Introdução Os recém-nascidos de muito baixo peso (<1.500g) necessitam de aporte adequado de nutrientes para desenvolver-se satisfatoriamente. O leite humano é fundamental na sua alimentação, já que possui uma composição nutricional balanceada que inclui nutrientes essenciais, tais como os ácidos graxos poli-insaturados, incluindo os ácidos ômega 3 e 6, de extrema importância para o desenvolvimento do sistema nervoso central e retina. No entanto, apenas o leite materno pode não ser suficiente para suprir essas necessidades, necessitando, portanto, de suplementação. Atualmente, surgiu a possibilidade de adequação do leite humano acrescido de um concentrado com liofilizado do próprio leite humano, que foi desenvolvido neste trabalho. Objetivos determinar a quantidade de lipídios totais e o perfil lipídico do Leite Humano Baseline (LHB) e dos Concentrados com o liofilizado de Leite Humano em diferentes momentos (Leite Humano concentrado no período imediato - LHCI, Leite Humano concentrado no período de 3 meses - LHC3m e Leite Humano concentrado no período de 6 meses - LHC6m). Métodos Foram obtidas 50 amostras de leite humano de mulheres doadoras do Banco de Leite Humano da FMRP-USP que aceitaram participar do estudo. A quantificação de lipídeos ocorreu pelo Analisador de Leite Humano MIRIS® e o perfil de ácidos graxos foi determinado por CG-FID, SHIMADZU®. Resultados Houve uma concentração de lipídios totais no LHCI em relação ao LHB; a concentração (% em relação ao total de ácidos graxos) dos ácidos foi de: palmítico (C16:0) 22,30% LHB, 21,46% LHCI, 21,54% LHC3m e 21,95% LHC6m (p<0,01); oleico (C18:1n-9) 30,41% LHB, 30,47% LHCI, 30,55% LHC3m e 29,79% LHC6m (p=0,46); linoléico (C18:2 n-6) 19,62% LHB, 19,88% LHCI, 19,49% LHC3m e 19,45% LHC6m (p=0,58); araquidônico (C20:4 n-6) 0,35% LHB, 0,16% LHCI, 0,13% LHC3m e 0,15% LHC6m (p<0,01); ?- linolênico (C18:3 n-3) 1,32% LHB, 1,37% LHCI, 1,34% LHC3m e 1,34% LHC6m (p=0,14); docosahexaenóico (C22:6 n- 3) 0,10% LHB, 0,06% LHCI, 0,05% LHC3m e 0,06% LHC6m (p<0,01). Há modificações específicas em alguns ácidos graxos, no entanto a somatória de ácidos graxos por suas classes estruturais não apresentam diferenças. Conclusão Apesar das pequenas diferenças encontradas no perfil lipídico, o concentrado com leite humano liofilizado é um alimento que pode vir a atender as necessidades do recém-nascido, já que houve aumento dos lipídios totaise preservação dos nutrientes essenciais presentes apenas no leite materno, mas estudos clínicos ainda precisam ser feitos para avaliar a segurança e eficácia do concentrado como alimentação dos recém-nascidos de muito baixo peso. / Introduction Very low birth weight infants (<1,500 g) require adequate nutrient intake to develop satisfactorily. Human milk is essential in its diet, since it has a balanced nutritional composition that includes essential nutrients, such as polyunsaturated fatty acids, including omega 3 and 6 acids, of extreme importance for the development of the central nervous system and retina. However, only breast milk may not be sufficient to meet these needs, thus requiring supplementation. Nowadays, the possibility of adapting the human milk plus a concentrate with lyophilized from the human milk itself, which was developed in this work, has arisen. Objectives To determine the amount of total lipids and the lipid profile of Human Baseline Milk (LHB) and Concentrates with human milk lyophilized at different times (Human Milk Concentrated in the immediate period - LHCI, Concentrated Human Milk in the 3-month period - LHC3m and Human Milk Concentrated in the 6-month period - LHC6m). Methods Fifty samples of human milk from women donors of the Human Milk Bank of FMRP-USP who were accepted to participate in the study were obtained. Lipid quantification was performed by the MIRIS® Human Milk Analyzer and the fatty acid profile was determined by CG-FID, SHIMADZU®. Results There was a total lipid concentration in LHCI relative to LHB; the concentration (% of total fatty acids) of the acids was: palmitic (C16: 0) 22.30% LHB, 21.46% LHCI, 21.54% LHC3m and 21.95% LHC6m (p <0) , 01); oleic acid (C18: 1n-9) 30.41% LHB, 30.47% LHCl, 30.55% LHC3m and 29.79% LHC6m (p = 0.46); linoleic acid (C18: 2 n-6) 19.62% LHB, 19.88% LHCI, 19.49% LHC3m and 19.45% LHC6m (p = 0.58); arachidonic (C20: 4 n-6) 0.35% LHB, 0.16% LHCl, 0.13% LHC3m and 0.15% LHC6m (p <0.01); ?-linolenic acid (C18: 3 n-3) 1.32% LHB, 1.37% LHCI, 1.34% LHC3m and 1.34% LHC6m (p = 0.14); (C22: 6 n-3) 0.10% LHB, 0.06% LHCl, 0.05% LHC3m and 0.06% LHC6m (p <0.01). There are specific modifications in some fatty acids, however the sum of fatty acids by their structural classes do not present differences. Conclusion Despite the small differences found in the lipid profile, the concentrate with freeze-dried human milk is a food that can meet the needs of the newborn, since there was an increase in total lipids and preservation of essential nutrients present only in breast milk, butclinical studies still need to be done to evaluate the safety and efficacy of concentrate as a feed for very low birth weight infants.

