京都大学 / 新制・課程博士 / 博士(農学) / 甲第23934号 / 農博第2483号 / 新制||農||1089(附属図書館) / 学位論文||R4||N5369(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 廣岡 博之, 教授 松井 徹, 教授 吉田 天士 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The regulation of glucagon-like peptide-1 and peptide YY hormone levels are regulated based on different influential factors, but primarily levels are dependent upon ingested food content. As meals today become more fat-enriched, there is greater requirement for evaluation of these hormones that regulate insulin and satiety levels within the body. We have shown that the gene expression transcript production of glucagon-like peptide-1 and peptide YY are modulated by different concentrations, and times of short-chain fatty acids and long-chain fatty acids. Although the peptide hormone levels have the influential physiological role on effector tissue, the regulation of these hormones begins at the transcript levels. Recent research indicates that glucagon-like peptide-1 and peptide YY hormones are altered in response to different free-fatty acids. The present investigation generally demonstrated an overall decrease in both hormones after chronic exposure to fatty acids. Intestinal secretin tumor cell line (STC-1 cells) was used as a representative for intestinal L-cells. Quantitative real-time PCR analysis was used to determine the changes in RNA transcripts. Overall, there was a decrease in the 3-hour timeline, which continued to decrease in the 16-hour and 24-hour timelines for glucagon-like peptide-1. Peptide YY transcript expression in 3-hours increased significantly after exposure to propionate, a significant decrease after exposure to acetate, and no significant increase or decrease after exposure to butyrate. However, there was a significant decrease in peptide YY once reaching 24-hour exposure. It was determined there is a threshold for different concentrations of free-fatty acids to influence glucagon-like peptide-1 and peptide YY production, which was present in the different concentrations of butyrate. Lastly, exposure to both concentrations of linolenic acid caused a significant decrease in glucagon-like peptide-1 and peptide YY.

Les cellules à mucus intestinales sécrètent des mucines, principalement MUC2, qui forment un gel protecteur. Malgré la place importante des acides gras à chaîne longue (AGCL) dans l'alimentation et leurs liens avec des pathologies où les cellules à mucus sont altérées il n'existe pas d'étude sur leurs impacts sur ces cellules. Mes travaux ont consisté à mettre en lumière ces interactions et à étudier leurs conséquences. In vitro, nous montrons que les AGCL saturés augmentent l'expression de MUC2 et son relarguage alors que les AGCL insaturés les inhibent et que l'acide palmitique favorise la différenciation cellulaire. In vivo, chez des ratons gavés avec de l'huile de palme, la production de MUC2 est augmentée et la perméabilité paracellulaire colique diminuée. Les huiles de colza ou de tournesol ne modifient pas la production de MUC2 et la perméabilité est identique aux contrôles malgré l'augmentation de l'expression de l'occludine. Dans un modèle de syndrome de l'intestin irritable, nous montrons que le gavage avec l'huile de palme, malgré la diminution de l'occludine, restaure la perméabilité et augmente le nombre de cellules à mucus. L'huile de colza et l'huile de tournesol ne corrigent pas le défaut de perméabilité. Parallèlement, deux études chez la souris ont mis en évidence l'impact de certains régimes sur le nombre des cellules à mucus. Ce travail a permis d'identifier les AGCL comme des nutriments capables de moduler les cellules à mucus, de faire émerger une nouvelle piste thérapeutique pour restaurer les populations de cellules à mucus et la perméabilité et d'ouvrir la réflexion sur les impacts de l'augmentation des populations de cellules à mucus chez l'individu sain / Intestinal goblet cells secrete mucins, mainly MUC2, which form a protective mucus gel. Despite the important place of long chain fatty acids (LCFA) in the diet and their links with pathologies where goblet cells are altered, there is no study dealing with their impact on goblet cells. The aim of my work was to highlight these interactions and to study their consequences. In vitro, we show that saturated LCFA increase MUC2 expression and release whereas unsaturated LCFA inhibit these process and that palmitic acid promotes cellular differentiation. In vivo, rat pups receiving oral administration of palm oil show a decrease of colonic paracellular permeability and an increase of MUC2 production. On the opposite, rapeseed and sunflower oils do not change MUC2 production and, intestinal permeability is the same as controls despite the increase of occludin expression. In an animal model of irritable bowel syndrome, palm oil effects are found again. When subjected rat pups to a maternal deprivation stress a few days after birth, they develop an intestinal hyperpermeability and a goblet cell depletion. We show that oral administration of palm oil concomitantly to the stress is able to, despite a decrease of occludin expression, restore the permeability at the level of non-stressed animals and to increase goblet cell number. Rapeseed oil and sunflower oil are not able to correct the increase of intestinal permeability. In parallel, studies in mice show that some types of diets can be associated with an increase of the number of proximal and distal goblet cells. To conclude, this work (i) helped to identify LCFA as nutrients able to modulate intestinal goblet cell number and physiology, (ii) put forward a new therapeutic track, via palmitic acid use, to restore goblet cell populations and intestinal permeability in pathologies associated with these kind of alterations and finally (iii) offered new tracks of reasoning about physiological and pathophysiological impacts of the increase of intestinal goblet cell number in a healthy subject

