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.

A formulação de dietas para ruminantes pode alterar o perfil de ácidos do leite, potencializando o valor nutritivo e nutracêutico do mesmo. Estas alterações podem, no entanto, aumentar a susceptibilidade do leite à oxidação, perdendo qualidade em sabor e tempo de prateleira. O objetivo deste trabalho consistiu em avaliar o efeito do fornecimento de dieta com alto teor de soja integral extrusada, associada ou não à suplementação com selênio orgânico, sobre a produção, a composição, a concentração de CLA, o perfil de ácidos graxos e a estabilidade oxidativa do leite. Vinte e quatro vacas holandesas foram agrupadas em três blocos, de acordo com o número de lactações e alimentadas com dieta total contendo 56% de volumoso e 44% de concentrado. Os animais foram distribuídos aleatoriamente nos seguintes tratamentos: A) dieta controle, B) dieta contendo 21% de soja extrusada e C) dieta contendo 21% de soja extrusada + 5 mg de selênio orgânico. As dietas foram balanceadas para obtenção dos mesmos níveis de energia e proteína. O fornecimento de soja extrusada não afetou o consumo de matéria seca, o peso vivo dos animais e a produção de leite. As vacas alimentadas com soja extrusada produziram leite com menor (P<0,01) porcentagem de gordura, menor (P<0,01) concentração de ácidos graxos de cadeias curta e média e maior (P<0,01) concentração de ácidos graxos de cadeia longa, quando comparadas às vacas alimentadas com a dieta controle. Com relação ao teor de saturação, produziram leite com menor (P<0,01) concentração de ácidos graxos saturados e maior (P<0,01) concentração de ácidos graxos poliinsaturados e de ácido linoléico conjugado (CLA). A concentração de colesterol total foi maior (P<0,01) no soro sanguíneo das vacas alimentadas com soja extrusada, quando comparado ao controle. A suplementação com selênio aumentou (P<0,05) significativamente a concentração do mineral no leite e retardou a oxidação do mesmo, avaliada pelo índice de peróxido (P<0,01). O fornecimento da dieta contendo 21% de soja alterou o perfil de ácidos graxos do leite, diminuindo a concentração de ácidos graxos saturados e aumentando a concentração de ácidos graxos poliinsaturados e ácido linoléico conjugado, no entanto, produziu um leite mais susceptível à oxidação. O enriquecimento do leite com selênio conseguiu minimizar este efeito influenciando positivamente a estabilidade do leite. / The manipulation of ruminant diet can modify the fatty acid profiles increasing its nutritive and therapeutic value. However, this change in fatty acid composition can increase milk oxidation and can affect its flavor and shelf life. The objective of this study was to evaluate the effect of extruded soybean diet and organic selenium supplementation on milk composition, CLA content, fatty acid profiles and oxidative stability of milk. Twenty four Holstein cows were grouped into three blocks according to number of parturition and were fed diets containing 56% forage and 44% concentrate. The animals were allocated to three treatments: A) control diet, B) 21% extruded soybean diet and C) 21% extruded soybean diet + 5 mg of organic selenium. The diets were formulated to provide the same energy and protein levels. The extruded soybean did not affect the dry matter intake, body weight and milk production. The cows fed extruded soybean diet produced milk with lower (P<0,01) concentration of fat, lower (P<0,01) concentration of short and medium chain fatty acids and higher (P<0,01) concentration of long chain fatty acids when compared to control group. Regarding to saturation level, those animals produced milk with lower (P<0,01) concentration of saturated fatty acids and higher (P<0,01) concentration of polyunsaturated fatty acids and CLA content. The serum concentration of cholesterol was higher (P<0,01) for treatments containing extruded soybean when compared to control. The organic selenium supplementation increased (P<0,05) milk selenium concentration and delayed milk oxidation measured by peroxide value (P<0,01). The inclusion of 21% of extruded soybean in dairy cattle diet resulted in production of milk fat with modified fatty acid profile with lower concentration of saturated fatty acids and higher concentration of polyunsaturated fatty acids and CLA content. However the milk fat was more susceptible to oxidation. The organic selenium supplementation was efficient to overcome this problem and therefore could maintain the stability of milk.

