Master of Science / Department of Agronomy / Dallas E. Peterson / During 2015 and 2016, five site years of research were implemented in double crop soybean after winter wheat at experiment fields in Kansas near Manhattan, Hutchinson, and Ottawa to assess various non-glyphosate herbicide treatments at three different application timings for control of Palmer amaranth (Amaranthus palmeri S. Wats.) and common waterhemp (Amaranthus rudis Sauer). Spring-post (SP) treatments with residual control of Palmer amaranth and waterhemp were applied in the winter wheat at Feekes 4 and resulted in less than 50% control of Palmer amaranth and waterhemp at the time of double crop soybean planting. Pre-harvest treatments were applied two weeks before winter wheat harvest. 2,4-D resulted in highly variable Palmer amaranth and waterhemp control whereas flumioxazin resulted in comparable control to PRE treatments that contained paraquat plus a residual herbicide. Excellent Palmer amaranth and waterhemp control was observed at 1 week after planting (WAP) double crop soybean with a preemergence (PRE) paraquat application; however, reduced control of Palmer amaranth and waterhemp was noted at 8WAP due to extended emergence. Palmer amaranth and waterhemp control was 85% or greater at 8WAP for most PRE treatments that included a combination of paraquat plus residual herbicides. PRE treatments that did not include the combination of paraquat and residual herbicides did not provide acceptable control. A second set of field experiments were established in 2015 and 2016 near Manhattan, Hutchinson, and Ottawa to assess residual Palmer amaranth and waterhemp control with very-long-chain-fatty acid (VLFCA) inhibiting herbicides. Acetochlor (non-encapsulated and encapsulated), alachlor, dimethenamid-P, metolachlor, S-metolachlor, and pyroxasulfone as well as the microtubule inhibiting herbicide pendimethalin were applied at three different field use rates (high, middle, and low) based on labeled rate ranges for soybean as PRE treatments in a non-crop scenario after the plot was clean tilled with a field cultivator. The experiment was conducted one time in 2015 and four times in 2016 at two different locations for a total of five site years of data. PRE applications were made June 1, 2015, near Manhattan. PRE applications in 2016 were made in April at locations near Hutchinson and Ottawa; the second run of the experiment was applied in June at the same locations on a different set of plot areas. At Manhattan pyroxasulfone, S-metolachlor, and dimethenamid-P resulted in the highest Palmer amaranth control at 4WAT. At Hutchinson, pyroxasulfone resulted in superior Palmer amaranth control compared to dimethenamid-P and pendimethalin at 4WAT and 8WAT. At Ottawa, acetochlor, S-metolachlor, and pyroxasulfone resulted in higher waterhemp control than alachlor and pendimethalin at 4WAT and 8WAT.

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

In both the legume symbiont Sinorhizobium meliloti and the mammalian pathogen Brucella abortus, the inner membrane BacA protein is essential for host persistence. In free-living S. meliloti and B. abortus loss of the BacA protein also results in an increased resistance to the glycopeptide bleomycin and a ~ 50% decrease in the lipopolysaccharide (LPS) very-long-chain-fatty-acid (VLCFA) content. Consequently, it was proposed that BacA may be involved in transport of peptides into the cell and/or that BacA may be involved in the VLCFA modification of the LPS. During this work it was determined that the increased resistance observed in an S. meliloti DbacA mutant to bleomycin and to the truncated eukaryotic peptide Bac7(1-16), is independent of the VLCFA modification. These data support a model for BacA having multiple non-overlapping functions. Using flow cytometry studies with fluorescently labelled forms of bleomycin and Bac7(1-16) it was found that the BacA protein plays a role in the uptake of bleomycin. However, BacA was shown to be essential for the uptake of Bac7(1-16). Additionally, it was determined that two symbiotically defective bacA site directed mutants with known reductions in their VLCFA could still take up Bac7, suggesting that the BacA function that leads to the VLCFA modification could also play a key role in host persistence. To investigate further the role of BacA in the VLCFA modification and where in the cell envelope the lipid A is modified with the VLCFA, the role of the putative lipid trafficking protein MsbA2 was investigated. Interestingly, it was discovered that S. meliloti lacking the MsbA2 protein, is unable to enter host cells and induces a plant defence response more characteristic of a pathogen. To investigate the importance of the VLCFA modification during the symbiosis S. meliloti mutants lacking either the AcpXL (VLCFA acyl carrier protein) or LpxXL (VLCFA acyl transferase protein) were characterized in the host. Although not essential for host persistence, loss of each of the proteins did result in distinct defects, suggesting the VLCFA modification is important during the symbiosis. Since there are hundreds of nodule specific cysteine-rich peptides produced by the host plant Medicago truncatula, the BacA mediated uptake of one of these peptides combined with the VLCFA modification may account for the essential role of the BacA protein in the legume symbiosis.

