Organisms adapt to their environments by adjusting their biochemistry and physiology; such adaptation is limited by resource availability and physiological constraints. The freshwater crustacean Daphnia magna inhabits a wide range of environments and must survive and reproduce within a range of temperatures. One limit to low-temperature adaptation is thought to be the availability of unsaturated fatty acids necessary to maintain proper fluidity of cellular membranes. D. magna maintained at 10 ºC on a diet poor in unsaturated fatty acids have been observed to produce clutches that fail to develop. However, this has not been observed on a diet rich in unsaturated fatty acids or at a higher temperature regardless of diet. Clonal variation is commonly seen in D. magna life history traits, including heat tolerance, and was also investigated. D. magna were kept at two temperatures and fed two algal diets that differ in unsaturated fatty acid content. To investigate the role of fatty acid composition on the reproductive success of D. magna, fatty acids were extracted from adults and eggs. Of the twenty-one clones studied, no clonal variation was seen in the ability to produce successful clutches at 10 °C on a diet poor in unsaturated fatty acids. Gas chromatography revealed significant differences in 20-carbon fatty acids and suggest a parent-offspring conflict over a limited resource.

No description available.

No / Age is characterized by deficits in synaptic function identified by decreased performance of aged animals in spatial learning tasks and reduced ability of animals to sustain long-term potentiation (LTP). Several cellular and molecular events are correlated with these deficits, many of which are indicative of cell stress. Thus there is evidence of age-related neuroinflammatory stress and oxidative stress and these have been linked with microglial activation which is likely to be primarily responsible for the age-related increase in production of proinflammatory cytokines and reactive oxygen species. It is significant that agents which decrease microglial activation are commonly associated with restoration of function. We set out to examine whether the n-3 polyunsaturated fatty acid docosapentaenoic acid (DPA), which is a metabolite of eicosapentaenoic acid (EPA), could modulate the age-related increase in microglial activation and the associated increase in oxidative changes and therefore impact on synaptic function in aged rats. We demonstrate that DPA possesses neurorestorative effects and is capable of downregulating microglial activation. The data show that it also decreases the coupled activation of sphingomyelinase and caspase 3, probably as a result of its ability to decrease age-related oxidative changes, and consequently attenuates the age-related decrease in spatial learning and LTP.

No / Nutritionally important PUFAs (polyunsaturated fatty acids) mediate some of their bioactivities through formation of oxygenated metabolites. These bioactive lipids are formed by COX (cyclo-oxygenase), LOX (lipoxygenase) and cytochrome-P450-catalysed reactions, as well as non-enzymatic lipid peroxidation. These reactions produce numerous species, some of which can be formed through more than one pathway. MS-based lipidomics offers the selectivity and sensitivity required for qualitative and quantitative analysis of multiple lipid species, in a variety of biological systems, and can facilitate the study of these mediators.

The important factor in regulation of metabolic processes is regulatory proteins, which are able to react by feed-back to energetic state of the cell. Big attention is focused on the AMP activated kinase (AMPK) and NAD+ activated deacetylase SIRT1. These enzymes interact together and their stimulation increases mitochondrial biogenesis and fatty acid oxidation. Due to this it functions beneficially against the onset of obesity, insulin resistance and ageing. Fasting, exercise and some antidiabetogenic drugs act by these regulators. n-3 polyunsaturated fatty acids (PUFA) are also known because of their stimulative effects on mitochondrial biogenesis and -oxidation. Previous work of our group have showed that intake of higher dose of n-3 polyunsaturated fatty acids (PUFA) in diet lead to increase in activity of AMPK in white adipose tissue. New results presented in this thesis show that SIRT1 is essential for increase in expression of stimulators of -oxidation (PPAR etc) in response to n-3 PUFA in diet. n-3 PUFA futher improve the metabolic profile synergistically with calorie restriction probably through SIRT1.