Les cours d’eau de tête de bassin versant, malgré leurs tailles réduites, jouent des rôles importants pour le fonctionnement des milieux situés en aval, et rendent de nombreux services écologiques. Depuis l’émergence du River Continuum Concept, le fonctionnement de ces cours d’eau est considéré comme reposant principalement sur les apports allochtones de matière organique détritique par opposition aux écosystèmes aval de plus grande taille dans lesquels la production primaire autochtone est considérée comme prépondérante. Bien que les détritus soient, par définition, des ressources de moindre qualité pour leurs consommateurs que les végétaux vivants, le rôle des facteurs qui limitent la production biologique et les processus fonctionnels dans ces écosystèmes détritiques reste encore peu exploré. Par exemple la carence en éléments minéraux essentiels (notamment en azote et en phosphore) dans les détritus peut limiter le développement des organismes vivants dans les cours d’eau. De ce fait, la disponibilité en éléments minéraux dans la colonne d’eau, en stimulant la croissance, l’activité des décomposeurs et la qualité élémentaire des ressources par le processus d’immobilisation microbienne, pourrait contrôler pour partie les processus écologiques en jeu dans ces écosystèmes. En étudiant in situ la réponse des communautés microbiennes aux nutriments le long d’un gradient d’occupation des sols, nos travaux ont mis en évidence un effet positif des éléments dissous (N et P) sur la décomposition des litières et les activités enzymatiques des décomposeurs, et ce même pour les niveaux en nutriments les plus élevés du gradient, pour lesquels des effets inverses étaient attendus. Indépendamment, dans des cours d’eau anciennement soumis à de forts dépôts acidifiants, nous avons pu observer une récupération du processus de décomposition des litières. Néanmoins, cette restauration s’accompagnant d’un appauvrissement marqué en N, des perturbations du fonctionnement de ces cours d’eau pourrait se manifester à terme. Le rôle de la production primaire dans les cours d’eau de tête de bassin versant a ensuite été questionné. En effet, bien qu’étant mineur quantitativement, l’apport de carbone de forte qualité pourrait jouer un rôle non négligeable sur le fonctionnement des cours d’eau de tête de bassin versant. En particulier, l’importance et le rôle des apports en acides gras polyinsaturés (AGPI) via les biofilms phototrophes ont été questionnés. En se basant sur un suivi de terrain sur 15 cours d’eau des Vosges le long d’un gradient d’acidification, nos recherches ont tout d’abord pu mettre en évidence la présence de biofilms diatomiques riches en AGPI, malgré le caractère très forestier de ces cours d’eau. Dans un second temps, une expérience en conditions contrôlées a permis d’observer le rôle positif des apports, mêmes faibles, de ressources diatomiques sur la croissance et la survie du crustacé amphipode Gammarus fossarum. Sans cet apport, ces organismes se sont avérés incapables de maintenir leurs niveaux internes d’AGPI, suggérant qu’une consommation de producteurs primaires autochtones était indispensable à ces organismes détritivores. Nos résultats ont ainsi pu mettre en évidence les rôles fondamentaux que peuvent jouer la disponibilité en éléments dissous (N et P) et la qualité du carbone dans les ressources sur la production biologique et sur le processus de décomposition des litières dans les cours d’eau de tête de bassin versant. Ces résultats appellent désormais à investiguer plus profondément les interactions entre ces deux facteurs limitants, et à comprendre dans quels contextes l’un des facteurs pourrait suppléer l’autre. En particulier, il sera intéressant de rechercher comment diverses perturbations anthropiques (xénobiotiques, réchauffement climatique etc…) pourraient affecter l’intensité ou la nature de ces limitations / Despite their reduced sizes, headwater streams play important roles in the functioning of downstream ecosystems and provide numerous ecological services. Since the River Continuum Concept, the functioning of these streams has been considered as mainly relying on allochthonous supplies of detrital organic matter for their functioning, in contrast with downstream ecosystems of larger size where primary production is considered to be predominant. Despite that detritus represent, by definition, a resource of lower quality for its consumers than living plants, the nature of the factors limiting the biological production and functional processes in these detrital ecosystems remains sparsely explored. First, the lack of essential elements (in particular nitrogen and phosphorus) in detritus could be a major constraint for the development of living organisms in streams. As a result, the availability of mineral elements in the water column, by stimulating the growth and activity of decomposers, but also by increasing the elemental quality of resources through microbial immobilization, could control ecological processes in a higher extent than previously believed in these ecosystems. By studying in situ, the response of microorganisms to nutrients availability along a land-use gradient, our results have shown a positive effect of the dissolved elements (N and P) on the decomposition of leaf litters and the