A presente pesquisa foi conduzida utilizando-se 288 galinhas poedeiras da linhagem Hisex White com 32 semanas de idade, pelo período de 10 semanas, com o objetivo de estudar o enriquecimento da gema do ovo em ácidos graxos a partir de rações suplementadas com óleo de peixe (OP) ou alga marinha (AM) em cinco níveis de DHA (120, 180, 240, 300 e 360 mg/100 g dieta). Foi aplicado modelo fatorial 2 x 5, inteiramente casualizado, com três repetições de oito aves por tratamento, de modo a constituir os grupos: OP120, OP180, OP240, OP300, OP360, AM120, AM180, AM240, AM300 e AM360. Um grupo controle submetido à ração basal de milho e soja (CON) e outro, acrescido de AM, contendo 420 mg de DHA/100 g dieta (AM420) foram também utilizados. Os seguintes parâmetros foram avaliados: postura, peso do ovo, consumo alimentar, conversão alimentar, qualidade externa e interna dos ovos e qualidade organoléptica dos mesmos. O peso médio do ovo assinalado para o grupo CON (63,30 g) decresceu linearmente de forma significativa (Y = - 0,0051X + 63,8560, R2 = 0,97) em resposta à suplementação de níveis crescentes de DHA da fonte OP, atingindo valor mínimo de 62,13 g no grupo OP360. O índice de postura das aves de 88,10% (CON) não foi influenciado pelas fontes de DHA na ração. A qualidade interna do ovo, avaliada em unidades Haugh, apresentou melhora significativa (P<0,05) com a adição de teores crescentes de AM igual ou superior a 1% na dieta das aves. Quanto aos teores de DHA na gema do ovo de aves suplementadas com OP, foi observado aumento significativo de 22,64 mg/ gema (CON), para 187,91 mg/ gema no grupo OP360. Os PUFAs n-3 apresentaram acréscimo significativo no contraste entre CON (62,16 mg/gema) e OP360 (218,62 mg/gema). Para a fonte AM, as médias de DHA também mostraram linearidade (Y = 0,23X + 1,27, R2 = 0,86), oscilando entre 22,64 mg/gema (CON) e 149,75 mg/gema (AM420), enquanto que o total de PUFAs n-3 oscilou de 104,18 mg/gema (AM120) a 175,32 mg/gema (AM420). O inverso ocorreu com os ácidos araquidônico, linoléico e PUFAs n-6 que mostraram decréscimos significativos (P<0,05) com o aumento de OP na dieta, variando, respectivamente, de 98,71 mg, 987,70 mg e 1108,92 mg/ gema na dieta CON a 38,87 mg, 734,22 mg e 802,79 mg/ gema, para o grupo OP360. O percentual de incorporação de DHA na gema dos ovos decresceu linearmente com o aumento dos níveis de DHA na ração suplementada com OP e AM, de 85,11% (OP120) e 65,28% (AM120) para 49,45% (OP360) e 34,06% (AM420). Melhora significativa (P<0,05) foi consignada na relação n-6/n-3, variando de 17,50 no grupo CON para 3,72 e 6,36 para tratamentos OP360 e AM420, respectivamente. A avaliação sensorial não detectou qualquer sabor ou odor estranho nos ovos dos diferentes tratamentos ou fontes utilizados, exceto para a análise qualitativa do grupo OP360 onde foi detectado grau de intensidade regular de sabor de peixe, diferindo significativamente (P<0,05) do grupo CON. / This experiment was conducted using two hundreds eighty eight 32-wk-old Hisex White laying hens for a period of ten weeks, with the objective of studying the fatty acid enrichment of the egg yolk of hens fed diets supplemented with fish oil (OP) or marine algae (AM) to provide five levels of DHA (120, 180, 240, 300 and 360 mg/100 g diet) for each source. A 2 X 5 completely randomized factorial design with three replicates of eight birds per treatment was applied in order to have the following groups: OP120, OP180, OP240, OP300, OP360, AM120, AM180, AM240, AM300 and AM360. A control group submitted to a corn/soy basal diet (CON) and another one supplemented with AM at the level of 420 mg of DHA/100 g diet (AM420) were also used. Egg production, egg weight, feed intake, feed conversion, external and internal egg quality and the organoleptic quality of the eggs were evaluated. The egg weight mean obtained for group CON (63.30 g), decreased linearly (Y = - 0.0051X + 63.8560, R2 = 0.97) as the supplemented DHA levels from the OP source increased, reaching the lowest egg weight mean of 62.13 g for the group OP360. The egg production of the control group hens (88.10%) was not influenced by the source of DHA. The internal quality of the egg evaluated by Haugh units presented significant improvement (P<0.05) with the increase of the AM source to 1% or more. The amounts of DHA into the egg yolk in birds fed OP diets, were significantly increased from 22.64 mg/egg yolk (CON), to 187.91 mg/egg yolk (OP360). The egg yolk PUFAs n-3 of the control group (62.16g) increased significantly as compared to the OP360 group (218.62 mg/yolk). The AM source showed DHA means also linear (Y = 0.23X + 1.27, R2 = 0.86), ranging from 22.64 mg/yolk (CON) to 149.75 mg/yolk (AM420), while the PUFAs n-3 ranged from 104.18 mg/yolk (AM120) to 175.32 mg/yolk (AM420). The araquidonic acid, linoleic acid and PUFAs n-6 showed significant decrease (P<0.05) with the increase of OP into the diet, ranging from 98.71 mg, 987.70 mg and 1108.92 mg/egg yolk for the CON group to 38.87 mg, 734.22 mg and 802.79 mg/egg yolk, for the OP360 group. The percentage of DHA incorporation into the egg yolk decreased linearly as the DHA levels increased into the diet. Thus, for the OP and AM sources, mean values of 85.11% (OP120) and 65.28% (AM120) decreased to 49.45% (OP360) and 34.06% (AM420). Significant improvement (P<0.05) was found in the ratio n-6/n-3, ranging from17.50 (CON) to 3.72 (OP320) and 6.36 (AM420). The sensorial evaluation of eggs did not detect any different flavor or odor in eggs among treatments or sources, except for the qualitative analysis of group OP360 which showed fish flavor, significantly different (P<0.05) from the control group.