L'acide arachidonique (AA, C20 :4, n-6) et l'acide docosahexaènoïque (DHA, C22 :6, n-3) sont présents en quantités importantes dans les membranes phospholipidiques. Lors d'un stress oxydant, ces acides gras polyinsaturés sont métabolisés en composés de structure tetrahydrofuranique appelés isofuranes (IsoFs) pour l'AA et neurofuranes (NeuroFs) pour le DHA. Nous nous sommes intéressés à la synthèse de ce type de composés afin d'accéder à de nouveaux biomarqueurs du stress oxydant. Dans ce même objectif, nous nous sommes également penchés sur la synthèse de potentielles structures analogues dérivant de l'acide adrénique (AdA, C22 :4, n-6), que nous avons nommées dihomo-isofuranes (dihomo-IsoFs). / Arachidonic acid (AA, C20 :4, n-6) and docosahexaenoïc acid (DHA, C22 :6, n-3) are phospholipidic membranes polyinsaturated fatty acids. During and oxidative stress, these are metabolized into tetrahydrofuran-containing compounds, known as isofurans (IsoFs) for AA and neurofurans (NeuroFs) for DHA. We were interested in the total synthesis of these compounds in the aim of getting access to novel biomarkers of the oxidative stress. Towards the same goal, we envisaged the total synthesis of potential analog structures coming from adrenic acid (AdA, C22 :4, n-6), that we named dihomo-isofurans (dihomo-IsoFs).

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Monika Novotná Supervisor of Diploma Thesis: Mgr. Monika Kuchařová, Ph.D. Title of Diploma Thesis: The determination of fatty acid levels in the tissues of healthy, suddenly deceased persons and polymorbid patients The aim of the thesis was to determine the levels of 14 fatty acids in tissues of suddenly deceased, otherwise healthy individuals and in the group of polymorbid, chronic patients. It was a comparison of fatty acid levels in seven tissues of the human body: subendocardial left ventricular tissue, liver parenchyma tissue, kidney cortex, adrenal tissue, skeletal muscle, abdominal subcutaneous adipose tissue, and brain tissue. Each group included 10 deceased patients. The theoretical part incudes fatty acids as the main component of lipids. It deals mainly with the group of polyunsaturated fatty acids and their relation to pathologies in the human body. Gas chromatography, thanks to which we analyzed the fatty acid spectrum, is also described. The experimental part consists of the basic characteristics of the research groups, the description of the workflow and the results. Statistically processed data are divided into categories by individual fatty acids and other monitored...

