Macrophage-derived inflammation contributes to chronic inflammation in adipose tissue in obesity and is also linked to the development of skeletal muscle (SM) insulin resistance. The long-chain n-3 PUFA have been shown to modulate cytokine secretion from macrophages, though subsequent effects on SM inflammation and function are unknown. A model of macrophage conditioned media (MCM) was used to examine effects of n-3 PUFA on macrophage inflammation and consequent effects on SM cells. Treatment of RAW 264.7 macrophages with long-chain n-3 PUFA decreased LPS-induced MCP-1 and IL-6 gene expression and MCP-1 secreted protein. In turn, MCM from n-3 PUFA-treated macrophages decreased TNF-α and IL-6 gene expression in LPS-stimulated L6 SM cells, but did not affect insulin-stimulated pAkt content. Long-chain n-3 PUFA did not affect gene expression of inflammatory signaling intermediates NF-κB and TLR4. Overall this thesis suggests that long-chain n-3 PUFA are important nutritional strategies for reducing macrophage-derived inflammation, with ensuing benefits in SM inflammation. / NSERC-CGS, Ontario Graduate Scholarship

The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

The blood-brain barrier, formed by brain microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. It was previously demonstrated in our laboratory that knock down of fatty acid transport proteins FATP-1 and CD36 attenuated apical to basolateral monounsaturated fatty acid transport across human brain microvessel endothelial cells (HBMEC). Arachidonic acid (AA; 5,8,11,14 - cis-eicosatetraenoic acid) is a conditionally essential, polyunsaturated fatty acid [20:4(n-6)] and a major constituent of brain lipids. We examined transport of AA across confluent monolayers of HBMEC. Control cells or HBMEC with knock down of FATP-1 or CD36 were cultured on Transwell® plates and incubated apically with [3H]AA and incorporation of [3H]AA into the basolateral medium was determined temporally. [3H]AA was rapidly incorporated into the basolateral medium with time in control cells. Surprisingly, knock down of FATP-1 or CD36 did not alter [3H]AA movement into the basolateral medium. The increased permeability mediated by AA was likely caused by a metabolite of AA produced de novo and was confirmed by an increased movement of fluorescent dextran from apical to basolateral medium. HBMECs expressed PGE2 synthase, cyclooxygenase-1 and -2, PGE2 receptors, tight junction proteins and prostaglandin transporters. The AA-mediated increase in membrane permeability was not attenuated by cyclooxygenase inhibitor drugs (NSAIDs). Incubation of the HBMEC monolayers with exogenous PGE2 resulted in attenuation of the AA-mediated permeability increases. The results indicate that AA increases the permeability of the HBMEC monolayer likely via increased production of metabolites or by-products of the lipoxygenase or epoxygenase pathways. These observations may explain the rapid influx of AA into the brain previously observed upon plasma infusion with AA.

Nutrigenomics studies the genome-wide influence of nutrients to understand the association between nutrition and human health. Studies in animal models and humans have demonstrated that dietary n-3 polyunsaturated fatty acids (PUFA) from fish oil may be beneficial in inflammatory bowel diseases (IBD). This thesis aimed to test the hypothesis that dietary n-3 PUFA eicosapentaenoic acid (EPA) reduced and n-6 PUFA arachidonic acid (AA) increased colitis in the interleukin- 10 gene-deficient (Il10–/–) mouse model of IBD, and that these PUFA altered the intestinal bacteria community during colitis development using genome-wide expression and bacterial profiling. Using a combined transcriptomic and proteomic approach, the time-course study defined the onset and progression of colitis in Il10–/– mice. Histopathology, transcript and protein changes before and after colitis onset involved in innate and adaptive immune responses suggested delayed remodelling processes in colitic Il10–/– mice and 11 weeks of age as suitable time point to study the effects of dietary PUFA on colitis development. Comparing the transcriptome and proteome profiles associated with colon inflammation of mice fed with the AIN-76A or oleic acid (OA) diet showed that OA was an appropriate control for unsaturated fatty acids in multi-omic studies. The PUFA intervention study indicated that dietary EPA-induced lipid oxidation might have a potential anti-inflammatory effect on inflamed colon tissue partially mediated through activation of peroxisome proliferator-activated receptor alpha (PPARα). Unexpectedly, dietary AA decreased the expression of inflammatory and stress colonic genes in Il10–/– mice. Altered intestinal bacteria community observed in Il10–/– mice before and after colitis onset was associated with the lack of IL10 protein led to changes in intestinal metabolic and signalling processes. Interestingly, dietary EPA and AA seemed to change intestinal bacteria profiles during colitis development. The role of PPARα in the colon was further examined in a concluding study which identified vanin1 as a likely new PPARα-target gene which may also be involved in lipid metabolism. These findings using a state-of-the-art approach combining transcriptomics, proteomics and physiology provide a basis for future research on molecular mechanisms underlying the effects of dietary PUFA, and might contribute to the development of fortified foods that improve intestinal health and wellness.