Der globale Prävalenzanstieg allergischer Erkrankungen wird mit der westlichen Ernährung und einem sich ändernden Fettsäurespektrum assoziiert. Die omega-3 Fettsäure Docosahexaensäure (DHA) wurde bereits bei verschiedenen chronisch-entzündlichen Erkrankungen erfolgreich therapeutisch eingesetzt. Die dabei zugrunde liegenden Wirkmechanismen sind jedoch nicht vollständig geklärt. Deshalb wurde hier der molekulare Mechanismus der DHA-vermittelten Hemmung der IgE-Produktion in humanen B-Zellen sowie der verminderten Differenzierung IgE-produzierender Plasmazellen in vitro untersucht. Analysen der beteiligten Signaltransduktionswege offenbarten eine Reduktion der IL-4-abhängigen STAT6-Phosphorylierung und der CD40-vermittelten NFkappaB-Translokation, was zu einer Inhibition des IgE-Klassenwechsels auf dem Niveau des epsilon-Keimbahntranskriptes sowie der Aktivierungsinduzierten Desaminase führte. Weiterhin wurde in einer randomisierten, kontrollierten Doppelblindstudie die Supplementierung mit hochdosierter DHA bei Patienten mit atopischem Ekzem hinsichtlich klinischer und immunologischer Parameter geprüft. Dabei führte DHA zu einer Reduktion des Schweregrades der Erkrankung und zu einer verminderten IgE-Produktion anti-CD40/IL 4-stimulierter Blutzellen ohne Beeinflussung des Serum-IgE-Spiegels. Schließlich wurden die lokalen Prozesse nach DHA-Verabreichung in einem Mausmodell für proteininduzierte Dermatitis analysiert. Dabei war die Reduktion der klinischen Ekzemausprägung mit der verminderten Zahl dermaler CD8+ T-Zellen verbunden. Andere histologische Parameter und das Serum-IgE blieben jedoch unbeeinflusst. Durch die Fähigkeit von DHA, in den IgE-Klassenwechsel in B-Zellen einzugreifen, stellt die Supplementierung mit DHA somit eine mögliche präventive Maßnahme gegen allergische Erkrankungen dar. Ebenso ist DHA in der Lage, den Schweregrad des atopischen Ekzems durch die positive Beeinflussung lokaler inflammatorischer Prozesse signifikant zu verbessern. / The prevalence of allergic diseases has increased worldwide. Westernised diet with its changing polyunsaturated fatty acid (PUFA) proportions is considered to contribute to this development. The omega-3 PUFA Docosahexaenoic acid (DHA) has been reported to be antiinflammatory, but its way of action is not completely understood. Initially, the molecular mechanisms of DHA impact on IgE production in human B cells were examined in vitro. Thereby, DHA inhibited IgE production and the differentiation of IgE secreting cells. This was mediated through direct inhibition of the immunoglobulin isotype switching process by decreasing epsilon germline transcript and activation induced desaminase transcription. Analysis of involved signalling pathways revealed an inhibition of IL-4 driven STAT6 phosphorylation and a reduced NFkappaB translocation into nucleus upon CD40 ligation. Next, in a randomised, double bind, controlled clinical study the efficacy of high-dose DHA supplementation in atopic eczema was determined by investigating the impact on clinical and immunological parameters. In the DHA, but not in control group a clinical improvement of atopic eczema and a reduction of CD40/IL-4 mediated IgE synthesis of peripheral blood cells were observed whereas serum IgE levels remained unchanged. Finally, in a mouse model the impact of oral DHA application on allergen induced dermatitis as well as the underlying local mechanisms were investigated. Thereby, the DHA mediated reduction of clinical skin score was associated with decreased dermal CD8+ T cell numbers, whereas other histological parameters or serum IgE values were not affected. In summary the results indicate that dietary DHA may be effective in prevention of allergic diseases by interference with the IgE switching process and improve the clinical outcome of atopic eczema by its positive impact on local inflammatory processes.

