, suggesting induction over a longer period rather than activation of peroxisomal B–oxidation. New metabolites of phenylbutyrate were identified in the urine of a vervet monkey. These new metabolites originated from monooxygenase, N–phenylacetyl–glutamine synthases and B–oxidation byproducts. Recently discovered metabolites in humans and rats were also verified and confirmed in the vervet monkey. We therefore propose that treatment with PBA, on account of its beneficial effects of restoring VLCFA levels and reducing oxidative stress, could be considered a novel approach for the treatment of X–ALD. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2011.