No description available.

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

La NADPH oxydase du phagocyte est une enzyme impliquée dans la défense immunitaire contre les pathogènes. Après activation du phagocyte, cette enzyme produit des ions superoxyde par réduction du dioxygène par le NADPH. Elle est constituée de quatre sous- unités cytosolubles (p47phox ; p67phox ; p40phox et Rac), et deux membranaires (gp91 ; p22phox). Son activation fait intervenir un processus complexe qui met en jeu des changements d’interaction entre les protéines la constituant et qui permet l’assemblage des six sous- unités. Afin d’obtenir des informations sur les processus d’assemblage et d’activation, j’ai reconstitué le complexe dans un système cell free à l’aide de protéines recombinantes pour pouvoir contrôler tous les paramètres. Dans ce travail nous avons comparé les modes d’activation de p47phox par phosphorylation, par mutation substitutionelle sérine - aspartate en position S303,S304 et S328 pour mimer la phosphorylation et enfin par addition d’acide arachidonique (AA) activateur connu de l’enzyme in vitro mais aussi in vivo. Bien qu’il ai été montré que ces trois méthodes ouvrent la protéine vers une conformation ayant des propriétés similaires, nous avons trouvé que les effets de ces méthodes d’activation sont significativement différents. Ainsi, les changement de conformation observés par dichroisme circulaire, sont dissemblables. Pour p47phox, l’addition de AA déstructure la protéine. La phosphorylation induit un déplacement bathochrome des bandes de CD qualitativement similaire, alors que les mutations S-D de p47phox provoquent un déplacement opposé. Pour le complexe p47phox-p67phox l’addition d’AA destructure le mélange tandis que la mutation induit relativement peu de changement. Nous avons mesuré les constantes de dissociation Kd du complexe p47phox-p67phox. Alors que pour les protéines « sauvages », le Kd est faible (4±2 nM), les mutations de p47phox ainsi que l’addition d’AA augmentent cette valeur jusqu’à environ 50 nM, montrant une diminution de l’affinité entre p47phox-p67phox. De même, sur le complexe entier, l’effet de la phosphorylation de p47phox est différent de la mutation. Nous avons mesuré les valeurs de EC50 relatives à p67phox pour les différentes formes de p47phox. L’activation de p47phox par phosphorylation diminue l’EC₅₀, alors que les doubles ou triple mutations augmentent sa valeur. Nous avons confirmé que la phosphorylation et la mutation sont insuffisantes pour activer l’enzyme. La présence de AA est indispensable pour le fonctionnement du complexe. L’ordre de fixation des sous unités cytosoliques semble indifférent mais il faut que tous les composants soient présents lors de l’ajout de AA. Enfin, la délétion de p47phox dans la partie C-terminale (aa 343 à 390, domaine d’interaction avec p67phox) il n’y a plus de formation du dimère mais l’enzyme fonctionne normalement. Ces résultats apportent des éléments nouveaux sur le rôle de la dimérisation p47 phox-p67 phox, non indispensable à l’activité du système et sur le rôle mineur de la phosphorylation dans l’activation de la NADPH oxydase in vitro. / The NADPH oxidase of phagocytes is an enzyme involved in the innate defense of organisms against pathogens. After phagocyte activation, this enzyme produces superoxide ions by reduction of dioxygen by NADPH. It is constituted of four cytosolic sub-units (p47phox ; p67phox ; p40phox et Rac) and two membrane proteins (gp91 ; p22phox). Its activation takes place through a complex process that involves protein-protein interaction changes leading to assembly and functionning of the catalytic core. In order to obtain information on this process, I have reconstituted the enzyme in a cell free systeme using recombinant proteins, to be able to fully control all the measurement conditions. In this work, we have compared different activation modes of p47phox i) phosphorylation; ii) substitution serine - aspartate by mutations at positions S303, S304 and S328 to mimic phosphorylation; iii) addition of arachidonic acid (AA), a well known activator molecule in vitro. It has been shown that these three activating methods transform p47phox to an open configuration with similar characteristics. However, we have found that the effects of these methods are significantly different. Indeed, the conformational changes observed by circular dichroism are different. For p47phox, the addition of AA destructures the protein. Its phosphorylation induces a bathochromic displacement of the bands, whereas the mutations S-D lead to an opposite displacement. For the dimer p47phox-p67phox , the addition of AA destructures the proteins while mutations induce hardly no changes. We have measured the dissociation constant Kd of the complex p47phox-p67phox. For wild type proteins, Kd value is low (4±2 nM), while mutations of p47phox as well as addition of AA increase its value up to 50 nM, showing a decrease of affinity between p47phox and p67phox. Moreover, on the whole complex, the effect of phosphorylation of p47phox is different from mutations. We have shown that the EC50 values relative to p67phox are sensitive to the various modifications of p47phox. Phosphorylation of p47phox decreases EC₅₀, while double or triple mutations increase its value. We have confirmed that phosphorylation and mutation are not sufficient to activate the enzyme. The presence of AA is a prerequisite for the functionning of the complex, i.e. production of superoxide. The binding order of the cytosolic proteins seems random but it is necessary that all the components be present during the activation by AA. Finally, deletion of the C terminal part of p47phox (aa 343 to 390, interaction domain with p67phox) leads to the absence of dimer formation but does not affect the enzyme activity. These results bring new information on the role of dimerisation of p47-p67 and on that of phosphorylation in the activation of NADPH oxidase in vitro.