enzymatic activities of the decomposers, even for the highest nutrient levels of the gradient, for which adverse effects were expected. Independently, in streams formerly subjected to acidification, we observed a recovery of the process of leaf litter decomposition. Nevertheless, this recovery was accompanied by a marked N reduction in the ecosystem, huge changes in stream functioning might be expected in the medium to long term. The role of autochthonous primary production was then questioned. Indeed, while being quantitatively minor, this contribution of high quality carbon may play a significant role in the functioning of headwater streams. In particular, the importance and role of polyunsaturated fatty acid (PUFA) intakes via phototrophic biofilms was questioned. Based on a field survey of 15 rivers in the Vosges Mountains along an acidification gradient, our results first showed the presence of diatomic biofilms rich in PUFA, despite these streams are clearly in a forested context. In a second step, an experimentation carried out under controlled conditions permitted to observe the positive role of diatomic resources, even in low concentrations, on the growth and survival of the crustacean amphipod Gammarus fossarum. Without this input, these organisms were unable to maintain their internal levels of PUFAs, suggesting that the consumption of autochthonous primary producers might be essential for these detritivore organisms. All our results permit to highlight the fundamental roles that can be played by the availability of dissolved elements (N and P) and the carbon quality of the resources on the biological production and on leaf litter decomposition in headwater streams. These results stress the need for more profound investigation of the interactions between these two limiting factors, and for understanding in which contexts one factor could supplement the other. In particular, it will be interesting to investigate to what extent various anthropogenic disturbances (xenobiotics, global warming, etc...) could affect the intensity or the nature of these limitations

Dans une stratégie de diversification des apports en acides gras polyinsaturés très longue chaîne n-3 (AGPI TLC n-3), essentiels à la santé de l'Homme et majoritairement apportés par les produits de la mer, l'objectif de la thèse était de mieux comprendre les mécanismes cellulaires pouvant expliquer la faible teneur en AGPI TLC n-3 présente dans le muscle de bovin, ceci en explorant trois voies métaboliques de ces acides gras potentiellement limitantes (biosynthèse, captage facilité et peroxydation non-enzymatique) par des approches de qPCR ou de transcriptomique. Les principaux résultats montrent que le foie et les muscles de bovin possèdent tout le matériel génétique nécessaire à la synthèse des AGPI TLC n-3 et pourraient donc assurer leur synthèse, sauf chez le mâle entier où l'expression du gène de l'élongase 5 est réprimée par la présence d'hormones sexuelles mâles. D'autre part, les teneurs musculaires en AGPI TLC n-3 plus élevées avec un régime base d'herbe par rapport à de l'ensilage de maïs et dans un muscle glycolitique par rapport à un muscle oxydatif ne semblent pas s'expliquer par une modulation de l'expression des gènes impliqués dans leur biosynthèse, ni dans leur captage facilité et dans la régulation endogène de leur lipoperoxydation. En conclusion, ce travail de thèse a permis de considérablement faire progresser la compréhension des mécanismes impliqués dans la régulation des dépôts d'AGPITLC n-3 dans les muscles de bovin. Toutefois, ces régulations sont certainement plus complexes et probablement multifactorielles. De nombreuses pistes restent encore à explorer avant d'envisager augmenter la teneur en AGPI TLC n-3 dans la viande bovine. / N-3 very long chain polyunsaturated fatty acids (n-3 VLC PUFA) are essential for human health but are almost exclusively present in seafood. Thus, in a strategy of diversification of n-3 VLC PUFA sources for human, the aim of this thesis was to better understand the cellular mechanisms that may explain the low n-3 VLC PUFA content in bovine muscle, by exploring three potentially limiting metabolic pathways of these fatty acids (biosynthesis, facilited uptake and non-enzymatic peroxidation) by qPCR or transcriptomic approaches. The main results indicate that the liver and muscles of cattle possess all the genetic material necessary for the synthesis of n-3 VLC PUFA and may therefore ensure their synthesis, except in entire males where gene expression of the elongase 5 is suppressed by the presence of male sex hormones. On the other hand, the higher n-3 VLC PUFA content in muscle of cattle with a grass based diet compard to corn silage and in glycolytic muscle compared to oxidative muscle do not seem to be explained by a modulation of gene expression of proteins involved in their biosynthesis or in their facilited uptake or in the endogenous regulation of their lipoperoxidation. In conclusion, this thesis has greatly advance our understanding of mechanisms involved in the regulation of n-3 VLC PUFA deposits in muscles of cattle. However, these regulations are certainly more complex and probably multifactorial. Many tracks remain to be explored before considering increasing n-3 VLC PUFA content in beef.