Les dernières données de consommation alimentaire de la population française démontrent que les apports nutritionnels en acide eicosapentaénoïque (EPA) et en acide docosahexaénoïque (DHA) sont nettement inférieurs aux recommandations fixées par l’Agence nationale de sécurité sanitaire, de l’alimentation, de l’environnement et du travail (ANSES). Compte tenu de leurs rôles physiologiques, l’amélioration de leur biodisponibilité est devenue une priorité nutritionnelle. La maitrise des formes de vectorisation des acides gras polyinsaturés à longue chaine de la série n-3 (AGPI-LC n-3) représente une piste d’intérêt afin de répondre à cette problématique. C’est dans ce contexte que s’inscrit ce travail de thèse, visant à étudier l’influence de la forme moléculaire (phospholipides (PL) vs triglycérides (TG)) et/ou supramoléculaire (phase continue, émulsion, liposomes) de vectorisation des AGPI-LC n-3 sur leur devenir métabolique.La démarche expérimentale adoptée a consisté, dans un premier temps, à caractériser les différentes formes de vectorisation des AGPI-LC n-3 par un couplage de méthodes d’analyse physico-chimiques (microscopie optique, granulométrie, mesure des produits primaires et secondaires d’oxydation, lipolyse enzymatique). Les données obtenues ont permis de poser des hypothèses mécanistiques pour l’étude de la biodisponibilité chez le rat. Ainsi, dans un deuxième temps, l’étude in vivo a permis d’aborder (1) l’absorption intestinale des acides gras (AG) suite à un apport en bolus de lipides et (2) l’accrétion tissulaire suite à un régime de 8 semaines.Les résultats indiquent que la forme moléculaire de vectorisation des AGPI-LC n-3 (TG marins vs PL marins, en phase continue) n’influence pas leur absorption intestinale mais plutôt leur incorporation dans les lipides lymphatiques, principaux transporteurs des AG alimentaires dans l’organisme. A l’inverse, la structuration des PL marins en milieu aqueux (émulsion ou liposomes), permet d’améliorer l’absorption de l’EPA et du DHA, de façon spécifique à la nature de la structuration supramoléculaire. Les résultats de l’accrétion tissulaire montrent qu’au terme des 8 semaines de régime, l’enrichissement tissulaire en AGPI-LC n-3 s’effectue de façon différente selon leur forme moléculaire de vectorisation. Ainsi, la consommation de TG marins favorise l’incorporation des AGPI-LC n-3 dans les lipides hépatiques, alors que la supplémentation des régimes en PL marins permet d’orienter le DHA vers le cerveau et la rétine.L’ensemble de ces résultats montre que le choix de la forme moléculaire de vectorisation des AGPI-LC n-3 (PL vs TG) peut être un moyen d'orienter leur incorporation tissulaire, alors que leur structuration supramoléculaire (émulsion ou liposomes), une stratégie pour moduler leur absorption intestinale. / The latest data from the cross-sectional national French dietary study show that dietary intakes of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are significantly lower than those recommended by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES). Considering their physiological roles, improving their dietary intake has became a nutritional priority. The control of the different forms of n-3 long chain polyunsaturated fatty (n-3 LC-PUFA) intake represents an interesting way to address this issue. In this context, this thesis aims at studying the influence of the molecular form (phospholipids (PL) vs triglycerides (TG)) and/or supramolecular form (continuous phase, emulsion, liposomes) of n-3 LC-PUFA towards their bioavailability.The experimental approach consisted in characterizing the different forms of n-3 LC-PUFAs by coupling various physicochemical methods (optical microscopy, granulometry, measurement of primary and secondary oxidation products, enzymatic lipolysis). The data obtained made it possible to propose some mechanistic hypothesis for the bioavailability studies conducted in rat. Thus, in a second step, the in vivo study made addresses (1) the intestinal absorption of fatty acids following a single dose of lipids and (2) the lipid tissue accretion following an 8-week diet.The data obtained from the intestinal absorption study indicate that the molecular form of n-3 LC-PUFA targeting (marine TG vs marine PL, in continuous phase) does not influence their intestinal absorption but rather their incorporation into lymphatic lipids, which are the main carriers for dietary fatty acids in the organism. Conversely, structuring marine PL (emulsion or liposomes) improves the EPA and DHA absorption, but differently according to the supramolecular form. The results concerning tissue accretion show that, after 8-week diet, the tissue enrichment in n-3 LC-PUFA is different according to the molecular form of targeting. The consumption of marine TG favours the incorporation of n-3 LC-PUFA into hepatic lipids, while the supplementation of diets with marine PL targets DHA preferentially to brain and retina.All these results show that the molecular form of targeting (PL vs TG) of n-3 LC-PUFA can be a way for managing their tissue incorporation, whereas the supramolecular organization (emulsion or liposomes) can be envisaged to monitor their intestinal absorption.