Introdução Os recém-nascidos de muito baixo peso (<1.500g) necessitam de aporte adequado de nutrientes para desenvolver-se satisfatoriamente. O leite humano é fundamental na sua alimentação, já que possui uma composição nutricional balanceada que inclui nutrientes essenciais, tais como os ácidos graxos poli-insaturados, incluindo os ácidos ômega 3 e 6, de extrema importância para o desenvolvimento do sistema nervoso central e retina. No entanto, apenas o leite materno pode não ser suficiente para suprir essas necessidades, necessitando, portanto, de suplementação. Atualmente, surgiu a possibilidade de adequação do leite humano acrescido de um concentrado com liofilizado do próprio leite humano, que foi desenvolvido neste trabalho. Objetivos determinar a quantidade de lipídios totais e o perfil lipídico do Leite Humano Baseline (LHB) e dos Concentrados com o liofilizado de Leite Humano em diferentes momentos (Leite Humano concentrado no período imediato - LHCI, Leite Humano concentrado no período de 3 meses - LHC3m e Leite Humano concentrado no período de 6 meses - LHC6m). Métodos Foram obtidas 50 amostras de leite humano de mulheres doadoras do Banco de Leite Humano da FMRP-USP que aceitaram participar do estudo. A quantificação de lipídeos ocorreu pelo Analisador de Leite Humano MIRIS® e o perfil de ácidos graxos foi determinado por CG-FID, SHIMADZU®. Resultados Houve uma concentração de lipídios totais no LHCI em relação ao LHB; a concentração (% em relação ao total de ácidos graxos) dos ácidos foi de: palmítico (C16:0) 22,30% LHB, 21,46% LHCI, 21,54% LHC3m e 21,95% LHC6m (p<0,01); oleico (C18:1n-9) 30,41% LHB, 30,47% LHCI, 30,55% LHC3m e 29,79% LHC6m (p=0,46); linoléico (C18:2 n-6) 19,62% LHB, 19,88% LHCI, 19,49% LHC3m e 19,45% LHC6m (p=0,58); araquidônico (C20:4 n-6) 0,35% LHB, 0,16% LHCI, 0,13% LHC3m e 0,15% LHC6m (p<0,01); ?- linolênico (C18:3 n-3) 1,32% LHB, 1,37% LHCI, 1,34% LHC3m e 1,34% LHC6m (p=0,14); docosahexaenóico (C22:6 n- 3) 0,10% LHB, 0,06% LHCI, 0,05% LHC3m e 0,06% LHC6m (p<0,01). Há modificações específicas em alguns ácidos graxos, no entanto a somatória de ácidos graxos por suas classes estruturais não apresentam diferenças. Conclusão Apesar das pequenas diferenças encontradas no perfil lipídico, o concentrado com leite humano liofilizado é um alimento que pode vir a atender as necessidades do recém-nascido, já que houve aumento dos lipídios totaise preservação dos nutrientes essenciais presentes apenas no leite materno, mas estudos clínicos ainda precisam ser feitos para avaliar a segurança e eficácia do concentrado como alimentação dos recém-nascidos de muito baixo peso. / Introduction Very low birth weight infants (<1,500 g) require adequate nutrient intake to develop satisfactorily. Human milk is essential in its diet, since it has a balanced nutritional composition that includes essential nutrients, such as polyunsaturated fatty acids, including omega 3 and 6 acids, of extreme importance for the development of the central nervous system and retina. However, only breast milk may not be sufficient to meet these needs, thus requiring supplementation. Nowadays, the possibility of adapting the human milk plus a concentrate with lyophilized from the human milk itself, which was developed in this work, has arisen. Objectives To determine the amount of total lipids and the lipid profile of Human Baseline Milk (LHB) and Concentrates with human milk lyophilized at different times (Human Milk Concentrated in the immediate period - LHCI, Concentrated Human Milk in the 3-month period - LHC3m and Human Milk Concentrated in the 6-month period - LHC6m). Methods Fifty samples of human milk from women donors of the Human Milk Bank of FMRP-USP who were accepted to participate in the study were obtained. Lipid quantification was performed by the MIRIS® Human Milk Analyzer and the fatty acid profile was determined by CG-FID, SHIMADZU®. Results There was a total lipid concentration in LHCI relative to LHB; the concentration (% of total fatty acids) of the acids was: palmitic (C16: 0) 22.30% LHB, 21.46% LHCI, 21.54% LHC3m and 21.95% LHC6m (p <0) , 01); oleic acid (C18: 1n-9) 30.41% LHB, 30.47% LHCl, 30.55% LHC3m and 29.79% LHC6m (p = 0.46); linoleic acid (C18: 2 n-6) 19.62% LHB, 19.88% LHCI, 19.49% LHC3m and 19.45% LHC6m (p = 0.58); arachidonic (C20: 4 n-6) 0.35% LHB, 0.16% LHCl, 0.13% LHC3m and 0.15% LHC6m (p <0.01); ?-linolenic acid (C18: 3 n-3) 1.32% LHB, 1.37% LHCI, 1.34% LHC3m and 1.34% LHC6m (p = 0.14); (C22: 6 n-3) 0.10% LHB, 0.06% LHCl, 0.05% LHC3m and 0.06% LHC6m (p <0.01). There are specific modifications in some fatty acids, however the sum of fatty acids by their structural classes do not present differences. Conclusion Despite the small differences found in the lipid profile, the concentrate with freeze-dried human milk is a food that can meet the needs of the newborn, since there was an increase in total lipids and preservation of essential nutrients present only in breast milk, butclinical studies still need to be done to evaluate the safety and efficacy of concentrate as a feed for very low birth weight infants.