Si l'intérêt nutritionnel des acides gras polyinsaturés (AGPI) n-3 dans la prise en charge et la prévention des dysfonctions associées au syndrome métabolique, est bien établi. Les mécanismes d'action spécifiques sous-jacents aux effets bénéfiques de cette famille d'acides gras sont encore en cours d'étude. L'objectif de ces travaux était d'explorer le rôle de l'acide alpha-linolénique ALA ou 18 :3 n-3, dans la modulation des voies affectant l'homéostasie de molécules bioactives dérivant du métabolisme secondaire des acides aminés (le monoxyde d'azote -NO- et le glutathion). L'hypothèse sous-jacente est que ces modulations pourraient expliquer, du moins en partie, le rôle des AGPI n-3 dans le maintien des fonctions biologiques contrôlées par ces métabolites (telles que la fonction endothéliale et le statut oxydant) et impliquées de près dans la physiopathologie du syndrome métabolique. Notre intérêt a porté particulièrement sur la voie de régulation génique via le PPARα et sur son implication dans le contrôle des gènes du métabolisme des acides aminés par l'ALA. Nous avons exploré chez la souris de type sauvage et invalidée pour le PPARα, l'effet de l'apport alimentaire d'ALA dans le cadre de régime normo- ou hyper-lipidiques sur les voies du métabolisme secondaire de l'arginine et de la cystéine. En parallèle nous nous sommes focalisés sur les effets de l'ALA au niveau vasculaire en utilisant un modèle de cellules endothéliales bovines en cultures. De ce travail de thèse s'est dégagé que l'ALA module effectivement le métabolisme secondaire de l'arginine et de la cystéine. L'apport d'ALA (à hauteur de 11% et 42% de l'apport énergétique) augmente la production de NO sans affecter l'expression hépatique des enzymes contrôlant l'utilisation de l'arginine (NOS et ARG). L'apport d'ALA (11%) augmente le pool hépatique du glutathion, alors que les plus forts apports d'ALA (42%) modulent l'expression des principales enzymes impliquées dans les voies d'utilisation de la cystéine (γGCL et CDO). Le PPARα ne semble pas être directement impliqué dans les effets observés de l'ALA, néanmoins, l'invalidation du PPARα rend le métabolisme secondaire des acides aminés plus sensible à la nature des acides gras alimentaire. Une meilleure biodisponibilité du NO et du glutathion suite à l'apport alimentaire d'ALA serait bénéfique pour la physiopathologie du syndrome métabolique. Il semble donc intéressant, à l'issus de ce travail, d'élaborer des études nutritionnelles validant ces effets de l'ALA chez l'homme dans une perspective de recommandations nutritionnelles. / *

<p>Insulin sensitivity is related to the fatty acid profile of skeletal muscle. The aim of this thesis was to investigate whether physical activity and dietary fat quality, independent of each other, influence the fatty acid composition of the skeletal muscle lipids. In an intervention study where middle-aged men were exercising for six weeks, and in a cross-sectional study comparing sedentary with endurance trained young men, it was demonstrated that the fatty acid composition of skeletal muscle lipids differed between physical active and inactive men. In brief, a lower proportion of palmitic acid (16:0) and total n-6 polyunsaturated fatty acids (PUFA) and a higher proportion of stearic (18:0) and oleic acid (18:1n-9) and total n-3 PUFA in the muscle phospholipids were associated with physical activity, despite similar fatty acid composition of the diet. In the second study, that included a larger training volume, differences in the fatty acid profile were also found in the skeletal muscle triglycerides. </p><p>In contrast, after short-term supra-maximal exercise we found no significant changes in the proportion of the fatty acids in skeletal muscle. </p><p>Furthermore, after a treatment period of three months, with diets with various dietary fat quality, the proportions of saturated fatty acids (14:0, 15:0 and 17:0) were higher and the proportion of 18:1 n-9 lower in subjects with a high intake of saturated fatty acids compared with subjects with a high intake of monounsaturated fatty acids. In addition subjects given n-3 supplementation had a higher proportion of total n-3 PUFA and lower n-6 PUFA in the skeletal muscle phospholipids than controls. Differences similar to those observed in the phospholipids were found in the triglycerides. </p><p>In summary, these results suggest that regular aerobic physical activity and dietary fat quality influence the fatty acid composition of the skeletal muscle lipids, which may affect insulin sensitivity and glucose homeostasis. </p>