Les récepteurs nucléaires LXRα et LXRβ sont activés par la fixation de dérivés oxygénés du cholestérol. Ils régulent l’expression de nombreux gènes appartenant au métabolisme du cholestérol et des acides gras, et jouent un rôle important dans l’inflammation et l’immunité innée. L’activation de LXR inhibe le développement de l’athérosclérose, en augmentant l’efflux de cholestérol des macrophages ainsi que le transport inverse jusqu’au foie et l’excrétion biliaire. De plus, LXR diminue la biosynthèse et la captation du cholestérol dans les tissus périphériques. Enfin, les agonistes synthétiques de LXR administrés à des souris diminuent significativement l’inflammation dans les lésions athérosclérotiques, notamment en inhibant la sécrétion de certaines cytokines inflammatoires. Néanmoins LXR régule également la lipogenèse et la synthèse d’acides gras mono-insaturés, et l’administration d’agonistes de LXR s’accompagne également d’effets indésirables liés à cette régulation, comme une accumulation dérégulée d’acides gras dans le foie et une augmentation du taux de LDLs circulantes. Plusieurs autres mécanismes restent encore à être explorés, comme la synthèse d’acides gras polyinsaturés et les conséquences sur le métabolisme cellulaire. Nos travaux identifient une nouvelle voie régulée entièrement par LXR, le métabolisme des acides gras polyinsaturés. Le récepteur nucléaire LXR régule l’ensemble des enzymes FADS1, FADS2 et ELOVL5, responsables de la synthèse d’acides gras polyinsaturés oméga-6 et oméga-3. Cette régulation s’accompagne d’une incorporation d’acide arachidonique dans les phospholipides, via la régulation de LPCAT3, ce qui prépare les macrophages à une synthèse accrue de dérivés inflammatoires issus de l’acide arachidonique, comme la Prostaglandine E2, suite à une stimulation au lipopolysaccharide. La régulation de cette voie par LXR a également un effet sur le développement de l’athérosclérose, augmentant les taux d’acides gras polyinsaturés oméga-6 et oméga-3 dans les plaques d’athérome. Nos résultats montrent donc que LXR régule la synthèse des acides gras polyinsaturés en plus des acides gras mono-insaturés et de la lipogenèse et que cette régulation a des conséquences sur le profil lipidique des macrophages in vitro et in vivo ainsi que sur leur réponse inflammatoire. / The nuclear receptors LXRα and LXRβ are activated by oxygenated metabolites of cholesterol. They regulate the expression of numerous genes belonging to cholesterol and fatty acids metabolism, and play a central role in inflammation and innate immunity. LXR activation inhibits atherosclerosis development, by increasing cholesterol efflux from macrophages as well as reverse cholesterol transport and biliary excretion. In addition, LXR decreases cholesterol uptake and biosynthesis. Synthetic LXR agonists fed to mice significantly decrease inflammation in atherosclerotic lesions, by inhibiting several inflammatory cytokines. However, LXR also regulate lipogenesis and monounsaturated fatty acids synthesis, and LXR agonists supplementation is accompanied by side effects due to this regulation, such as a deregulated accumulation of fatty acids in the liver and an increase in circulating LDLs. Other mecanisms still need to be characterized, such as polyunsaturated fatty acids synthesis and the consequences on cell metabolism. Our work identify a new pathway regulated by LXR, the metabolism of polyunsaturated fatty acids. The nuclear receptor LXR regulates all enzymes responsible for omega-6 and omega-3 polyunsaturated fatty acids synthesis, FADS1, FADS2 and ELOVL5. This regulation is accompanied by an increase in arachidonic acid incorporation in phospholipids, via LPCAT3 regulation, which subsequently primes human macrophages for an increased inflammatory metabolites secretion derived from arachidonic acid, such as Protaglandin E2, following a LPS stimulation. The regulation of this pathway by LXR has an effect on atherosclerosis, increasing omega-6 and omega-3 ployunsaturated fatty acids in atheroma plaques. Our results show therefore that LXR regulates polyunsaturated fatty acids synthesis in addition to monounsaturated fatty acids and lipogenesis, and that this regulation has direct consequences on lipid profile of macrophages in vitro and in vivo as well as on their inflammatory response.