Diversos estudos indicam que touros europeus (Bos taurus) apresentam uma maior susceptibilidade ao estresse térmico com um consequentemente aumento do estresse oxidativo. Este, por sua vez, leva a uma diminuição na qualidade espermática. O espermatozoide possui, fisiologicamente, uma grande quantidade de ácidos graxos poli-insaturados (PUFAs) em sua membrana. As ligações duplas entre carbonos desses PUFAs confere a membrana plasmática uma maior fluidez, necessária aos processos fisiológicos do espermatozoide como motilidade e fertilização. No entanto essas ligações são mais facilmente oxidadas e, portanto, mais susceptíveis a peroxidação lipídica. Sendo assim, a suplementação com PUFAs promoveria uma melhoria na qualidade espermática, porém, tornaria o espermatozoide ainda mais susceptível à peroxidação lipídica. A vitamina E é um antioxidante chave na prevenção do estresse oxidativo através da interceptação do radical hidroxila, protegendo os PUFAs da membrana espermática contra a peroxidação lipídica. Portanto, uma alternativa para melhorar a qualidade espermática de touros europeus submetidos ao estresse térmico, seria a suplementação com PUFAs associada a um tratamento com vitamina E para prevenir um possível efeito deletério oxidativo desses ácidos graxos. Estudos sugerem uma importante relação entre os testes convencionais de avaliação de sêmen e fertilidade. No entanto, a limitação destes testes também tem sido reconhecida, principalmente em casos de infertilidade idiopática. Estudos demonstram que os testes funcionais de avaliação do sêmen, podem ser uma alternativa importante para a avaliação da capacidade fecundante de uma determinada amostra seminal. A interação entre espermatozóide e zona pelúcida ocorre através da ligação de receptores acrossomais às proteínas ZP3 e ZP2 do oócito. A membrana perivitelínica de ovos de galinha possui uma homologia com essas proteínas, permitindo sua ligação com espermatozóides de diversas espécies. Sendo assim, o teste de ligação espermática a essa membrana parece ser bastante eficiente para avaliar a capacidade fecundante do sêmen. No presente estudo, 16 touros Bos taurus foram submetidos à insulação testicular durante 4 dias e submetidos a um arranjo fatorial 2x2, sendo os fatores: dieta rica em PUFAs (Megalac&reg;, 4 Kg ao dia) e tratamento com Vitamina E (Monovin E&reg;, 3000 UI a cada 13 dias) durante 60 dias. As amostras seminais criopreservadas/descongeladas foram submetidas à avaliação da análise computadorizada da motilidade (CASA; Hamilton Thorne, Ivos 12.3, USA), integridade de membrana (Eosina Nigrosina), integridade acrossomal (Fast Green Rosa Bengala), atividade mitocondrial (33 diaminobenzidina), susceptibilidade ao estresse oxidativo (Substâncias reativas ao ácido tiobarbitúrico - TBARS), integridade de DNA (SCSA) e o teste de ligação de espermatozóides à membrana vitelínica de ovos de galinha, que foi previamente padronizado. O tratamento com vitamina E apresentou efeitos benéficos para algumas características espermáticas, sendo o contrário observado pelo tratamento com PUFAS, quando ambos foram analisados isoladamente. No entanto, a associação entre estes tratamentos não foi eficiente para melhorar a qualidade espermática assim como a capacidade fecundante avaliada através do teste de ligação espermática em membrana perivitelínica de ovos de galinha. / Several studies suggest a high susceptibility of European bulls (Bos taurus) to the heat stress, which will lead to an increase on oxidative stress and consequent impairment on sperm viability. Sperm membranes are rich in poly-unsaturated fatty acids (PUFAs). The carbon-carbon double bounds (insaturations) that characterize these PUFAs confer the necessary fluidity to the sperm membrane, essential to motility and fecundation capacity. However, these insaturations are more unstable and, therefore, more susceptible to the lipid peroxidation (i.e., attack of reactive oxygen species to the lipids). Hence, PUFA supplementation would improve certain sperm characteristics but increase the already high susceptibility to the oxidative stress. Vitamin E is a key antioxidante on oxidative stress prevention by intercepting hydroxyl radical and protecting sperm membrane PUFAS against lipid peroxidation. Therefore, an alternative to improve sperm quality in heat stressed european bulls would be a PUFA supplementation associated to a Vitamin E therapy in order to avoid a possible deleterious oxidative effects of these fatty acids. Estudies suggest an important relationship between conventional tests for sperm evaluation and fertility. However, limitations have been also verified, especially in cases of idiopathic infertility. Thus, tests to evaluate sperm functionality may be an interesting alternative to assess fecundation capacity of semen samples. The binding of sperm to the zona pellucida occurs initially by the interaction between acrosomal receptors and oocyte proteins ZP3 and ZP2. The perivitelline membrane of chicken eggs present homologies to zona pellucida proteins, which allows sperm from several species to bind to this membrane. Thus, the sperm binding test to the perivitelline membrane may be an efficient method to asses the fecundation capacity in semen of bulls. In the present study, 16 Bos taurus bulls were submitted to testicular insulation for 4 day. Animals were then submitted to a 2x2 factorial design, with the factor represented by a PUFA oral supplementation (Megalac&reg;, 4 Kg for day) and the treatment with Vitamin E (Monovin E&reg;, 3000 UI, each 13 days) for 60 days. Samples were cryopreserved/thawed and analyzed for motility variables by the computer assisted sperm analysis (CASA; Hamilton Thorne, Ivos 12.3, USA), membrane integrity (Eosin / Nigrosin), acrosome integrity (Fast Green / Bengal Rose), mitochondrial activity (33 diaminobenzidine), susceptibility to the oxidative stress (tiobarbituric acid reactive substances - TBARS), DNA integrity (SCSA) and the sperm binding test to the chicken egg perivitelline membrane, which was previously validated. Results demonstrated a beneficial effect of Vitamin E on some sperm characteristics, with the inverse effect observed for PUFA when both treatments were considered individually. However, the association between both treatments were not efficient to improve sperm quality as well as the fecundation capacity as evaluated by the sperm binding test to the chicken perivitelline membrane.