Les lipases, protéines ubiquitaires, sont les enzymes les plus étudiées et les plus utilisées dans l’industrie. Elles catalysent un très grand nombre de réactions, d’hydrolyse et de synthèse, conduisant à une grande diversité de molécules, acides, esters, amides…. Les domaines d’applications sont nombreux : les bio-énergies, les arômes, bio-lubrifiants, bio-plastifiants, émulsifiants, produits phytosanitaires et détergents, cosmétiques, synthons pour la chimie fine, produits pharmaceutiques… Aujourd’hui, grâce aux outils génétiques, il est possible de modifier leur activité, spécificité et thermostabilité pour les adapter idéalement aux contraintes industrielles. Dans ce travail de doctorat, nous nous sommes intéressés à quatre lipases d’intérêt industriel. Les 3 premières appartiennent à la famille des lipases de Candida rugosa (Lip1, Lip3 et Lip4). Bien que très homologues, leurs spécificités sont très différentes. Elles se distinguent de toutes les autres lipases par un site actif composé d’un long tunnel avec la triade catalytique à l’entrée de celui-ci. Cela en fait une enzyme particulièrement intéressante pour la conversion et la purification d’acides gras à longue chaîne. La quatrième est une nouvelle lipase identifiée chez la levure oléagineuse, Yarrowia lipolytica. Elle est très active sur les acides gras à longue chaîne, active à pH acide et présentant une grande énantiosélectivité sur des molécules d’intérêt pharmaceutique, les esters d’acide 2- halogéno-aryl acide acétique. Dans un premier temps, un nouveau système d’expression, une souche spécifique de Yarrowia lipolytica, a été étudié pour l’expression de variants construits par mutagenèse dirigée. Cette souche JMY1212 permet une intégration ciblée dans le génome de Y. lipoytica. Nous avons démontré qu’il s’agissait du premier système d’expression permettant de comparer statistiquement l’activité de variants directement à partir du surnageant de culture. Trois des lipases de Candida rugosa ont été clonées avec succès dans cette souche et leurs activités et spécificités vis-à-vis de la longueur de chaines des acides gras ont été étudiées. Lip1 et Lip3 présentent une spécificité pour les acides gras à longueur de chaine moyenne (C8-C10) alors que Lip4 préfère les C18:1. De, plus, pour la première fois, la purification, à partir d’un mélange d’esters éthyliques issu d’huile de poissons, d’acides gras poly-insaturés (PUFAs); acides cis-5, 8, 11, 14, 17-eicosapentaenoic (EPA) et cis-4, 7, 10, 13, 16, 19-docosahexaenoic (DHA), molécules bonnes pour la santé, a été réalisée avec les trois lipases séparées de C. rugosa. Quelle que soit l’enzyme, le rendement de récupération du DHA est supérieur à 93 % (97, 100 et 93 % pour Lip1, Lip3 et Lip4 respectivement. Une pureté maximale en DHA de ~60 % a été obtenue avec Lip3 et Lip4, à partir d’un mélange initial d’esters éthyliques contenant 25% de DHA. Une différence remarquable entre ces trois enzymes est que Lip4 est capable de mieux hydrolyser l’ester d’EPA (60% contre 14 et 16% pour Lip1 et Lip3). Lip4 est même capable d’hydrolyser le DHA (7% contre 3 et 0 % pour Lip1 et Lip3). La deuxième partie de ce travail a été consacrée à l’amélioration de l’énantiosélectivité des deux enzymes étudiées vis-à-vis de synthons d’intérêt dans l’industrie pharmaceutique, les esters de 2-bromo aryl acide acétique. La construction raisonnée d’un double variant de la lipase Lip2 de Y. lipolytica, D97AV232F, a permis d’obtenir une enzyme totalement énantiosélective (E >200). Celle-ci reconnaît l’énantiomère R alors que la lipase sauvage avait une faible préférence pour l’énantiomère S (E=5). Par ailleurs, cette exceptionnelle augmentation de l’énantiosélectivité s’accompagne d’une amélioration de l’activité de l’enzyme qui est ainsi multipliée par 4,5. Sur ce même mélange d’énantiomères, les 3 lipases de C. rugosa se sont avérées remarquables. Malgré leur grande homologie, leur spécificité est différente. Lip1 et Lip3 sont totalement S spécifiques (E>200), alors que Lip4 est R spécifique (E=15). Le docking moléculaire des énantiomères S et R dans le site actif des lipases Lip1 et Lip4 a permis de mieux comprendre ces différences de spécificité et de proposer des cibles de mutagenèse dirigée. L’encombrement et la nature de l’acide aminé présent en position 296 sont cruciaux pour la discrimination de l’enzyme / Lipases, ubiquitous proteins, are the most studied enzymes and the most used in industry. They catalyse a great number of reactions, hydrolysis and synthesis, leading to a great diversity of molecules, acids, esters, amides. There are numerous fields of applications: bio-energies, flavours, bio-lubricants, bio-plasticizers, emulsifiers, detergents, cosmetics, synthons for fine chemistry, and pharmaceutical products. Nowadays, thanks to genetic tools, it is possible to modify their activity, specificity and thermostability to ideally adapt enzymes for the industrial constraints. In this work, we were interested in four lipases of industrial interest. The third ones belong to the lipase family of Candida rugosa (Lip1, Lip3 and Lip4). Although they present high homology, their specificities are very different. They are distinct from the other lipases by the active site composed of a long tunnel with the catalytic triad at the entry of the tunnel. It leads to enzymes particularly interesting for the conversion and the purification of long chain fatty-acids. The fourth one is a new lipase identified from oleaginous yeast, Yarrowia lipolytica. It is one of the most active lipase on long chain fatty-acids; it is very active and stable at acid pH and presents a high enantioselectivity on molecules of pharmaceutical interest, the esters of 2- halogeno-aryl acetic acid. In this work, we first tested a new expression system, a specific strain of Y. lipolytica, for expression of variants obtained by site-directed mutagenesis. This strain JMY1212 enables integration to be targeted to a special locus of the Y. lipoytica genome. We demonstrated that it is the first expression system in which it is possible to compare statistically variant activities directly from the supernatant of the culture. Secondly, three lipases of C. rugosa were cloned successfully in this strain and