Le complexe NADPH oxydase est un élément essentiel de l’immunité inné. Présent dans les cellules phagocytaires (neutrophile), sa fonction est de produire massivement, dans le phagosome, des anions superoxyde et générer ainsi des espèces encore plus réactives de l’oxygène qui vont détruire acides nucléiques, lipides et protéines des bactéries phagocytées. Le cœur membranaire catalytique du complexe NADPH oxydase est constitué d’un hétérodimère membranaire, le cytochrome b₅₅₈ (Cyt b₅₅₈). Après activation de celui-ci par les partenaires protéiques cytosoliques p47phox, p67phox, p40phox et Rac, une succession de réactions de transferts d’électron de part et d’autre de la membrane a lieu au sein du Cyt b₅₅₈ pour aboutir à la réduction du dioxygène de manière très contrôlée. Afin de mieux comprendre cette régulation, nous nous sommes d’abord intéressés aux stéreoisomères trans de l’acide arachidonique, activateur naturel de cet enzyme (cis), sur le fonctionnement de la NADPH oxydase et avons abordé cette étude parallèlement sur du Cytb₅₅₈ d’origine bovine présent dans des membranes de neutrophiles et dans des membranes de levures exprimant le Cytb₅₅₈ de manière hétérologue. Nous avons montré que la géométrie joue un rôle important sur l’activation du complexe enzymatique. Dans un deuxième temps, afin d’étudier le rôle de l’environnement membranaire sur le fonctionnement de la NADPH oxydase, nous avons déterminé les propriétés cinétiques et thermodynamiques de l’activité NADPH oxydase du Cytb₅₅₈ recombinant exprimé en levures, purifié, puis reconstitué en liposomes de composition lipidique variée. Après comparaison avec ces mêmes propriétés obtenues pour le Cytb₅₅₈ dans les membranes plasmiques et du réticulum endoplasmique de levures, nous avons montré que l’activité NADPH oxydase très sensible à la température peut être modulée par la composition et l’état physique de la membrane. / NADPH oxidase complex is a major actor of both antimicrobial host defense and inflammation by generating highly regulated superoxide anion, rapidly converted into reactive oxygen species (ROS). The NADPH oxidase complex consists of a heterodimeric integral membrane flavocytochrome b₅₅₈ and three cytosolic components p67phox, p47phox and p40phox, and the small GTP binding protein Rac. In response to a cellular stimulus, cytosolic proteins are recruited to the phagosomal membrane where they are assembled with the Cytb₅₅₈ to form the active NADPH oxidase. The aim of the work was to better understand the modulation of superoxide anion production by this enzyme. For this purpose, we performed experiments with both bovine neutrophil membranes and yeast membranes expressing the bovine recombinant Cytb₅₅₈. We first investigated the effect of the trans-isomerization of the cis-arachidonic acid, the activator of NADPH oxidase in vitro and showed that specific geometry of the activator plays an important role in the activation of the complex. We also studied the role of the membrane environment on the functioning of NADPH oxidase and determined the kinetics and thermodynamics of NADPH oxidase activity depending on the lipid composition of Cytb₅₅₈ proteoliposomes. Comparison with these properties obtained with recombinant Cytb₅₅₈ embedded into endoplasmic reticulum and plasma membranes, we showed that the NADPH oxidase activity is highly temperature dependent and can be modulated by the lipid environment and the physic state of the membrane.