<p>Polyunsaturated fatty acids can be bioactivated by two families of dioxygenases, which either contain non-heme iron (lipoxygenases) or heme (cyclooxygenases, linoleate diol synthases and α-dioxygenases).</p><p>Lipoxygenases and their products play important roles in the pathophysiology of plants and fungi. The only known lipoxygenase with catalytic manganese (Mn-lipoxygenase) is secreted by a devastating root pathogen of wheat, the Take-all fungus <i>Gaeumannomyces graminis</i>. Its mycelia also contains linoleate diol synthase (LDS), which can oxidize linoleic acid to sporulation hormones.</p><p>Mn-lipoxygenase belongs to the lipoxygenase gene family. Recombinant Mn-lipoxygenase was successfully expressed in the yeast <i>Pichia pastoris</i> with an expression level of 30 mg/L in fermentor culture. The tentative metal ligands of Mn-lipoxygenase were studied by site-directed mutagenesis. The results show that four residues His-274, His-278, His-462 and the C-terminal Val-602 likely coordinate manganese, as predicted by sequence alignments with Fe lipoxygenases.</p><p>Mn-lipoxygenase (~100 kDa) contains an Asp-Pro peptide bond in the N-terminal region, which appears to hydrolyze during storage and in the acidic media during Pichia expression to an active enzyme of smaller size, mini-Mn-lipoxygenase (~70 kDa). The active form of Mn-lipoxygenase can oxygenate fatty acids of variable chain length, suggesting that the fatty acids enter the catalytic site with the ω-end (“tail first”).</p><p>Mn-lipoxygenase is an <i>R</i>-lipoxygenase with a conserved Gly316 residue known as a determinant of stereospecificity in other <i>R/S</i> lipoxygenases. The Gly316Ala mutant showed an increased hydroperoxide isomerase activity and transformed 18:3n-3 and 17:3n-3 to epoxyalcohols.</p><p>The genome of the rice blast fungus, <i>Magnaporthe grisea</i>, contains putative genes of lipoxygenases and LDS. Mycelia of <i>M. grisea</i> were found to express LDS activity. This enzyme was cloned and sequenced and showed 65% amino acid identity with LDS from <i>G.graminis</i>. </p><p>Take-all and the rice blast fungi represent a constant threat to staple foods worldwide. Mn-lipoxygenase and LDS might provide new means to combat these pathogens.</p>

Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids required for proper embryonic development, specifically neurodevelopment. However, little is known regarding their conversion to other metabolites during embryogenesis. The oxidation of ARA gives rise to the biologically active eicosanoids and the oxidation of DHA gives rise to the biologically active docosanoids. The oxidation of ARA and DHA occurs through enzymatic processes, via lipoxygenase (LOX), or non-enzymatic processes, via radical-mediated lipid peroxidation. We hypothesize that oxidation of ARA and DHA via LOX is required for proper embryonic development. Additionally, we hypothesize that α-tocopherol, a potent lipid soluble antioxidant, mediates the conversion of ARA and DHA to their respective oxidized metabolites. Using zebrafish as a model of vertebrate embryogenesis, we found that the selective knockdown of either 12-LOX or 5-LOX decreased the production of docosanoids, altered fatty acid homeostasis, and increased the incidence of malformations and mortality in embryos by 24 hours post fertilization. α-Tocopherol deficiency also increased the incidence of malformations and mortality during embryogenesis, and in its absence, increased oxidized metabolites of ARA and DHA and decreased fatty acids concentrations. Therefore, oxidized metabolites of ARA and DHA perform crucial functions during embryonic development, but the production of oxidized fatty acids must be balanced with antioxidant bioavailability for proper embryogenesis. / Graduation date: 2012