their activities and specificities with respect to fatty acid chain lengths were studied. Lip1 and Lip3 have specificity for the fattyacids of medium chain (C8-C10) whereas Lip4 prefers C18: 1. Moreover, for the first time, purification, from a mixture of ethyl esters issued from fish oil, polyunsaturated fatty acids (PUFAs); cis-5, 8, 11, 14, 17- eicosapentaenoic acid (EPA) and cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (DHA), molecules with health benefits, was realised with the three C. rugosa lipases, separately. Whatever the enzyme the recovery of DHA is superior to 90 % (97, 100 and 93 % for Lip1, Lip3 and Lip4 respectively. The maximal DHA purity ~60 % was obtained with Lip3 and Lip4, with an initial ethyl ester mixture containing 25% DHA. A remarkable difference between these enzymes lies in the fact that Lip4 is able to better hydrolyse the EPA esters (60% against 13% and 16% respectively for Lip1 and Lip3). Lip4 is also able to hydrolyse DHA (7% against 3 and 0 % for Lip1 and Lip3 respectively). The third part of this work was devoted to the improvement of the enantioselectivity of the two enzymes studied with respect to the resolution of a racemic mixture of pharmaceutical industry, the R, S esters of 2-bromo aryl acetic acid. The rational construction of a double variant of Lip2 lipase from Y. lipolytica, D97A V232F was realized to obtain a total enantioselective enzyme (E > 200). This variant recognizes the enantiomer R whereas wild-type lipase had a weak preference for the enantiomer S (E=5). In addition, this exceptional increase in the enantioselectivity is accompanied by a 4.5 fold improvement of the activity. With the same mixture of enantiomers, the 3 lipases of C. rugosa proved to be remarkable from the point of view of enantioselectivity. In spite of their high homology, their specificity is different. Lip1 and Lip3 are completely specific for the S enantiomer, whereas Lip4 is R specific (E=15). The molecular docking of the S and R enantiomers in the active site of Lip1 and Lip4 lipases enables the observed differences in specificity to be better understood and targets for site-directed mutagenesis to be proposed. We demonstrated that the nature of the amino acid present in position 296 is crucial for the discrimination of these enzymes

Les acides gras polyinsaturés oméga-3 à longue chaîne (AGPIω3-LC), principalement représentés par l'acide eicosapentaénoïque (EPA) et l'acide docosahexaénoïque (DHA), présentent des effets bénéfiques vis-à-vis de l'athérosclérose. Leur action au niveau vasculaire est suggérée. Les mécanismes d'action sont mal compris du fait de la complexité d'action des AGPIω3-LC au niveau cellulaire. Les AGPIω3-LC affectent de nombreuses protéines y compris les facteurs de transcription, ce qui engendre la modulation de l'expression de nombreux gènes. La complexité s'accroît si l'on considère l'ensemble des métabolites oxygénés issus des AGPIω3-LC. Les métabolites issus de la peroxydation lipidique peuvent être produits en abondance au cours de l'athérogenèse mais leur bioactivité est quasiment inconnue. Des points majeurs restent à éclaircir comme l'identification des cibles cellulaires et moléculaires au niveau vasculaire et la bioactivité des métabolites peroxydés. Objectifs : étudier l'impact du DHA au niveau vasculaire et évaluer la bioactivité de certains métabolites issus de la peroxydation lipidique du DHA. Les travaux visant à étudier les propriétés athéro-protectrices du DHA n'ont jusqu'à présent pas permis d'obtenir une vision globale de son spectre d'action. La nutrigénomique a paru être une approche pertinente pour étudier les effets du DHA au niveau vasculaire. Une étude in vivo a été réalisée sur des souris athérosclérotiques LDLR-/- recevant ou non une supplémentation en DHA (2% de l'apport énergétique journalier). Le rôle athéro-protecteur du DHA a été confirmé dans une étude précédente rapportant une diminution de 35% de l'étendue des lésions au niveau aortique dans le groupe DHA. Au niveau protéique, nous avons montré une altération significative de l'expression de protéines intervenant principalement au niveau des métabolismes glucidique et lipidique mais aussi dans les défenses anti-oxydantes. Au niveau génique, la composante inflammatoire s'est avérée être une cible majeure du DHA au niveau vasculaire. La supplémentation en DHA a réduit l'expression de gènes impliqués dans l'adhésion cellulaire, la chimiotaxie et la présentation de l'antigène. Plusieurs gènes se sont avérés être des marqueurs phénotypiques des macrophages et l'analyse des régulateurs transcriptionnels montrait une implication probable de PPARγ, IFNγ et NFκB dans les modulations d'expression génique observées. Nos analyses suggèrent une orientation préférentielle des macrophages vers un phénotype réparateur de type M2 chez les souris recevant la supplémentation en DHA. Une analyse immunohistochimique au niveau aortique a révélé une plus grande abondance d'arginase I. Une approche plus ciblée alliant une étude in vitro sur un modèle de macrophages humains et l'utilisation de métabolites peroxydés spécifiques du DHA, les neuroprostanes, nous a permis de conforter notre hypothèse selon laquelle le DHA pourrait agir au moins en partie via ses métabolites peroxydés. L'exposition des macrophages à deux types de neuroprostanes (les 4-F4t et les 14-A4-Neuroprostanes) a permis de réduire significativement l'expression et la sécrétion de plusieurs médiateurs pro-inflammatoires. Des résultats préliminaires suggèrent que ces effets anti-inflammatoires des neuroprostanes seraient indépendants de PPARγ mais liés à une inhibition de la voie NFκB. L'étude in vivo et l'utilisation de la nutrigénomique nous ont permis d'explorer de façon ouverte et non biaisée l'impact du DHA au niveau vasculaire. Les résultats sont que le DHA active le métabolisme énergétique et les défenses anti-oxydantes et diminue l'état inflammatoire avec une implication probable du DHA dans l'orientation phénotypique des macrophages vers un phénotype réparateur de type M2. Les résultats obtenus in vitro sur macrophages primaires humains confirment que les métabolites peroxydés du DHA contribuent à ses propriétés anti-inflammatoires. / Numerous studies have