La maladie d’Alzheimer (MA) est un problème majeur de santé publique. Elle se traduit par des atteintes de la mémoire reposant sur des dysfonctionnements synaptiques induits par les oligomères de peptide ß-amyloïde (Aß). Ceux-ci activent la phospholipase A2 cytosolique (cPLA2) qui libère l’acide arachidonique (ARA) des phospholipides (PL) membranaires neuronaux. L’acyl-CoA synthétase 4 (ACSL4) peut limiter cette libération en favorisant la réincorporation d’ARA dans les PL. Dans l’alimentation occidentale, il constitue une part croissante des apports lipidiques. Contrairement à l’acide docosahexaénoïque (DHA), l’influence de l’ARA dans la MA a été peu étudiée. C’est pourquoi nous avons étudié la mobilisation de l’ARA et son effet sur la sensibilité au peptide Aß. Nous avons montré dans un premier temps qu’un apport alimentaire en ARA affecte la mémoire à court terme et sensibilise les capacités d’apprentissage au peptide Aß. Ces altérations sont associées à des diminutions d’expression des récepteurs AMPA et de l’ACSL4, une prolifération astrocytaire et une incorporation accrue en ARA dans les espèces de PL phosphatidylsérine et phosphatidyléthanolamine (PE). D’autre part, la différenciation des cellules HT22 nous a permis de montrer que l’ACSL4 intervient dans l’incorporation de l’ARA et dans l’équilibre ARA/DHA, notamment dans les espèces PE. Cela indique qu’un excès en ARA dans l’alimentation peut constituer un facteur d’aggravation de la MA et que les enzymes assurant sa mobilisation, comme la cPLA2 et l’ACSL4, peuvent moduler ce risque. La caractérisation de leurs niveaux d’expression pourrait permettre de définir des groupes d’individus à risque vis-à-vis de la MA / Alzheimer’s disease (AD) is a major public health problem. This disease is characterized by memory impairments which are caused by synaptic dysfunctions induced by the oligomers of amyloid-ß peptide (Aß). These activate the cytosolic phospholipase A2 (cPLA2) which releases arachidonic acid (ARA) from neuronal membrane phospholipids (PL) whereas acyl-CoA synthetase 4 (ACSL4) potentially counteracts this release by favoring ARA reincorporation into PL. Western diets contain growing amount of ARA. Contrary to docosahexaenoic acid (DHA), a few studies were devoted to the influence of ARA in AD. This is why we decided to study the mobilization of ARA and its effects on the sensitivity to Aß oligomers. First we showed that dietary ARA reduces short-term memory abilities and increases the deleterious effects of Aß on learning abilities. These alterations of cognitive abilities are associated to reductions of expression levels of AMPA receptors and ACSL4, an astrocyte proliferation, and greater incorporation of ARA in PL species phosphatidylethanolamine (PE) and phosphatidylserine. Secondly, we used differentiated HT22 to show that ACSL4 modulate ARA incorporation and ARA / DHA balance in the PE species. These results indicate that excessive dietary intake of ARA may be a worsening factor in AD and the enzymes regulating ARA mobilization, such as cPLA2 and ACSL4, can modulate this risk. The characterization of their enzymatic activities could allow the identification of groups of individuals at AD risk.