The ability to extrapolate nutritional intervention data from experimental rodent models to humans requires standardization of dietary design. The inability to translate the level of nutrients from animal models to humans has contributed to contradictory findings between species. It is hypothesized that dietary linoleic acid (LA) promotes chronic and acute diseases by enriching tissues with arachidonic acid (AA), its downstream metabolite. However, levels of LA in rodent diets are notoriously erratic making interspecies comparisons unreliable. Therefore, the ability to extrapolate the biological effects of dietary LA from experimental rodents to humans necessitates an allometric scaling model that is rooted within a human equivalent context. To determine the physiological effect of dietary LA on tissue AA, a mathematical model for extrapolating nutrients based on energy was designed to mimic human equivalent doses. C57BL/6J mice were divided into 9 groups fed a background diet equivalent to that of the US diet (including LA, ALA, AA, EPA, DHA) with supplemental doses of LA (up to 2.3x) or AA (up to 5x). Changes in the phospholipid fatty acid compositions were monitored in plasma and erythrocytes and compared to data from humans supplemented with equivalent doses of LA or AA. Increasing dietary LA had little effect on tissue AA, while supplementing diets with AA significantly increased tissue AA levels, recapitulating results from human trials. Thus, interspecies comparisons for dietary LA between rodents and humans can be achieved when rodents are provided human equivalent doses based on differences in metabolic activity as defined by energy consumption.

Insulin sensitivity is related to the fatty acid profile of skeletal muscle. The aim of this thesis was to investigate whether physical activity and dietary fat quality, independent of each other, influence the fatty acid composition of the skeletal muscle lipids. In an intervention study where middle-aged men were exercising for six weeks, and in a cross-sectional study comparing sedentary with endurance trained young men, it was demonstrated that the fatty acid composition of skeletal muscle lipids differed between physical active and inactive men. In brief, a lower proportion of palmitic acid (16:0) and total n-6 polyunsaturated fatty acids (PUFA) and a higher proportion of stearic (18:0) and oleic acid (18:1n-9) and total n-3 PUFA in the muscle phospholipids were associated with physical activity, despite similar fatty acid composition of the diet. In the second study, that included a larger training volume, differences in the fatty acid profile were also found in the skeletal muscle triglycerides. In contrast, after short-term supra-maximal exercise we found no significant changes in the proportion of the fatty acids in skeletal muscle. Furthermore, after a treatment period of three months, with diets with various dietary fat quality, the proportions of saturated fatty acids (14:0, 15:0 and 17:0) were higher and the proportion of 18:1 n-9 lower in subjects with a high intake of saturated fatty acids compared with subjects with a high intake of monounsaturated fatty acids. In addition subjects given n-3 supplementation had a higher proportion of total n-3 PUFA and lower n-6 PUFA in the skeletal muscle phospholipids than controls. Differences similar to those observed in the phospholipids were found in the triglycerides. In summary, these results suggest that regular aerobic physical activity and dietary fat quality influence the fatty acid composition of the skeletal muscle lipids, which may affect insulin sensitivity and glucose homeostasis.