reported beneficial effects of long chain omega-3 polyunsaturated fatty acids (LC-ω3PUFAs) on atherosclerosis and associated cardiovascular events. These fatty acids are mainly represented by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). LC-ω3PUFAs exert their athero-protective action mainly by a reduction of triglyceridemia, increased endothelial relaxation, and reduced inflammation. Their specific action at the vascular level is suggested. Their mechanisms of action are still only partially understood because of the complexity of action of LC-ω3PUFAs at the cellular level. LC-ω3PUFAs affect several membrane or cytosolic proteins, including transcription factors, which modulate numerous gene expression. The complexity is further increased when considering a wide range of oxygenated metabolites derived from LC-ω3PUFAs. Among them, the metabolites from lipid peroxidation may be produced in abundance during atherogenesis but their bioactivity is almost unknown. There are still major issues to be clarified, such as the identification of cellular and molecular targets at the vascular level ; the bioactivity of peroxidized metabolites that could play a key role in the prevention of atherosclerosis. The objective of this work was to study the impact of DHA at the vascular level and to evaluate the bioactivity of selected metabolites from DHA lipid peroxidation. Studies on anti-atherogenic properties of DHA have so far been carried out only in a targeted manner that does not provide a comprehensive and integrated view of its spectrum of action. Nutrigenomics appears to be a relevant approach to study the effects of DHA at the vascular level. In vivo study was performed on LDLR-/- atherosclerotic mice supplemented or not with DHA. The athero-protective effect of DHA has been confirmed in the previous study performed by our team reporting a 35% decrease of the atherosclerotic lesion in the DHA group. Aorta proteome study demonstrated a significant alteration of the expression of proteins involved mainly in the carbohydrate and lipid metabolism and also in antioxidant defenses. At the transcriptome level, the inflammatory component of atherosclerosis appears to be a major target of DHA at the vascular level. More precisely, DHA supplementation reduced the expression of genes involved in cell adhesion, chemotaxis and antigen presentation. Several genes were found to be phenotypic markers of macrophages and analysis of transcriptional regulators showed a possible contribution of PPARγ, IFNγ and NFκB to the observed modulation of gene expression. Our results suggest a preferential orientation of macrophages to a M2 type repair phenotype in mice receiving DHA supplementation. This was confirmed by immunohistochemical analysis at the aorta which revealed a greater abundance of arginase I. We chose a focused in vitro study of the action of specific DHA peroxidized metabolites: the neuroprostanes on a human macrophages. This study allowed us to confirm our hypothesis that DHA may act at least in part via its peroxidized metabolites. The macrophages exposition to 2 types of neuroprostanes (4-F4t and 14-A4-Neuroprostanes) has significantly reduced the expression and secretion of several pro-inflammatory mediators. Preliminary results suggest that these anti-inflammatory effects are PPARγ-independent but related to inhibition of NFκB pathway. In vivo study, and nutrigenomics approach, allowed us to explore the impact of DHA at the vascular level by an open and unbiased way. The results are that DHA activates energy metabolism and antioxidant defenses and reduces the inflammatory component of atherosclerosis with a possible involvement of DHA in the phenotypic direction of macrophages to a M2 type repair phenotype. The results obtained in vitro on primary human macrophages confirmed that DHA peroxidized metabolites contribute to its anti-inflammatory properties.

O diabetes mellitus tipo I (DMI) é uma doença crônica autoimune caracterizada por hiperglicemia devido à destruição das células beta pancreáticas produtoras de insulina. Ao final de 30 dias da indução do diabetes por estreptozotocina, os macrófagos peritoneais residentes dos animais diabéticos apresentaram aumento de RNAm de citocinas e quimiocinas inflamatórias, secreção de óxido nítrico, expressão de NLRP3, iNOS e PARP1 e da atividade da via glicolítica. Perfil pró-inflamatório também foi observado em macrófagos peritoneais de animais NOD (non-obese diabetic). Camundongos diabéticos deficientes em NLRP3 (NLRP3 KO) apresentaram diminuição na expressão de iNOS, PARP1 e na produção de NO em relação aos macrófagos dos animais diabéticos selvagens. O estado diabético tipo I influenciou o perfil dos macrófagos peritoneais residentes, causando aumento na produção de NO, via NLRP3-PARP1-iNOS, expressão de citocinas pró-inflamatórias, receptores de quimiocinas e da atividade glicolítica. O tratamento com DHA (ômega-3) ex-vivo reverteu este perfil e atenuou o quadro pró-inflamatório por diminuição da produção de NO e da expressão de citocinas pró-inflamatórias. / Type I diabetes mellitus (DMI) is a chronic autoimmune disease characterized by hyperglycemia due to the destruction of insulin-producing pancreatic beta cells. At the end of 30 days after type I diabetes induced by streptozotocin, macrophages from diabetic animals had increased expressions of inflammatory cytokines and chemokines, secretion of nitric oxide, expression of NLRP3, iNOS and PARP1, and glycolytic activity compared to the cells from control animals. Proinflammatory features was also observed in peritoneal macrophages of NOD (non-obese diabetic) animals. Macrophages from NLRP3 deficient diabetic mice (NLRP3 KO) had decreased expression of iNOS, PARP1 and of NO production when compared to cells from wild type animals. The type I diabetic state led to a proinflammatory feature in resident peritoneal macrophages by increasing NO production, via the NLRP3-PARP1-iNOS pathway, expressions of proinflammatory cytokines, chemokine receptors and glycolytic activity. In contrast, ex-vivo treatment with DHA (omega-3) reversed this profile and attenuated the proinflammatory state by reducing NO production and expression of proinflammatory cytokines.