Les acides docosahexaénoïque (DHA) et arachidonique (ArA), qui appartiennent respectivement aux familles n-3 et n-6, sont présents en grande quantité dans les tissus cérébraux, en particulier dans les phospholipides membranaires. Ces deux acides gras polyinsaturés (AGPI) jouent des rôles essentiels dans le fonctionnement cérébral, notamment dans le neurodéveloppement et la neuroinflammation. De plus, ces AGPI sont des précurseurs de plusieurs médiateurs lipidiques oxygénés impliqués dans divers processus physiologiques et pathologiques.Dans ce contexte, l'objectif de cette thèse était de caractériser les métabolites oxygénés dérivés du DHA et de l'ArA dans le cerveau, étape cruciale pour une meilleure compréhension de leurs rôles biologiques dans cet organe. Ainsi, une technique d'analyse à haute performance a été utilisée, la chromatographie liquide couplée à de la spectrométrie de masse en tandem (UHPLC-MS/MS), pour identifier les différents métabolites oxygénés dérivés du DHA et de l'ArA présents dans les cerveaux de rats exsanguinés et non exsanguinés, que ce soit en conditions basales ou inflammatoires. Ainsi, plusieurs produits oxygénés dérivés du DHA et de l'ArA ont été identifiés et quantifiés dans les cerveaux exsanguinés et non-exsanguinés, à la fois à l'état libre dans le cerveau mais également estérifiés dans les phospholipides. Les métabolites mono-hydroxylés sont les principaux dérivés oxygénés du DHA et de l'ArA, cependant, des quantités mesurables de produits di-hydroxylés, tels que le 8,15-diHETE et la protectine DX, ont également été détectées. L'exsanguination permet de différencier les métabolites oxygénés du tissu cérébral de ceux présents dans le sang. De plus, le métabolisme oxygéné du DHA et de l'ArA est impacté lors d'une inflammation cérébrale. En effet, dans ces conditions, la synthèse de certains métabolites est augmentée, notamment celle de la protectine D1, molécule connue pour ces fortes propriétés anti-inflammatoires. Ces résultats méritent de plus amples recherches dans des conditions pathophysiologiques, notamment lors de maladies neurodégénératives, afin d'observer leur impact sur le métabolisme oxygéné de ces AGPI / Docosahexaenoic (DHA) and arachidonic (ArA) acids, two PUFA which belong to n-3 and n-6 families respectively, are both present at high amount in brain tissues, and especially in membrane phospholipids. These two polyunsaturated fatty acids (PUFA) play important roles in brain functioning, notably in neurodevelopment and neuroinflammation. Moreover, these PUFA are precursors of various oxygenated lipid mediators involved in diverse physiological and pathological processes. In this context, the aim of this work was to characterize oxygenated metabolites derived from DHA and ArA in brain, crucial step for better understanding their biological roles in this organ. For this purpose, a high performance analytical approach was usd, the liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), in order to identify the different oxygenated metabolites derived from DHA and ArA in exsanguinated and non-exsanguinated brains, both in basal and inflammatory conditions. Several oxygenated products from DHA and ArA were identified and measured in exsanguinated and non-exsanguinated brain, both in the free form and esterified in phospholipids. Mono-hydroxylated metabolites were the main oxygenated derivatives from DHA and ArA, however, measurable amounts of di-hydroxylated products such as 8,15-diHETE and protectin DX, were also detected. Exsanguination allowed discriminating oxygenated metabolites from brain tissue against those in blood brain. Moreover, DHA and ArA oxygenated metabolism was impacted during brain inflammation. Indeed, in these conditions, the synthesis of some metabolites was increased, including that of protectin D1, molecule known for its strong anti-inflammatory properties. These results deserve further research in pathophysiological conditions, especially in neurodegenerative diseases, in order to observe their impact on the oxygenated metabolism of these PUFA