Introduction: Epoxyeicosatrienoic acids (EETs) are converted by the enzyme soluble epoxid hydrolase (sEH) to the biologically inactive dihydroxyeicosatrienoic acids (DHETs). EETs are significantly involved in the control of blood pressure, they influence vascular tone and renal transport mechanism. sEH inhibitor reduce blood pressure by increasing the bioavailability of EETs in many models of hypertension. Aim of the study: To determine that sEH inhibitor decreases blood pressure and improves the renal function during the development of malignant hypertension in transgenic rats after the induction of the mouse renin gene. Methods: Hypertension in Cyp1a1-Ren-2 transgenic rats was induced through a dietary administration of the natural xenobiotic indole-3-carbinol (I3C, 0.3 %) for 3 and 11 days. I3C activates the renin gene. At the same time, during a three-day induction of hypertension, the inhibitor of nitric oxide synthase L-NAME (600 mg/l) was administered in drinking water. The sEH inhibitor c-AUCB was given in drinking water at a dose of 13 or 26 mg/l, starting 48 hours before the initiation of I3C and L-NAME administration. Radiotelemetric measurement of blood pressure was performed and renal excretory parameters were monitored in the conscious animals. The effects on renal hemodynamics and...

The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (PUFA) with neuroprotective and cardioprotective properties. It is thought that some of the actions of EPA may be attributed to its elongated metabolite, the PUFA docosapentaenoic acid (DPA). Docosahexaenoic acid (DHA) and arachidonic acid (AA) are bioactive PUFA ubiquitously expressed in neural tissues. EPA and AA can be converted by cyclooxygenase (COX) to prostanoids and by lipoxygenase (LOX) to hydroxy fatty acids. PUFA can also be converted to ethanolamides in the brain. These mediators are involved in physiological and pathological processes in many bodily systems. The purpose of this study was to examine the production of eicosanoids, hydroxy fatty acids and fatty acid ethanolamides in young and aged rat brain following EPA or DPA enriched diets. The effects of specific PUFA on human platelet eicosanoid production were also investigated as these mediators play a role in adhesion and aggregation. Liquid chromatography coupled to tandem mass spectrometry (LC/ESI-MS/MS) assays were developed and used to measure lipid mediators in rat brain and human platelets. Ageing in rat brain was accompanied with several changes in the prostanoid and hydroxy fatty acid profiles. Supplementing the diet with EPA or DPA at a daily dose of 200 mg/kg for 8 weeks prevented these changes and decreased levels of PGE2. DPA changed the profile of hydroxy fatty acids synthesised in the brain tissue of young animals. This study has shown that levels of eicosapentaenoylethanolamide (EPA-EA) increase in the brain as a result of ageing and that this is accompanied by an increase in levels of anandamide. Feeding aged animals EPA or DPA further increased the levels of EPA-EA but prevented any change in the level of anandamide. Niacin is used to treat hypercholesterolaemia although it is associated with an unpleasant PGD2 mediated skin flush. This exploratory study has shown that human platelets treated with niacin did not show any changes in their prostanoid and hydroxy fatty acid profiles. Platelets treated with EPA showed increased production of TXB3 and 12-HEPE. Niacin augmented the effects of EPA on human platelet mediator synthesis. Overall, this study has demonstrated that EPA can change brain and platelet lipid mediator synthesis and has provided evidence that could explain some of the neuroprotective and cardioprotective actions of this PUFA.

No description available.

No / Bioactive lipid mediators derived from polyunsaturated fatty acids (PUFA) and exhibit a range of tissue and cell-specific activities in many physiological and pathological processes. Electrospray tandem mass spectrometry coupled to liquid chromatography (LC/ESI-MS/MS) is a sensitive, versatile analytical methodology for the qualitative and quantitative analysis of lipid mediators. Here we present an LC/ESI-MS/MS assay for the simultaneous analysis of twenty mono- and poly-hydroxy fatty acid derivatives of linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids. The assay was linear over the concentration range 1-100 pg/¿L, whilst the limits of detection and quantitation were 10-20 and 20-50 pg respectively. The recovery of the extraction methodology varied from 76-122% depending on the metabolite. This system is useful for profiling a range of biochemically-related potent mediators including the newly discovered resolvins and protectins, and their precursor hydroxy-eicosapentaenoic and hydroxy-docosahexaenoic acids, and, consequently, advance our understanding of the role of PUFA in health and disease. / Wellcome Trust, British Heart Foundation