La surveillance immunologique des tumeurs repose sur la capacité des cellules effectrices du système immunitaire à détecter et à éliminer les cellules cancéreuses. Nonobstant ce constat, la régression complète et spontanée de cancers établis n’est observée que dans de très rares cas. L’échec de la résolution des cancers par le système immunitaire pourrait résulter de la conjonction de plusieurs facteurs : i) une réponse immune inadéquate liée au manque d’immunogénicité des tumeurs, ii) l’incompétence du système immunitaire consécutif à des immunodéficiences acquises ou induites et iii) la sélection de variants tumoraux résistants capables de déjouer la surveillance opérée par le système immunitaire ou de subvertir ses effets. Ainsi, le développement de stratégies visant à potentialiser les réponses antitumorales de l’hôte revêt un enjeu crucial en cancérologie.Au laboratoire, notre travail de recherche a pour objectif de mieux caractériser les liens entre réponse immunitaire et cancer. Mon travail de thèse vise précisément à comprendre les mécanismes moléculaires impliqués dans la différenciation des lymphocytes T CD4 et à déterminer le rôle de ces cellules dans l’immunité antitumorale. Au cours de ma thèse, nous nous sommes particulièrement attachés à explorer les mécanismes moléculaires qui sous tendent la différenciation des populations lymphocytaires Th17, Th9 et TFh pour mieux appréhender et moduler leurs fonctions effectrices afin d’optimiser les réponses antitumorales. Ces travaux s’inscrivent dans une démarche d’application potentielle en immunothérapie anticancéreuse, un domaine de recherches qui connaît actuellement des avancées spectaculaires.Nous avons tout d’abord étudié l’influence de l’acide docosahexaénoïque (DHA), un acide gras à longue chaîne de la série n 3, sur la différenciation des cellules Th17. Nous avons mis en évidence le mécanisme moléculaire responsable de l’inhibition directe de la polarisation cellulaire Th17 par le DHA. L’activation de PPARγ par le DHA induit l’expression de SOCS3 qui agit comme un répresseur intrinsèque de la différenciation Th17. Dans deux modèles de cancers murins, nous avons également montré que l’activité anticancéreuse du DHA était dépendante de sa capacité à inhiber la sécrétion d’IL 17 par les cellules T CD4 in vivo. Nous avons ainsi caractérisé l’un des mécanismes impliqués dans l’effet anticancéreux du DHA.Dans un deuxième travail, nous avons caractérisé les effets de l’interleukine 1β sur le programme moléculaire des cellules Th9. Nous avons montré que les cellules Th9 différenciées en présence d’IL-1β possédaient de puissantes propriétés anticancéreuses dépendantes de l’IL-21 reposant sur l’activation du facteur de transcription IRF1. Au niveau moléculaire, nous avons démontré que l’IL-1β induisait la phosphorylation de STAT1 elle même responsable de l’activation d’IRF1 qui est alors capable d’interagir sur les promoteurs de l’Il9 et de l’Il21 pour induire l’expression de ces gènes dans les cellules Th9.Le dernier projet porte sur la régulation transcriptionnelle du facteur de transcription IRF1 sur la réponse T folliculaire auxiliaire et cherche à en évaluer les retombées potentielles en immunothérapie anticancéreuse. Notre étude met en évidence l’activation précoce d’IRF1 dans la différenciation TFh et suggère que ce facteur de transcription semble initier le développement de ces cellules. Des approches de transfert adoptif révèlent que les TFh semblent posséder des propriétés anticancéreuses capables de limiter efficacement la croissance des tumeurs dans des modèles murins. Enfin, après caractérisation phénotypique nous montrons que les cellules TFh sont présentes dans des tumeurs mammaires chez l’Homme et validons la présence d’IRF1 dans ces lymphocytes. / Immune surveillance of tumors is based on the ability of effector cells of the immune system to detect and eliminate the cancer cells. Notwithstanding, the complete and spontaneous regression of established cancers was observed only in very few cases. The failure of cancer resolution by the immune system could result from the combination of several factors: i) inadequate immune response related to a low tumor immunogenicity, ii) incompetent immune system consecutively to induced or acquired immunodeficiencies and iii) the selection of resistant tumor variants able to thwart immune surveillance or subverting immune responses. Developing novel cancer immunotherapy strategies leading to potentiation of the host antitumor responses is thus a key challenge in oncology.We aim to better characterize the relationships between immune response and cancer. My work is precisely to understand the molecular mechanisms involved in CD4 T cell differentiation and to determine the role of these cells in antitumor immunity. I am particularly committed to explore the molecular mechanisms underlying the Th17, Th9 and TFh cell differentiations. The goal is to better understand and adjust their effector functions to optimize antitumor responses. This work is part of a potential application in cancer immunotherapy approach, an area that is experiencing dramatic advances and is likely to grow in the years ahead.We first studied the influence of the n 3 polyunsaturated fatty acid docosahexaenoic acid (DHA) on Th17 cell differentiation. We unraveled the molecular mechanism responsible for the direct inhibition of Th17 cell polarization by DHA, explaining one way of DHA to exert its anticancer activity. TH17 cells induced in vitro displayed increased SOCS3 expression and diminished capacity to produce interleukin 17 following activation of PPARγ by DHA. In two different mouse cancer models, DHA prevented tumor outgrowth and angiogenesis in an IL 17 dependent manner. Altogether, our results uncover a novel molecular pathway by which PPARγ induced SOCS3 expression prevents IL 17 mediated cancer growth.Then, we characterized the effects of interleukin 1β (IL-1β) on Th9 cells molecular program. We found that the transcription factor IRF1 enhanced the effector functions of Th9 cells and dictated their anticancer properties. Under Th9 skewing conditions, IL-1β induced phosphorylation of the transcription factor STAT1 and subsequent expression of IRF1, which bound to Il9 and Il21 gene promoters and enhanced their secretion by Th9 cells. In addition, IL-1β induced Th9 cells exerted potent anticancer functions in an IRF1 and IL 21 dependent manner. Thus, our findings identify IRF1 as a target for controlling the function of Th9 cells.We are currently investigating the transcriptional regulation of IRF1 on follicular helper CD4 T (TFh) cell program. We address the question whether TFh cells could be beneficial in cancer immunotherapy. Our study highlights the early activation of IRF1 during the TFh cell polarization and suggests that IRF1 appears to initiate the development of these cells. Adoptive transfer approaches show that TFh lymphocytes seem to habor anticancer properties by limiting efficiently tumor outgrowth in mouse models of cancer. Finally, phenotypic characterization of TFh cells points out that they infiltrate human breast tumors and express IRF1.