La digestion anaérobie est un processus biologique dans lequel un consortium microbien complexe fonctionnant en absence d’oxygène transforme la matière organique en biogaz, principalement en méthane et en dioxyde de carbone. Parmi les substrats organiques, les lipides sont les plus productifs de méthane par rapport aux glucides et aux protéines; mais leur dégradation est très difficile, en raison de leur hydrolyse qui peut être l’étape limitante. Les algues peuvent être une source importante pour la production de méthane à cause de leur contenu en lipides potentiellement élevé. L’objectif de cette étude était, par conséquent, d’évaluer la production en méthane des microalgues en utilisant la technique du BMP (Biochemical méthane Potential) et d’identifier les limites de biodégradion des lipides dans la digestion anaérobie. Le plan expérimental a été divisé en plusieurs étapes: 1) Comparer le potentiel énergétique en méthane des macroalgues par rapport aux microalgues. 2) Faire le criblage de différentes espèces de microalgues d’eau douce et marines afin de comparer leur potentiel en méthane. 3) Déterminer l'impact des prétraitements sur la production de méthane de quelques microalgues ciblées. 4) Identifier les limites de biodégradation des lipides algaux dans la digestion anaérobie, en étudiant les étapes limitantes de la cinétique des lipides et de chacun des acides gras à longues chaines. Les résultats ont montré que les microalgues produisent plus de méthane que les macroalgues. Les BMP des microalgues d'eau douce et marines n'ont montré aucune différence en termes de rendement en méthane. Les résultats des prétraitements ont montré que le prétraitement thermique (microonde) semblait être plus efficace que le prétraitement chimique (alcalin). Les tests de contrôle du BMP faits sur l'huile de palme, l’huile de macadamia et l'huile de poisson ont montré que l'hydrolyse des huiles en glycérol et en acides gras à longues chaines n'était pas l'étape limitante dans la production de méthane. L'ajout de gras dans les échantillons de Phaeodactylum dégraissée a augmenté le rendement de méthane et cette augmentation a été corrélée à la quantité de matières grasses ajoutées. / Anaerobic digestion is a biological process in which an anaerobic microbial consortium converts organic matter into biogas, primarily methane and carbon dioxide. Among the organic substrates, lipids are the most productive of methane in comparison to carbohydrates and proteins; but their degradation is very difficult, due to their hydrolysis which can be the limiting step. Algae can be an important source for methane production because of their potentially high content of lipids. The objective of this study was, therefore, to evaluate the methane production of microalgae using the Biochemical methane Potential (BMP) technique and to identify the limit of biodegradation of lipids in the anaerobic digestion. The experimentation plan was divided into the following stages: 1) Compare the energy potential in methane of macroalgae versus microalgae. 2) Screen different species of freshwater and marine microalgae to compare their methane potential. 3) Determine the impact of mild pretreatment of targeted microalgae on the methane production. 4) Identify the limits of biodegradation of algal lipids in the anaerobic digestion by studying kinetics limiting steps of lipids and individual LCFA (Long Chain Fatty Acids). The results showed that microalgae produce more methane than macroalgae. The BMP of freshwater and marine microalgae showed no difference in terms of methane yield. The results of pretreatment showed that the thermal (microwave) pretreatment seemed to be more effective than the chemical (alkaline) pretreatment. A BMP control test done on palm oil, macadamia oil and fish oil showed that the hydrolysis of oils in glycerol and LCFA was not the limiting step in the production of methane. The addition of fat in the samples of defatted Phaeodactylum increased the methane yield and this augmentation was correlated to the quantity of fat added.

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

No description available.

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.