L’acyl-CoA oxydase 1 (ACOX1) est l’enzyme qui catalyse la première étape de la voie classique de la β-oxydation peroxysomale. Cette voie catabolise exclusivement les acides gras à très longue chaîne (AGTLC). Chez l’homme, la déficience en ACOX1 est à l’origine de la pseudo adrénoleucodystrophie néonatale (P-NALD), une maladie neurodégénérative rare caractérisée par une accumulation des AGTLC dans le plasma et les tissus, une hépatomégalie, un retard du développement moteur et une démyélinisation de la matière blanche cérébrale. Chez la souris, l’extinction du gène Acox1 provoque une accumulation des AGTLC dans le plasma, un retard de croissance, une stéatose hépatique et le développement d’une hépatocarcinogenèse avec l’âge. Cependant, ces souris ne développent pas de symptômes cérébraux contrairement aux patients P NALD. Au cours de ce travail, on a pu montrer sur des fibroblastes issus de patients atteints de P NALD qu’en absence d’activité ACOX1, les peroxysomes sont diminués en nombre et augmentés en taille avec un niveau de β-oxydation peroxysomale fortement réduit. L’accumulation des AGTLC suite à la déficience en ACOX1 dans ces cellules provoque, au niveau transcriptionnel, la perturbation de la voie de synthèse du cholestérol et déclenche une réaction inflammatoire caractérisée par l’activation de la voie de l’IL-1 et la sécrétion d’IL-6 et d’IL-8. Le rôle métabolique important que joue l’ACOX1 dans l’homéostasie énergétique cellulaire a pu être souligné chez l’homme et chez la souris. En effet, la déficience en ACOX1 dans les fibroblastes de patients P-NALD perturbe la morphologie de la mitochondrie qui apparaît anormale ainsi que le métabolisme énergétique mitochondrial caractérisé par une inhibition de PGC-1α par acétylation, une surexpression de l’activité du complexe V et une diminution du taux d’ATP mitochondrial. L’absence dans le foie de l’activité ACOX1, chez la souris Acox1-/-, se traduit par des perturbations, au niveau mitochondrial, dela biogenèse et du métabolisme énergétique. Ces perturbations mitochondriales se caractérisent par une diminution de l’activité du complexe IV de la chaîne respiratoire accompagnée d’une diminution de la respiration. Cependant, ces perturbations n’affectent pas le taux d’ATP total. Les altérations mitochondriales observées chez les souris Acox1-/- sont en grande partie corrigées par l’expression de l’ACOX1 humaine. Ceci montre le rôle indispensable de l’ACOX1 dans l’homéostasie de la fonction mitochondriale.L’ensemble des résultats obtenus au cours de ce travail confirme l’importance de l’activité acyl-CoA oxydase 1 pour la dégradation des AGTLC au niveau du système de β-oxydation peroxysomale et pour la biogenèse du peroxysome. L’accumulation des substrats non métabolisés en absence d’ACOX1 pourrait être à l’origine de la perturbation de la fonction mitochondriale montrant à quel point l’activité de l’ACOX1 est indispensable au métabolisme cellulaire / Acyl-CoA oxidase 1 (ACOX1) is the rate-limiting enzyme of the peroxisomal fatty acid β-oxidation pathway of very-long-chain fatty acid (VLCFAs). In humans, ACOX1 deficiency, also called pseudo-neonatal adrenoleukodystrophy, is an autosomal recessive and a severe form of the peroxisomal β-oxidation deficiency. Patients suffer from severe delayed motor development followed by a progressive neurological regression including progressive hypodensity of cerebral white matter, hepatomegaly and deafness and die during late-infantile period. Elevated plasma and tissues VLCFAs levels are detected in these patients. Mice lacking ACOX1 develop severe microvesicular steatohepatitis with increased intrahepatic H2O2 levels and hepatocellular regeneration. Liver cell proliferation in Acox1-/- mice leads to complete replacement of steatotic hepatocytes with hepatocytes that exhibit massive spontaneous peroxisome proliferation. Older mice develop hepatocellular carcinomas due to the sustained activation of peroxisome proliferator-activated receptor-alpha (PPARα). Contrary to humans, mice lacking ACOX1 have no apparent neurological disorder. Based on fibroblasts cell model from P-NALD patients, we show that ACOX1 deficiency lead to abolition of peroxysomal β-oxidation of cerotic acid (C26:0) and modification of peroxysomal morphology which appear reduced in number and enlarged in size. Moreover, accumulation of VLCFAs in ACOX1 deficiency in human fibroblasts interferes at the transcription level with cholesterol synthesis pathway. Furthermore, these cells show activation of interleukin-1b pathway with elevated production of interleukin-6 and interleukin-8 as an inflammatory response to metabolic disturbance due to VLCFAs accumulation. Furthermore, we show in this study that the ACOX1 deficiency in human fibroblasts and in mice liver leads to alteration of the mitochondrial ultra structure, changes in the expression and activity of mitochondrial chain complexes. These alterations of mitochondrial functions are accompanied by reduction in mitochondrial ATP levels in human fibroblasts and decreased mitochondrial respiration in ACOX1 deficient mice. Interestingly, the mitochondrial changes observed in Acox1-/- mice are restored by expression of human ACOX1 in liver suggesting an essential role of human and murine Acyl-CoA oxidase 1 activity in preventing mitochondrial and lipid disturbance.Together, the results presented in this work underscore the important role of ACOX1 in humans and mice to ensure peroxisomal β-oxidation, VLCFAs catabolism and to preserve peroxisomal morphology. Given mitochondrial perturbation in ACOX1 deficiency, it is clear that this enzyme plays a pivotal role in preventing VLCFAs accumulation and their cellular toxicity and guarantees mitochondrial normal morphology and function in response to energy demand

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.