Submitted by Michele Mologni (mologni@unoeste.br) on 2018-08-17T13:30:36Z No. of bitstreams: 1 Danielle Alves Silva.pdf: 1187936 bytes, checksum: 4436228a6d8425a8ba13dd03636f218a (MD5) / Made available in DSpace on 2018-08-17T13:30:36Z (GMT). No. of bitstreams: 1 Danielle Alves Silva.pdf: 1187936 bytes, checksum: 4436228a6d8425a8ba13dd03636f218a (MD5) Previous issue date: 2018-03-21 / The objective of this study was to compare the efficacy of two omega 3 oral formulations with different ratios of EPA, DHA and antioxidants, as an adjuvant in the treatment of dogs affected by KCS and to evaluate by fractal analysis the palpebral conjunctiva of the treated dogs. Forty-five dogs with KCS were evaluated monthly for 6 months by the Schirmer Tear Test (TLS), Fluorescein Test (TF), Tear Film Break-up Time (TBUT), Lissamine Green Test (LGT), cytology of the conjunctiva at the beginning, middle and end of the study, biopsy and fractal analysis of the conjunctiva at the beginning and end of the study. The dogs were randomly assigned into 3 groups (n = 15): T group (tacrolimus 0.03% topical), TO group (tacrolimus + omegas EPA/DHA) and TOA group (tacrolimus + omegas EPA/DHA + Antioxidants). The result demonstrated that there was a significant improvement in clinical signs in both groups. In TBUT, all groups presented increase during the treatment, and the TO group presented the best result at all times when compared to the other groups. At the end of the experiment, the groups T, TO and TOA presented cytological analysis, reduction of lymphocytes, neutrophils, metaplastic and squamous cells, and histopathological analysis, reduction of lymphocytes and neutrophils and increase of goblet cells highlighting the best performance at TO. In the fractal analysis, at the end of the experiment, the TO group presented the best result and the values close to the values found in the fractals of healthy eyes. The difference between the TO group and the other groups is in the higher EPA concentration, being a natural anti-inflammatory, which may be one of the causes of its better performance. We concluded that oral omega 3, which contains a higher proportion of EPA than DHA, has shown greater benefit in terms of the improvement of clinical signs and the inflammatory process in the treatment of KCS in dogs. / O objetivo deste estudo foi comparar a eficácia de duas formulações de ômega 3 por via oral, com diferentes proporções de EPA, DHA e antioxidantes, como adjuvante no tratamento de cães acometidos por CCS e avaliar por meio da análise fractal a conjuntiva palpebral dos cães tratados. Quarenta e cinco cães atendidos no HV da UNOESTE portadores de CCS foram avaliados mensalmente por 6 meses pelo Teste Lacrimal de Schirmer (TLS), Teste de Fluoresceína (TF), Tempo de Ruptura do Filme Lacrimal (TRFL), Teste de Rosa Bengala, citologia da conjuntiva no início, meio e fim do projeto, biopsia e análise fractal da conjuntiva no início e final do projeto. Os cães foram distribuídos aleatoriamente em 3 grupos (n=15): grupo T (tacrolimus 0.03% tópico), grupo TO (tacrolimus + ômegas EPA/DHA oral) e grupo TOA (tacrolimus + ômegas EPA/DHA + antioxidantes oral). Os resultados demonstraram que houve uma melhora significativa nos sinais clínicos em ambos os grupos. No TRFL todos os grupos apresentaram aumento no decorrer do tratamento, sendo que o grupo TO foi o que apresentou melhor resultado em todos momentos quando comparado aos demais grupos. Ao final do experimento, os grupos T, TO e TOA apresentaram na análise citológica, diminuição de linfócitos, neutrófilos, células metaplásicas e escamosas, e na análise histopatológica, diminuição de linfócitos e neutrófilos e aumento das células caliciformes, ressaltando o melhor desempenho ao TO. Na análise fractal, ao final do experimento, o grupo TO que apresentou melhor resultado e os valores próximos aos valores encontrados nos parâmetros fractais de olhos sadios. A diferença entre o grupo TO e os demais grupos está na concentração de EPA maior, sendo um anti-inflamatório natural, o que pode ser uma das causas de seu melhor desempenho. Concluímos que o ômega 3 oral que contém maior proporção de EPA do que DHA trouxe maior benefício quanto a melhora dos sinais clínicos e do processo inflamatório no tratamento de CCS em cães.