La résistance des cellules tumorales à la chimiothérapie constitue une cause majeure d’échec des traitements anticancéreux. Des études précliniques montrent que les acides gras polyinsaturés oméga-3 à longues chaînes (AGPIn-3LC), apportés par l’alimentation, améliorent l’efficacité des chimiothérapies sans majorer les effets secondaires. Cette thèse a eu pour but d’identifier les mécanismes moléculaires impliqués dans l’augmentation de la sensibilité des cellules tumorales mammaires au docétaxel. Nous avons montré que le docétaxel induit un mécanisme de résistance via l’activation des voies de signalisation PKC/ERK et Akt impliquées dans la prolifération et la survie cellulaires. La modification de l’environnement lipidique membranaire par la supplémentation en AGPIn-3LC inhibe ces voies de signalisation et augmente l’efficacité du docétaxel dans des lignées tumorales mammaires et dans un modèle préclinique de tumeurs mammaires autochtones chez le rongeur. De plus, dans ce modèle in vivo, nous avons identifié une autre cible moléculaire régulée par les AGPIn-3LC : l’épiréguline, membre de la famille EGF. Les AGPIn-3LC bloquent l’induction de l’épiréguline par le VEGF dans les cellules endothéliales et induisent un remodelage de la vascularisation tumorale. Outre un effet direct des AGPIn-3LC sur les cellules tumorales, les AGPIn- 3LC agissent sur le microenvironnement tumoral. Ces travaux de thèse apportent des arguments supplémentaires pour l’utilisation des AGPIn-3LC comme molécules adjuvantes pour lutter contre la résistance des tumeurs mammaires aux agents anticancéreux. / Chemotherapy-resistant tumor cells are a major cause of cancer treatment failure. Preclinical studies show that polyunsaturated omega-3 long chain fatty acids (AGPIn-3LC), provided by food, improve the efficacy of chemotherapy without increasing side effects. AGPIn-3LCs are incorporated in cancer and stromal cells. This thesis aimed to identify molecular mechanisms involved in the increased sensitivity of mammary tumor cells to docetaxel. We have shown that docetaxel induces a resistance mechanism via activation of PKC/ERK and Akt pathways involved in cell proliferation and survival. Modification of the membrane lipid environment by AGPIn-3LCs supplementation inhibits these signaling pathways and increases the efficacy of docetaxel in mammary tumor cell lines and in a preclinical rodent model of native mammary tumors. Moreover, in this mammary tumor model we have found another molecular target regulated by AGPIn-3LCs: epiregulin, a member of the EGF family. AGPIn-3LCs inhibit epiregulin-VEGF induced in endothelial cells and induce a remodeling of tumor vasculature. Furthermore, AGPIn-3LCs act on the tumor microenvironment directly. This thesis work provides additional arguments for the use of AGPIn-3LCs as adjuvant molecules to reduce the resistance of breast tumors to anticancer agents.

Les techniques d’électrophysiologie chez les animaux et d’imagerie fonctionnelle chez l’Homme ont permis d’étudier le contrôle hédonique de la prise alimentaire. Ce contrôle hédonique n’a cependant jamais été exploré chez l’Homme par l’étude des potentiels évoqués gustatifs (PEG), de meilleure résolution temporelle que l’imagerie fonctionnelle. Le premier objectif de la thèse a été de mettre au point une technique fiable et reproductible de recueil des PEG, en regard des aires cérébrales gustatives, en réponse à une stimulation sapide. Le deuxième objectif a été d’étudier les variations des PEG en fonction de la valeur hédonique de la prise alimentaire. Le travail de thèse a permis de mettre au point l’enregistrement des PEG, réalisés chez plus de 100 jeunes sujets sains. Les comparaisons effectuées entre les enregistrements cérébraux obtenus en réponse à l’eau seule ou à l’huile de paraffine, solutions non palatables, et après stimulation par une solution sapide ont permis d’apporter des arguments forts en faveur de l’origine gustative des potentiels évoqués enregistrés. L’analyse des PEG a permis de mettre en évidence des modifications de l’activation cérébrale en fonction du plaisir alimentaire, traduites par des changements de latence ou d’amplitude des PEG. Plusieurs situations connues pour faire varier le plaisir alimentaire ont été étudiées : avant/après repas ; stimulation par des solutions sucrées d’intensités différentes ou de valeurs énergétiques différentes ; stimulation par des acides gras. Des PEG en réponse aux acides gras à longue chaine (acides linoléiques) ont été enregistrés par ce biais, renforçant l’hypothèse du «gras» en tant que sixième saveur primaire. / Hedonic control of food intake has been studied using neurophysiological investigations in animals and functional imaging in humans. Gustatory evoked potentials (GEPs), a higher time resolution technique than functional imaging, have never been used for this purpose. The first aim of this thesis was to establish a reliable recording of GEPs in humans, in response to a sapid stimulus. The second aim was to determine the GEPs modifications according to the hedonic value of food intake. GEPs recording was performed in response to an intermittent stimulation of a sapid solution in more than 100 young healthy subjects. The comparisons between cerebral recordings in response to water or paraffin oil, non palatable solutions, and in response to sapid solutions (sucrose, sodium chlorure and fatty acids) allow us to advance strong arguments for the gustative nature of the recorded evoked potentials. GEPs analysis underlined changes in cerebral activation according to the hedonic value of the stimulus. These changes in cerebral activation were highlighted by modifications of GEPs latency or amplitude. Several physiological situations, marked by different pleasantness of food stimulation, were studied: before/after food intake, stimulation by sweet solutions with different concentrations or different caloric contents, stimulation by fatty acids. Moreover, GEPs in response to long chain fatty acids (linoleic acids) were recorded, reinforcing the hypothesis that fatty acids could be the sixth primary flavor.