Il existe deux populations d’adipocytes thermogéniques, les adipocytes bruns du tissu adipeux bruns et les adipocytes ‘’brites’’ qui apparaissent au sein du tissu adipeux blanc. Récemment, la caractérisation d’adipocytes bruns et brites fonctionnels chez l’homme adulte a permis d’envisager de nouvelles approches nutritionnelles et thérapeutiques pour traiter l’obésité. Mon projet de thèse a porté sur l’étude des effets des acides gras polyinsaturés ω6 et ω3 d’origine alimentaire sur la conversion des adipocytes blancs en brites, d’abord chez l’homme in vitro puis chez les rongeurs in vivo. Nous avons pu ainsi démontrer que l'acide arachidonique ω6 à l’origine de nombreux métabolites oxygénés, exerce 1) un effet inhibiteur sur la formation des adipocytes brites grâce aux prostaglandines E2 et F2α, et 2) un effet inducteur via la prostacycline. En effet, celle-ci active la conversion des adipocytes blancs en brites par une voie impliquant le récepteur membranaire IP et les récepteurs nucléaires PPARs. En nous basant sur les recommandations nutritionnelles actuelles chez l’Homme, qui ont pris en considération l’insuffisance de l’apport en acides garsω3 par rapport à l’excès des ω6, nous avons pu montrer chez la souris qu’une supplémentation en acides gras ω3 dans le régime alimentaire était capable d’inhiber l’effet néfaste des acides gras ω6 et d’activer le tissu adipeux brun. Nos résultats démontrent l’importance de la biodisponibilité de l’acide arachidonique dans la biologie du tissu adipeux et permettent de renforcer l’idée que le rééquilibrage du ratio ω6/ω3 est un outil de choix dans la prévention du surpoids et de l’obésité et les maladies métaboliques associées / There are two types of thermogenic adipocytes able to use fatty acids and glucose to produce heat. We distinguish brown adipocytes from the brown adipose tissue and ‘’brite’’ adipocytes which occur into the white adipose tissue. Recently, the characterization of functional brown and brite adipocytes in adult humans has led to the consideration of their use to treat obesity by increasing energy expenditure. My thesis project was to study the effect of dietary polyunsaturated fatty acids, on the conversion of white into brite adipocytes, in vitro and in vivo, in humans and rodents respectively. We demonstrated that arachidonic acid ω6, precursor of prostaglandins, has 1) an inhibitory effect on the recruitment of brite adipocytes via prostaglandins E2 and F2α and 2) an activatory effect via prostacyclin. In fact, prostacyclin induces the conversion of white into brite adipocytes through the IP receptor and the PPARs signaling pathways. Based on human present nutritional recommendations, we demonstrated that a supplementation of ω3 fatty acids in mice diet was able to inhibit the negative effect of ω6 fatty acids and activate brown adipose tissue. Our data highlights the importance of arachidonic acid bioavailability on the biology of adipose tissue and reinforce the idea that an equilibrate ω6/ω3 ratio is a tool that can be used to prevent overweight obesity and associated metabolic disorders