Essential fatty acids are necessary for growth, survival, and development of larval fishes, but there is limited information on the essential fatty acid requirements of larval southern flounder (Paralichthys lethostigma). The objectives of this study were to elucidate connections between dietary supply of docosahexaenoic acid (DHA) and arachidonic acid (ARA) and deposited fatty acids in the head or body, and then link diet and stored fatty acids in the head or body with larval quality traits. From 4-15 days posthatch (dph), southern flounder larvae were fed rotifers enriched with four different combinations of DHA-rich Algamac 3050 and ARA-rich Algamac ARA. Fatty acid concentrations in the head and body were measured at 15 dph, and relationships between fatty acids in head or body and in the diet were determined. Larval quality traits, including specific growth rate (SGR), survival, and eight behavioral performance variables were measured. Results showed that concentrations of DHA and ARA in the head and in the body were correlated with concentrations of DHA and ARA in the diet. Growth rate did not vary among the four diets, but survival was positively correlated with the amount of lipid in the diet. Responsiveness to a visual stimulus was positively correlated with the concentration of DHA in the diet, the ratio DHA:EPA in the head, and total energy content of the diet. Turning rate during routine swimming was correlated with body DHA. This study demonstrates the influence of DHA content, total lipid content, and energy levels in the diet of southern flounder and provides a foundation for future studies examining causal factors of recruitment variability or larviculture production success. / text

Thirty-five volunteers were randomized and twenty-nine completed the study. Mean plasma and red blood cell (RBC) total DHA concentrations, which were analyzed among all participants as a measure of adherence, increased significantly in the DHA-enriched treatment compared to control oil-control flour. The plasma and RBC n-6: n-3 ratio was reduced after consumption of HOCODHA-control flour compared to control oil- control flour. The present study failed to see differences in body composition with the HOCODHA-barley flour treatment versus control oil-control flour treatment. In conclusion, significant increases in plasma EPA and DHA levels, as well as the omega-3 index, provide evidence supporting the cardioprotective effects of HOCODHA. The present study demonstrated that in the context of current Western macronutrient intakes, altering the dietary fatty acid composition and adding β-glucan had no major effect on body composition during the 28 days controlled dietary intervention. / February 2017

A inseminação artificial é utilizada como uma importante ferramenta para o melhoramento reprodutivo, tanto com sêmen resfriado ou congelado. O primeiro experimento teve como objetivo identificar um tampão de pH para resfriamento de sêmen de pôneis por 48 horas a 5°C e 15°C. O efeito de cinco tampões (TES (ácido N-tris (hidroximetil) metil-2-aminoetanosulfônico), PIPES (ácido piperaxín-N,N'-bis(2-etanosulfônico)), BES (ácido N,N-Bis(2-18hidroxietil)-2-aminoetanosulfônico), MES (ácido 4-morfolinoetanosulfônico) e HEPES (ácido N-2-hidroxietilpiperazina-N’-2-etanosulfônico)) foi avaliado em um diluente composto de leite em pó desnatado e glicose. Os testes do diluente com os tampões BES, MES e TES foram conduzidos com o sêmen fresco de oito pôneis e no sêmen resfriado por 48 h a 15°C. Uma amostra de cada grupo foi utilizada para análise da motilidade, pH, osmolaridade, teste hiposmótico, atividade mitocondrial (MTT) e integridade de membrana através das sondas fluorescentes. O pH e a osmolaridade dos diluentes sem o sêmen também foram avaliados. Os dados foram analisados por análise de variância e pelo teste de Tukey, quando P < 0.05 foi significante. A osmolaridade do sêmen diluído não variou entre diluentes e foi para o BES 350.91 ± 11.24 mOsm, MES 350.41 ± 11.76 mOsm e TES 350 ± 12.96 mOsm. O pH do sêmen após diluição nos respectivos diluentes variou entre as horas avaliadas (P < 0.05) e não variou com o passar do tempo o que evidencia o efeito tamponante dos diluentes. A osmolaridade dos diluentes foi similar entre os tampões BES (366 ± 5.47 mOsm), MES (370 ± 6.12 mOsm) ou TES (371 ± 5.47 mOsm) a fresco e sem adição de sêmen. Já o pH variou (P < 0.05) de acordo com o tampão. No experimento a 5°C ocorreu decréscimo na porcentagem de motilidade total, progressiva e local do sêmen resfriado após 24 h e 48 h comparado a avaliação a fresco em todos os grupos. No entanto, a porcentagem de espermatozoides móveis a fresco, 24 h e 48 h a 5°C entre tratamentos foi similar. A porcentagem de espermatozoides reativos ao teste hiposmótico, e com atividade mitocondrial não diferiu entre tratamentos. No experimento a 15°C o vigor e motilidade total, progressiva e local foram similares entre os tampões BES, MES e TES em cada período avaliado. A osmolaridade do sêmen diluído não variou entre diluentes e foi para o BES 360.93 ± 10.52 mOsm, MES 361.47 ± 6.79 mOsm e TES 361.56 ± 7.68 mOsm. Devido as características individuais de cada tampão o pH do sêmen diluído variou entre os diluentes com os tampões utilizados. A porcentagem de espermatozoides reativos ao teste hiposmótico, com atividade mitocondrial e membrana intacta observada com CFDA e PI não diferiu entre tratamentos. Evidenciou-se que tanto BES, MES ou TES podem ser utilizados no armazenamento e transporte do sêmen de pôneis durante 48 h a 5°C ou 15°C. O segundo experimento teve como objetivo foi avaliar a influência da suplementação de ácidos graxos poli-insaturados sobre a qualidade de sêmen de pôneis da raça Brasileira a fresco e após congelamento. Oito pôneis receberam sua dieta convencional não suplementada (grupo controle) ou dieta convencional e 70 g de farinha de alga Schizochytrium sp rica em DHA (grupo PUFA). O efeito da Vitamina E (1 mM, DL-α-tocoferol), adicionada ao diluente de congelamento para sêmen também foi avaliado sobre a qualidade seminal, antes e após o congelamento do sêmen. O sêmen foi coletado a cada 15 dias durante 60 dias. Os garanhões tratados passaram a controle e vice-versa após um intervalo de sessenta dias. O sêmen foi avaliado a fresco e após o congelamento. Motilidade e vigor foram avaliados a fresco. Após o descongelamento motilidade, funcionalidade de membrana, integridade de membrana e análise computadorizada da motilidade foram avaliados. Os valores médios para os parâmetros avaliados a fresco no grupo PUFA e controle foram similares. Os valores médios da motilidade total, progressiva e local, hiposmótico, CFDA/PI e análise computadorizada de sêmen suplementados com