La bactérie Helicobacter pylori est responsable de l’infection la plus répandue dans la population mondiale. Cette bactérie est considérée comme le principal agent étiologique de la gastrite chronique, de l’ulcère duodénal et du cancer gastrique non-héréditaire. La thérapeutique prescrite pour l’éradication de cette infection est inefficace pour un nombre de plus en plus élevé de patients, dû à l’induction constante de souches résistantes aux antibiotiques habituellement prescrits.H. pylori a aussi d’autres façons d’assurer sa survie dans le milieu gastrique que l’induction de résistances aux antibiotiques. En particulier en intéragissant avec le cholestérol des cellules épithéliales gastriques. De ce fait, l’utilisation de molécules inhibitrices de la croissance de H. pylori, autres que les antibiotiques classiques, est une stratégie importante pour combattre cette infection.L’objectif majeur de ce travail a été de mettre en évidence de nouvelles molécules inhibitrices de la croissance et de la viabilité de H. pylori, permettant ainsi le développement de solutions alternatives à la thérapeutique classiquement utilisée. Les acides gras polyinsaturés et l’acide docosahexaenoic (DHA) se mettre en lumière pour réunir incontestable propriété anti-inflammatoires et anti-tumoral. Nous avons émis l’hypothèse que le DHA influence la survie de H. pylori, et peut moduler la disponibilité des acides gras et du cholestérol cellulaires.Nos résultats montrent que le DHA inhibe la croissance de H. pylori in vitro et affecte sa capacité à coloniser la muqueuse gastrique dans le modèle souris. Dans ces conditions, le DHA diminue la réponse inflammatoire gastrique induite par l’infection. Au niveau des cellules épithéliales gastriques, des modifications du profil des acides gras et du cholestérol avec des conséquences sur le métabolisme et la signalisation cellulaire sont observées. De plus, un traitement antibiotique classique combiné à une administration de DHA aux souris infectées diminue de façon drastique la récidive de l’infection. En conclusion, cette étude démontre un effet inhibiteur du DHA sur l’infection par H. pylori et sa récidive. Ces résultats justifient la proposition du DHA comme coadjuvant thérapeutique, constituant ainsi une stratégie prophylactique alternative de l’éradication de l’infection par H. pylori. / H. pylori infection is extremely common worldwide and is recognized as a major etiological factor in chronic active gastritis, gastric duodenal ulcers and gastric cancer development. H. pylori eradication treatment has not changed to a large extent in the last decades and can raise some concern mainly due to recurrence of infection, and most importantly, acquired resistance to classically used antibiotics. In this context, the use of compounds other than antibiotics that could decrease H. pylori infection in a safe way could provide an alternative to tackle this problem. It is known that H. pylori extracts cholesterol from host cell-membrane rafts, modifies it into an α-glycosylated form, and uses this mechanism to increase its survival. The main aim of this thesis work was to explore the role of different non-antibiotic molecules in inhibiting H. pylori growth. Among molecules known to affect in vitro H. pylori growth and viability are certain polyunsaturated fatty acids (PUFAs). Within the many molecules available, we concentrated our efforts on the study of docosahexaenoic acid (DHA). We also pursued the hypothesis that DHA affects survival of H. pylori by modulating the host epithelial cell levels of fatty acids and cholesterol availability.Our results show that DHA inhibits H. pylori growth both in vitro and in vivo, and attenuates the host inflammatory response. Additionally, we demonstrate that DHA induces morphological and cell wall protein composition changes that altogether decrease bacteria-gastric epithelial cell adherence, inflammation and survival. Also, we demonstrated that DHA alters cholesterol levels in epithelial cells, thereby influencing H. pylori ability to uptake and use epithelial cholesterol. This will ultimately impair H. pylori survival. Importantly, the combination of DHA and antibiotic standard treatment decreased the recurrence of H. pylori infection in a mouse model. Our results have gathered important evidence to pave the way for DHA use in the clinical setting and in prophylactic/preventive strategies against H. pylori infection.