Em equinos é descrita uma variabilidade na qualidade do sêmen congelado, relacionada principalmente a variações consideráveis na composição da membrana plasmática do espermatozoide. Neste contexto, estudos investigam alternativas para aumentar a fertilidade ao se usar sêmen congelado, e a adição de ácidos graxos polinsaturados ao diluente de congelamento é sugerida. O objetivo deste estudo foi avaliar o efeito da adição de diferentes níveis de ácidos graxos poli-insaturados: ácido eicosanóico e ácido docosahexaenóico, em meio diluente específico para a espécie sobre as características do espermatozoide pós-descongelamento. Foi utilizado sêmen de quatro garanhões (quatro ejaculados por garanhão) da raça Crioula. O sêmen foi diluído em diluente de congelamento comercial a base de gema de ovo, glicerol e dimetilformamida (Botu-Crio©) ajustando a concentração para 200 x 10⁶ espermatozoides viáveis/mL (grupo controle) e, na sequência, os demais tratamentos: adição de ácido docosahexaenóico nas doses 25μm e 50μm /mL e ácido eicosanóico nas doses 25μm e 50μm /mL. Após o descongelamento foram realizadas análises de cinética espermática no sistema de Análise Computadorizada para Avaliação Espermática, das seguintes variáveis: motilidade total, motilidade progressiva, motilidade rápida, motilidade lenta, motilidade local, velocidade de trajeto, velocidade progressiva, velocidade curvilinear, amplitude do deslocamento lateral da cabeça, frequência de batimentos, retilinearidade e linearidade; e avaliação da integridade física através do uso de sondas fluorescentes, e funcionalidade de membrana pelo teste hiposmótico. Não ouve diferença nas variáveis avaliadas. Este é o primeiro estudo que descreve a adição de ácido eicosanóico ao sêmen equino. Concluí-se a adição de ácido docosahexaenóico e eicosanóico nas concentrações testadas não alterou as variáveis avaliadas no sêmen de garanhões da raça Crioula. / In equines, a variability in the quality of frozen semen is described, mainly related to the considerable variations in the composition of the sperm plasma membrane. In this context, studies investigate alternatives to increase fertility when using frozen semen, and the addition of polyunsaturated fatty acids to the freezing diluent is suggested. The objective of this study was to evaluate the effect of the addition of different levels of polyunsaturated fatty acids: eicosanoic acid and docosahexaenoic acid, in specific diluent medium for the species on the characteristics of the post-thawing spermatozoa. Semen was used of four stallions (four ejaculates per stallion) of the Crioula breed. The semen was diluted in commercial freezing diluent based on egg yolk, glycerol and dimethylformamide (Botu-Crio ©) by adjusting the concentration to 200 x 10 vi viable spermatozoa / mL (control group) and, in sequence, the other treatments: addition of docosahexaenoic acid at 25μm and 50μm / mL and eicosanoic acid at 25μm and 50μm / mL. After thawing, sperm kinetics analyzes were performed in the Computerized Analysis System for Sperm Evaluation, of the following variables: total motility, progressive motility, rapid motility, slow motility, local motility, path velocity, progressive velocity, curvilinear velocity, lateral head displacement amplitude, beating frequency, linearity and linearity; and evaluation of physical integrity through the use of fluorescent probes, and membrane functionality by the hyposmotic test. No difference in the variables evaluated. This is the first study to describe the addition of eicosanoic acid to equine semen. We concluded that with the addition of docosahexaenoic and eicosanoic acid at the concentrations tested did not alter the variables evaluated in the semen of Crioula stallions.

Investigating the biosynthesis of long-chain (C20–24) polyunsaturated fatty acids (LC-PUFA), physiologically important compounds including arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in fish is crucial to identify dietary requirements for essential fatty acids (EFA). Moreover, knowledge of the C20–24 LC-PUFA biosynthetic capability of farmed fish species enables us to understand their ability to utilise commonly used raw materials such as vegetable oils, which naturally lack LC-PUFA but include C18 PUFA that are metabolic precursors of LC-PUFA. Studies have shown that the potential of a species for LC-PUFA biosynthesis is associated with the complement and function of fatty acyl desaturase (fads) and elongase of very long chain fatty acid (elovl) genes existing in that species. The present study therefore aimed to investigate these genes in the African catfish (Clarias gariepinus), the most commercially important farmed fish in sub-Saharan Africa. A fads2, a fads6 and four elovl (elovl2, elovl4a, elovl4b, elovl8) cDNAs were cloned and functionally characterised by heterologous expression in yeast. The Fads2 was a bifunctional desaturase enzyme with ∆6∆5 and ∆8 activities, and thus catalysing all the desaturation reactions required for ARA and EPA biosynthesis from C18 precursor fatty acids. Moreover, the C. gariepinus Fads2 enzymes also desaturated 24:5n-3 to 24:6n-3, a ∆6 desaturation required for the biosynthesis of DHA through the so-called “Sprecher pathway”. Functional characterisation of Fads6 by heterologous expression in yeast did not reveal its function. With regards to elongases, the C. gariepinus Elovl2 demonstrated the ability to elongate C20 and C22 PUFA and thus complements the Elovl5 with elongase capability towards C18 and C20 PUFA. The Elovl8 was capable of only limited elongation of C18 and C20 PUFA. Elovl4a and Elovl4b, enable the biosynthesis of very long-chain (>C24) fatty acids, compounds with major roles in vision and fertility of vertebrates. The present study confirmed that C. gariepinus possess all the enzymatic capabilities required for the biosynthesis of ARA, EPA and DHA and, therefore, its physiological EFA requirements could be satisfied with dietary provision of C18 PUFA.