Uma das razões da menor fertilidade dos touros europeus criados em regiões tropicais em relação a touros Bos indicus é uma maior índice de estresse oxidativo provocado por uma maior produção de espécies reativas de oxigênio (ROS), não compensada pela proteção antioxidante. Por outro lado, sabe-se que a célula espermática é extremamente susceptível ao estresse oxidativo devido à alta quantidade de ácidos graxos poli-insaturados (PUFA) em sua membrana plasmática, o que, no entanto, é muito importante para que o espermatozoide seja fértil e resistente ao choque frio. Sendo assim, tratamentos que interfiram no processo oxidativo, podem ser importantes para aumentar a produtividade destes animais, tanto à campo como em centrais de inseminação artificial. O presente experimento objetivou avaliar qual ROS seria a mais lesiva para touros europeus submetidos ao estresse térmico visando determinar um possível tratamento antioxidante direcionado para estes animais. Em um segundo momento visou então verificar a eficiência da interação entre uma dieta rica em PUFAs e o tratamento antioxidante sistêmico direcionado na qualidade espermática do sêmen ejaculado e epididimário (fresco e criopreservado) de touros europeus submetidos ao estresse térmico. Para isso, quatro touros Bos taurus adultos foram submetidos a insulação testicular (bolsa escrotal por 5 dias). Sessenta dias após a insulação o sêmen foi coletado por eletroejaculação. O sêmen de cada animal foi dividido em 4 alíquotas s submetidas à indução com quatro sistemas geradores de ROS: Ânion Superóxido (xantina/xantina oxidase), peróxido de hidrogênio, radical hidroxila (Ascorbato + Sulfato de Ferro) e malondialdeído (MDA; produto da peroxidação lipídica). As amostras foram incubadas por 1 hora e avaliadas através da análise espermática computadorizada (CASA), eoxina/nigrosina (integridade da membrana plasmática), fast-green/rosa bengala (integridade de acrossomo), 3, 3´ diaminobenzidina (atividade mitocondrial), ensaio da cromatina espermática (fragmentação de DNA) e substâncias reativas ao ácido tiobarbitúrico (TBARS; peroxidação lipídica). Os resultados indicaram ser o MDA, a substância mais deletéria aos animais Bos taurus submetidos ao estresse térmico aguda. Desta forma, 16 touros foram então submetidos á insulação testicular e divididos em 4 grupos: Controle (n=4; aplicação de óleo mineral; placebo); Grupo Vitamina E: (n=4; 5 ml de Monovin® E a cada 13 dias); Grupo PUFA: (n=4; 4 kg/dia Megalac® E1), Grupo PUFA+Vitamina E: (n=4; combinação entre os tratamentos dos grupos PUFA e Vitamina E). O sêmen destes animais foi coletado no dia da inserção da bolsa escrotal, no dia da retirada, 30 e 60 dias após. Os resultados indicaram que a vitamina E foi eficiente para a melhora nos danos causados pelo estresse térmico no DNA espermático e na mitocôndria, mas apenas nas amostras coletadas do epidídidmo. Da mesma forma, foi possível observar que a combinação entre a Vitamina E e a suplementação com PUFA foi eficiente na melhora dos padrões de motilidade espermática. Os resultados do presente estudo indicam que a combinação entre um tratamento antioxidante com Vitamina E e a suplementação com PUFA pode ser uma alternativa interessante para evitar os danos causados pelo estresse térmico agudo em touros europeus. No entanto, possivelmente este tratamento poderia ser ainda mais eficiente caso seja administrado de forma preventiva. / One reason for the lower fertility of European bulls bred in tropical regions is a higher rate of oxidative stress caused by increased production of reactive oxygen species (ROS) not compensated by antioxidant protection . On the other hand , it is known that the sperm cell is extremely susceptible to oxidative stress due to the high amount of polyunsaturated ( PUFA) on their plasma membrane. However the presence of these PUFAs is fundamental for the sperm to be fertile and resistant to cold shock. Thus, treatments that interfere with the oxidation process may be important to increase the productivity of these animals. This study aimed to evaluate which would be the most damaging ROS for European bulls subjected to heat stress to determine a possible antioxidant targeted treatment for these animals. In a second step we sought to verify the efficiency of the interaction between a diet rich in PUFAs and targeted-antioxidant treatment on sperm quality of ejaculated and epididymal sperm quality in European bulls subjected to heat stress. Four Bos Taurus bulls were submitted to testicular insulation (5 days). Sixty days after insulation semen was collected by electroejaculation. Semen from each animal was divided into 4 aliquots and submitted to the induction with four ROS generating systems: superoxide anion (xanthine / xanthine oxidase), hydrogen peroxide, hydroxyl radical (ascorbate + Ferrous Sulfate) and malondialdehyde (MDA; lipid peroxidation product). The samples were incubated for 1 h and assessed by computerized sperm analysis (CASA), eoxina / nigrosin (membrane integrity), fast-green/Bengal rose (acrosome integrity) , 3 , 3 \' diaminobenzidine (mitochondrial activity), sperm chromatin structure assay (DNA fragmentation) and thiobarbituric acid reactive substances (TBARS, lipid peroxidation). The results indicated that the MDA was the most deleterious substance to Bos Taurus semen subjected to an acute heat stress. Thus, 16 bulls were then subjected to testicular insulation and divided into 4 groups: control (n= 4; application of mineral oil; placebo); Group Vitamin E: (n= 4, 5 ml of Monovin ® E every 13 days); PUFA group: (n= 4; 4 kg/day Megalac ® E1 ); Group PUFA+Vitamin E (n=4; combination of groups PUFA and Vitamin E treatments). Semen was collected from these animals on the day of insertion of the thermal bag, on the day of withdrawal, 30 and 60 days after. The results indicated that vitamin E was effective for the improvement in damages caused by heat stress in sperm DNA and mitochondria, but only in samples collected from epididymis. Similarly, the combination of vitamin E and PUFA supplementation was effective in improving sperm motility patterns . The results of this study indicate that the combination of an antioxidant treatment with vitamin E and PUFA supplementation may be an interesting alternative to avoid the damage caused by acute heat stress in European bulls. However, possibly, this treatment would be more effective if performed preventively.