Cette thèse s’inscrit dans le cadre du projet SMILE de Microphyt qui vise à proposer une gamme d’ingrédients naturels issus de microalgues afin de répondre à deux enjeux sociétaux majeurs : le surpoids/obésité et le déclin des fonctions cognitives liées à l’âge (DCLA). Les acides gras polyinsaturés oméga-3 (AGPI-w3) et la fucoxanthine (Fx) ont des activités prouvées contre les voies métaboliques liées à la prise de poids et aux DCLA. C’est dans ce contexte que les efforts se sont tout d’abord focalisés vers la sélection de deux espèces de microalgues brunes dont le critère principal a été la productivité en ces molécules d’intérêts. Deux souches des espèces Tisochrysis lutea et Phaeodactylum tricornutum ont alors été sélectionnées du fait de leurs fortes teneurs et productivités en AGPI-w3 et Fx, pour être par la suite cultivées à l’échelle de 5000 L dans des photobioréacteurs (PBR) industriels Camargue©. Dans un second temps, les travaux ont porté sur l’optimisation des procédés post-culture pour les deux espèces, c’est-à-dire, de la récolte de la biomasse à l’obtention d’un extrait, afin de garantir la sécurité, la stabilité et l’efficacité des produits finis. L’étude des procédés de récolte/concentration de la culture a montré leur influence sur les classes lipidiques et l’apparition d’acides gras sous forme libre (AGL), engendrés par les stress mécaniques et thermiques subis par la biomasse lors de cette étape. La formation de ces AGL a pu être limitée par l’utilisation de la filtration tangentielle et de la centrifugation dans certaines conditions. De plus, le lien entre procédé et activité a été souligné via une étude sur modèle cellulaire CaCo2 : les AGL pourraient être à l’origine d’une cytotoxicité accrue des extraits. Enfin, l’optimisation des conditions de co-extraction des AGPI-w3 et de la Fx a abouti à la sélection d’un solvant éco-compatible (éthanol) et a mis en lumière le fort impact de la granulométrie et du ratio solvant/biomasse sur les rendements d’extractions. / This thesis is part of Microphyt's SMILE project, which aims to propose a range of natural ingredients derived from microalgae. The main objective of this project is to respond to two major societal issues: overweight/obesity and the decline of age-related cognitive functions (DCLA). Omega- 3 polyunsaturated fatty acids (PUFAs) and fucoxanthin (Fx) have proven activities against the metabolic pathways associated with weight gain and DCLA. In this context, the first efforts were initially directed towards the selection of two species of brown microalgae with their productivity in these molecules of interest as the main criterion. Two strains of the species Tisochrysis lutea and Phaeodactylum tricornutum were then selected as raw materials rich in PUFAs and Fx and were grown at a 5,000 L scale in Camargue© photobioreactors. The work then focused on optimizing the downstream processing steps to the cultivation of microalgae in order to guarantee the safety, stability and efficiency of the final products. Harvesting processes had, in particular, through the mechanical stress generated, an impact on lipid classes and the appearance of free fatty acids (FFA) in the studied species. A CaCo2 cell model study has suggested that FFA may be responsible for increased cytotoxicity of extracts. The formation of FFA during the concentration stage could be limited by the use of tangential filtration and centrifugation under certain conditions. Finally, the optimization of the co-extraction conditions of PUFAs and Fx resulted in the selection of an eco-friendly solvent (ethanol) and highlighted the strong impact of particle size and solvent/biomass ratio on extraction yields.

Vários estudos indicam uma maior susceptibilidade ao estresse térmico de touros europeus quando criados em regiões tropicais, levando a efeitos deletérios à reprodução (e.g., diminuição da qualidade espermática, degeneração testicular). Tal fato pode causar expressivo prejuízo econômico visto que, nestas regiões, a estação de monta é realizada no verão. Um dos principais mecanismos propostos para explicar este efeito seria um aumento do estresse oxidativo. Assim, uma alternativa promissora seria o tratamento com antioxidantes e ácidos graxos poli-insaturados. O presente estudo tem como objetivo: Avaliar os possíveis efeitos benéficos da suplementação oral com ácidos graxos poli-insaturados (com vs. sem) e a ação protetora do tratamento sistêmico com vitamina E (com vs. sem) contra os danos oxidativos causados pelo estresse térmico testicular em touros taurinos insulados através das avaliações ultrassonográfica, morfométrica testicular (i.e, volume, temperatura, consistência, circunferência) e histológica dos testículos. Para isso, foram utilizados 16 touros Bos taurus taurus jovens (em torno de 2 anos de idade) divididos em 4 grupos (fatorial 2x2) com perfil espermático normal. Todos os animais foram submetidos à insulação testicular por um período de 96 horas. Foi realizada a ultrassonografia testicular a cada 13 dias por todo o período e mais dois meses após o período da insulação. Como controles foram avaliados os testículos dos touros antes da insulação. Concomitante à insulação, os animais foram divididos aleatoriamente em 4 lotes que foram submetidos a um arranjo fatorial 2x2, sendo um dos fatores a dieta rica em PUFA (Ácidos graxos poli-insaturados Megalac E&reg;) todo o dia durante dois meses pós insulação. Já o tratamento sistêmico com Vitamina E (Monovin E&reg;) foi realizado através da injeção subcutânea de 5 ml de &alpha;-tocoferol a cada 13 dias pós insulação com duração de dois meses pós insulação. Aos animais que não receberam a vitamina E, foi administrado o mesmo volume em placebo (óleo inerte). No final deste período, os animais foram castrados e os testículos utilizados para a histologia. Os resultados foram analisados através do SAS system for Windows e indicaram que tanto a suplementação oral de ácidos graxo poli-insaturados como o tratamento sistêmico com vitamina E, não alteraram as características ultrassonográficas, morfométricas e histológicas testiculares de touros de origem europeia submetidos a insulação testicular. / Several studies indicate an increased susceptibility to the heat stress in European bulls raised under tropical conditions, which leads to deleterious effects on reproduction (e.g., testicular degeneration, impaired sperm quality). This may cause significant economic losses since, in these regions, breeding season usually occurs during summer. The main mechanism proposed to explain such event is the oxidative stress. Therefore, an alternative to overcome this effect would be the treatment with antioxidant combined with a supplementation with poly-unsaturated fatty acids (PUFA). The present study aimed to evaluate the possible beneficial effect of the oral PUFA supplementation combined with a protective antioxidant treatment to overcome the deleterious effects of the heat stress on testicular echogenicity, morphometry (i.e., volume, circumference, consistency) and histology. Towards this aim, 16 young but sexually mature Bos taurus taurus (around 2 years old). All animals were submitted to testicular warming for 96 hours. Simultaneously to the insulation, animals were randomly allocated into 4 groups in a 2x2 fatorial design. One of the factors was a PUFA supplementation (supplemented vs. nonsupplemented; Megalac E&reg;) everyday for two months. The other factor was a systemic treatment with a subcutaneous administration of 5 mL of &alpha;-tocopherol (treated vs. non treated) every 13 days for two months. Animal that did not receive the treatment were injected with 5 mL of inert oil. Testicular ultrasound evaluation was performed each 13 days from the beginning until two months after testicular warming. At the end of the period experimental period, animals were castrated and the testicles were submitted to histological evaluation. Data were statistically analyzed using the SAS system for Windows. Results indicated that oral PUFA supplementation combined with the systemic -tocopherol treatment had no influence on testicular echogenicity, morphometry and hystology in European bulls submitted to testicular warming.