Made available in DSpace on 2015-07-22T20:50:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-11-24 / Introdução: A fração lipídica das membranas celulares consiste de ácidos graxos poliinsaturados (PUFAS) e o uso crônico do álcool altera sua composição, modificando a permeabilidade. Portanto, a administração de PUFAS pode ser um potencial tratamento para evitar a compulsão pelo álcool. Metodologia: Este foi um estudo placebo controlado, duplo-cego, randomizado, onde, "PUFAS”, “Naltrexone”, “Naltrexone + PUFAS" e "Placebo", foram administrados por 90 dias, sendo aplicadas escalas para avaliar o craving pelo álcool (OCDS) e a severidade da dependência do álcool (SADD) no início e 90 dias após a administração das substâncias. Resultados: Após 3 meses de seguimento, houve uma melhora significativa ao longo do tempo em relação aos "dias de ingesta", SADD e OCDS em todos os grupos (p <0,001) dentre os 43 pacientes que completaram o estudo. A comparação entre os grupos quanto aos "dias de ingesta" não demonstrou diferença estatisticamente significante (F = 0,71, p = 0,69). O mesmo efeito foi observado para a compulsão (OCDS) (F = 1,08, p = 0,37) e escala de severidade da dependência (SADD) (F = 0,73, p = 0,53). Conclusões: A administração de n-3 e n-6 PUFAS por 3 meses não diferiu significativamente do placebo na redução da quantidade de ingesta de álcool, ou dos escores de OCDS e SADD em um grupo de pacientes dependentes de álcool. Estudos posteriores com mais participantes, ou com tratamento mais prolongado são necessários para avaliar o possível benefício da administração de ácidos graxos poli-insaturados para pacientes dependentes de álcool, quer como monoterapia ou em combinação com o Naltrexone. / TEDE / BV UNIFESP: Teses e dissertações

Em equinos é descrita uma variabilidade na qualidade do sêmen congelado, relacionada principalmente a variações consideráveis na composição da membrana plasmática do espermatozoide. Neste contexto, estudos investigam alternativas para aumentar a fertilidade ao se usar sêmen congelado, e a adição de ácidos graxos polinsaturados ao diluente de congelamento é sugerida. O objetivo deste estudo foi avaliar o efeito da adição de diferentes níveis de ácidos graxos poli-insaturados: ácido eicosanóico e ácido docosahexaenóico, em meio diluente específico para a espécie sobre as características do espermatozoide pós-descongelamento. Foi utilizado sêmen de quatro garanhões (quatro ejaculados por garanhão) da raça Crioula. O sêmen foi diluído em diluente de congelamento comercial a base de gema de ovo, glicerol e dimetilformamida (Botu-Crio©) ajustando a concentração para 200 x 10⁶ espermatozoides viáveis/mL (grupo controle) e, na sequência, os demais tratamentos: adição de ácido docosahexaenóico nas doses 25μm e 50μm /mL e ácido eicosanóico nas doses 25μm e 50μm /mL. Após o descongelamento foram realizadas análises de cinética espermática no sistema de Análise Computadorizada para Avaliação Espermática, das seguintes variáveis: motilidade total, motilidade progressiva, motilidade rápida, motilidade lenta, motilidade local, velocidade de trajeto, velocidade progressiva, velocidade curvilinear, amplitude do deslocamento lateral da cabeça, frequência de batimentos, retilinearidade e linearidade; e avaliação da integridade física através do uso de sondas fluorescentes, e funcionalidade de membrana pelo teste hiposmótico. Não ouve diferença nas variáveis avaliadas. Este é o primeiro estudo que descreve a adição de ácido eicosanóico ao sêmen equino. Concluí-se a adição de ácido docosahexaenóico e eicosanóico nas concentrações testadas não alterou as variáveis avaliadas no sêmen de garanhões da raça Crioula. / In equines, a variability in the quality of frozen semen is described, mainly related to the considerable variations in the composition of the sperm plasma membrane. In this context, studies investigate alternatives to increase fertility when using frozen semen, and the addition of polyunsaturated fatty acids to the freezing diluent is suggested. The objective of this study was to evaluate the effect of the addition of different levels of polyunsaturated fatty acids: eicosanoic acid and docosahexaenoic acid, in specific diluent medium for the species on the characteristics of the post-thawing spermatozoa. Semen was used of four stallions (four ejaculates per stallion) of the Crioula breed. The semen was diluted in commercial freezing diluent based on egg yolk, glycerol and dimethylformamide (Botu-Crio ©) by adjusting the concentration to 200 x 10 vi viable spermatozoa / mL (control group) and, in sequence, the other treatments: addition of docosahexaenoic acid at 25μm and 50μm / mL and eicosanoic acid at 25μm and 50μm / mL. After thawing, sperm kinetics analyzes were performed in the Computerized Analysis System for Sperm Evaluation, of the following variables: total motility, progressive motility, rapid motility, slow motility, local motility, path velocity, progressive velocity, curvilinear velocity, lateral head displacement amplitude, beating frequency, linearity and linearity; and evaluation of physical integrity through the use of fluorescent probes, and membrane functionality by the hyposmotic test. No difference in the variables evaluated. This is the first study to describe the addition of eicosanoic acid to equine semen. We concluded that with the addition of docosahexaenoic and eicosanoic acid at the concentrations tested did not alter the variables evaluated in the semen of Crioula stallions.