DHA e o grupo controle pós-descongelamento não diferiram. A associação da suplementação de DHA e Vitamina E adicionada ao diluente não potencializou a capacidade antioxidante do diluente durante o congelamento. A associação da suplementação de DHA e Vitamina E adicionada ao diluente resultou em diminuição da motilidade total e progressiva comparada ao grupo não suplementado (controle). A suplementação de 70 g de farinha de alga Schizochytrium sp rica em DHA e ou a inclusão de 1 mM de vitamina E ao diluente de congelamento de sêmen em pôneis da raça Brasileira não foi eficiente para promover melhoria na viabilidade seminal após a coleta ou mesmo após o congelamento. / Artificial insemination with cooled and frozen semen is an important tool for breeding industry. The first experiment aimed to identify a pH buffer for cooling semen of ponies for 48 h at 5°C and 15°C. The effect of five buffers (TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid), PIPES (piperaxin-N, N'-bis (2-ethanesulfonic acid)), BES (N, N-Bis (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)) was evaluated in a extender composed of skimm milk powder and glucose. Analysis with the BES, MES and TES buffers were performed in fresh semen of eight ponies and in cooled semen for 48 h at 15°C. A sample from each group was used for analysis of motility, pH, osmolarity, hyposmotic test, mitochondrial activity (MTT) and membrane integrity through fluorescent probes. The pH and osmolarity of the extenders without semen were also evaluated. Data were assessed by analysis of variance and Tukey's test, when P <0.05 was considered significant. Osmolarity of the diluted semen did not differ between extenders and was for BES 350.91 ± 11.24 mOsm, MES 350.41 ± 11.76 mOsm and TES 350 ± 12.96 mOsm. The pH of the semen after dilution in the respective extenders varied between the hours (P <0.05) and did not change over time as evidenced by the buffering effect of the extenders. Extender osmolarity was similar between BES (366 ± 5.47 mOsm), MES (370 ± 6.12 mOsm) or TES (371 ± 5.47 mOsm) buffers after dilution and without addition of semen. The pH varied (P <0.05) according to the buffer. In the experiment at 5°C all treatments showed a decrease in the percentage of total, progressive and local motility of the cooled semen after 24 h and 48 h compared to the fresh. However, in the percentage of motile sperm to fresh, 24 h and 48 h at 5°C between treatments was similar. Percentage of spermatozoa reactive to the hyposmotic test, and with mitochondrial activity did not differ between treatments. In the experiment at 15°C total, progressive and local vigor and motility were similar between BES, MES and TES buffers in each period. Osmolarity of the diluted semen did not differ between extenders and was for BES 360.93 ± 10.52 mOsm, MES 361.47 ± 6.79 mOsm and TES 361.56 ± 7.68 mOsm. Due to the individual characteristics of each buffer, diluted semen pH varied between the extenders with the buffers used. Percentage of spermatozoa reactive to the hyposmotic test, with mitochondrial activity and intact membrane observed with CFDA and PI did not differ between treatments. We concluded that BES, MES or TES can be used in the storage and transport of ponies semen for 48 h at 5°C or 15°C. The second experiment aimed to evaluate the influence of polyunsaturated fatty acid supplementation in the fresh and after freezing semen of Brazilian ponies. Eight ponies received their conventional diet (control group) or conventional diet and 70 g of DHA-rich Schizochytrium sp algae meal (PUFA group). The effect of Vitamin E (1 mM, DL-α-tocopherol) added to the freezing diluent for semen was also evaluated on seminal quality, before and after freezing. Semen was collected every 15 days during 60 days. Stallions were reversed in treatments after an interval of sixty days. Semen was evaluated fresh and after freezing. Motility and vigor were estimated in the fresh semen. After thawing motility, membrane functionality, membrane integrity and computed motility analysis were evaluated. Mean values for fresh parameters evaluated in the PUFA and control groups were similar. Mean values of total, progressive and local motility, hyposmotic, CFDA/PI and computerized analysis of semen supplemented with DHA and post-thaw control group did not differ. The combination of DHA and Vitamin E supplementation added to the diluent did not potentiate the antioxidant capacity of the diluent during freezing and resulted in decreased total and progressive motility compared to the non-supplemented group. Supplementation of 70 g of DHA-rich microalgae Schizochytrium sp and the addition of 1 mM vitamin E to the semen freezing diluent in Brazilian ponies was not efficient to promote improvement in seminal viability after collection or even after freezing.

Colon cancer is the second leading cause of cancer death in the United States. Epidemiological data indicate that the consumption of dietary fiber and fish/marine products favorably modulate colon tumorigenesis. Docosahexaenoic acid (DHA, 22:6n-3) from fish oil, and butyrate, a fiber fermentation product generated in colon, protect against colon tumorigenesis in part by inducing apoptosis. We have shown that DHA is incorporated into mitochondrial membrane phospholipids, which enhances oxidative stress and mitochondrial membrane potential (MP) dissipation. To elucidate the subcellular origin of oxidation induced by DHA and butyrate exposure, young adult mouse colonocytes (YAMC) were treated with 0200 ??M DHA, linoleic acid (LA, 18:2n-6) or no fatty acid (control) for 72 h with or without 5 mM butyrate for the final 6-24 h. Real time analysis of cellular membrane lipid oxidation, as indicated by oxidation of a lipophilic vital dye, mitochondrial permeability transition (MPT), as characterized by MP dissipation, and cytosolic ROS production, as depicted by hydrophilic ROS reactive fluorophore accumulation, were measured by living cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells showed a 29% increase in lipid oxidation (p<0.01), compared to no butyrate treatment, which could be blocked by a mitochondria targeted antioxidant, MitoQ (p <0.05), whereas LA treatment did not show an effect. In the absence of butyrate, DHA treatment, compared to LA, increased resting MP by 14% (p <0.01). In addition, butyrate-induced MP dissipation was greater (20%) in DHA primed cells as compared to LA (10%). This effect was blocked by pre-incubation with MPT inhibitors, cyclosporin A or bongkrekic acid at 1 ??M. These data suggest an increase in mitochondrial lipid oxidation and the resultant change in MP may contribute to the induction of apoptosis by DHA with butyrate as shown previously.