Foram utilizadas 144 galinhas poedeiras da linhagem Shaver White por um período de quatro semanas, com o intuito de verificar o efeito da adição de 3% dos óleos de soja, milho, canola, linhaça, salmão ou da mistura de sardinha e atum na dieta das aves, sobre a composição dos ácidos graxos na gema dos ovos e no plasma sanguíneo das aves, bem como o tempo de incorporação total de cada ácido graxo na gema dos ovos durante o período experimental. Paralelamente foram avaliados, o desempenho das aves e a qualidade externa dos ovos. Para análise estatística dos resultados empregou- se modelo fatorial 6X6 em blocos casualizados sendo as aves distribuídas em 72 gaiolas. A adição de 3% de óleo de salmão ou de milho à ração determinou redução significativa no consumo alimentar. O tratamento que recebeu 3% de óleo de salmão promoveu menor peso da casca dos ovos (g) bem como menor espessura da casca (mm). A inclusão dos óleos de linhaça, soja ou milho à dieta, determinou aumento dos ácidos graxos poliinsaturados na gema e no plasma das galinhas. A adição de óleo de sardinha e atum proporcionou maiores concentrações de ácidos graxos saturados na gema. Os teores de PUFAs n-3 foram maiores nas gemas do tratamento a base de óleo de sardinha, enquanto que no plasma a maior concentração foi observada no tratamento com óleo de linhaça. O óleo de linhaça proporcionou o maior teor de ácido &alpha;-linolênico incorporado à gema e no plasma sanguíneo. As quantidades de EPA na gema e no plasma mostraram-se maiores no grupo que recebeu 3% de óleo de sardinha e atum que, por sua vez, também foram responsáveis pelos mais elevados teores de DHA na gema dos ovos. Já com relação ao plasma, as maiores concentrações de DHA foram observadas nos grupos alimentados com óleos de salmão e sardinha/atum. Os PUFAs diminuíram na gema até o oitavo dia experimental, enquanto que os PUFAs n-3 aumentaram até este dia. Os teores de ácido &alpha;-linolênico foram crescentes até o décimo dia de experimento, enquanto que as concentrações de EPA e DHA aumentaram até o oitavo dia de experimento. / Hundred forty-four Shaver White laying hens were used over a 4 week experimental period to investigate the effect of 3% of soybean oil, corn oil, canola oil, flaxseed oil, salmon oil or tuna and sardine oil added to the diets, upon the fatty acid egg yolk composition, blood plasma levels and incorporation time of each fatty acid into the egg yolk. Reproductive performance of hens and external egg quality were evaluated. Hens were allocated into 72 cages and the experimental design was a 6X6 randomized factorial model. Hens fed 3% salmon or corn oil diet showed a significant reduction of food consumption, eggshell weight (g) and eggshell thickness (mm). The addition of flaxseed, soybean or corn oil into the diet increased the polyunsaturated fatty acids levels into the egg yolk and in the blood plasma. Adding tuna and sardine oil into the diet increased the concentration of yolk saturated fatty acids. The levels of PUFAs n-3 were increased in the tuna and sardine oil treatment, while the flaxseed oil increased the plasma fatty acids. The deposition of &alpha;-linolenic fatty acids was higher in the group fed flaxseed oil The percentage of EPA into the yolk and plasma was higher for the hens fed 3% tuna and sardine oil diet, as well as the levels of yolk DHA. The concentration of DHA into the plasma was higher for the salmon and tuna/sardine oil treatments. The PUFAs yolk decreased during the first eight days of experiment, while the PUFAs n-3 increased during the same period. The concentration of &alpha;-linolenic acid increased until ten days of experiment, while the percentage of EPA and DHA increased up to the eighth experimental day.

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.