Made available in DSpace on 2014-06-11T19:33:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-17Bitstream added on 2014-06-13T19:04:39Z : No. of bitstreams: 1 faitarone_abg_dr_botfmvz.pdf: 754134 bytes, checksum: 45dd71e6381045c73eb898cfbd217a7f (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / O presente experimento teve duração de 112 dias e teve por objetivo avaliar o desempenho e qualidade dos ovos de poedeiras leves alimentadas com dietas suplementadas com óleos vegetais. As aves foram distribuídas em um delineamento experimental inteiramente casualisado com 10 tratamentos - (T1 – Controle; T2 – inclusão de 2,5% de óleo de linhaça; T3 – inclusão de 2,5% de óleo de canola; T4 – inclusão de 2,5% de óleo de soja; T5 – inclusão de 5% de óleo de linhaça; T6 – inclusão de 5% de óleo de canola; T7- inclusão de 5% de óleo de soja; T8 – inclusão de 2,5% de óleo de linhaça + 2,5% de óleo de soja; T9 – inclusão de 2,5% de óleo de canola + 2,5% de óleo de soja e T10 – inclusão de 2,5% de óleo de linhaça + 2,5% de óleo de canola) - e seis repetições de oito aves por parcela experimental, perfazendo um total de 480 aves. Foram avaliadas o consumo de ração, a produção de ovos, o peso dos ovos, a massa de ovos, a conversão alimentar por dúzia e por quilograma de ovos produzidos, a gravidade específica dos ovos, a resistência da casca à quebra, a cor de gema, as percentagens de gema, albúmen e casca, a espessura de casca e o índice gema. Não foram observadas diferenças significativas entre os tratamentos para peso e massa de ovos, conversão alimentar por dúzia de ovos produzidos, gravidade específica dos ovos, resistência da casca à quebra, espessura de casca, percentagens de gema, albúmen e casca. Concluiuse que a suplementação de óleos vegetais, na ração de poedeiras comercias leves não promoveu melhoras no desempenho e na qualidade dos ovos / The present experiment lasted 112 days and aimed to weigh up the performance and eggs quality of light laying hens fed with diets supplemented with vegetal oil. The light laying hens were distributed on an completely randomized design with 10 treatments (T1 - control, T2- inclusion of 2.5% of linseed oil in the diet, T3- inclusion of 2.5% of canola oil in the diet; T4-inclusion of 2.5% of soybean oil in the diet; T5- inclusion of 5% of linseed oil in the diet; T6- inclusion of 5% of canola oil in the diet; T7-inclusion of 5% oil soy in the diet; T8- inclusion of 2.5% of linseed oil +2.5% soybean oil in the diet; T9- inclusion of 2.5% canola oil + 2.5% soybean oil in the diet and T10- inclusion of 2.5% of linseed oil + 2.5% of canola oil in the diet) and six repetition of eight birds by experimental sector, adding up to 480 birds. The ration consumption, the eggs production, the weight and mass, the alimentary conversion per dozen and per kilogram of produced eggs, specific gravity of the eggs, the strength egg shell to break, the yolk color, proportion of yolk, albumen and shell, shell thickness and yolk index were weighed up. Significant differences among the treatments for weight and eggs mass, food conversion by produced dozen eggs, specific eggs gravity, resistance to shell break, shell thickness, percentages of yolk, albumen and shell were not observed. It was concluded that the vegetal oils supplementation on the given diets to light laying commercial hens doesn’t affect the performance and the eggs quality