Sterols serve two important biological functions in animals - they act as cellular membrane components, and as the precursor to steroid hormones. Insects require a dietary source of sterol because they cannot synthesize sterols de novo. Cholesterol is the most common sterol in plant-feeding insects, but because plants contain very little cholesterol, plant-feeding insects must convert plant sterols into cholesterol. In this dissertation I investigate the effect of common and novel plant sterols and steroids found in a transgenic tobacco line on several caterpillar species. I also explore the metabolism of these sterols and steroids, and use a microarray approach to identify genes involved in sterol use and metabolism in plant-feeding insects. I also study cholesterol homeostasis using a grasshopper species. Modified tobacco plants containing a novel sterol profile negatively affected performance three different caterpillar species, especially in the second generation. Insects reared on modified plants contained less total sterols and cholesterol than those on control plants having normal sterol profile. Similar results were found using artificial diets containing atypical steroids, e.g., cholestanol and cholestan-3-one, identified in the tobacco plants that were fed to my experimental caterpillars. More importantly, the sterol/steroid ratio, but not their absolute amount in the diets, determined the negative effects. Caterpillar species could convert stigmasterol, a common plant sterol, into cholesterol. They could also convert cholestan-3-one into cholestanol and epicholestanol, although this ability varied among different species. A microarray study, that focused on gene expression in midgut tissue, indicated that stigmasterol, cholestanol and cholestan-3-one could induce different gene expression level, and that cholestan-3-one caused a the largest pool of genes to be regulated. The genes possibly involved in the metabolism of stigmasterol and cholestan-3-one were reported. These findings are important in directing further research on the potential application of plant sterol modification to control pests in agricultural systems. Insect herbivores could behaviorally regulate the intake of several nutrients, but they could not regulate their sterol intake. They did, however, practice cholesterol homeostasis, by postingestively regulating tissue sterol levels, even when feeding on diets with high cholesterol content. Collectively, the results from this dissertation provide unique insight into cholesterol regulation, which is difficult to achieve in mammals that are capable of synthesizing their own sterols.

Phytosterols are major components of food and are structurally related to cholesterol, but differ from it by virtue of a carbon substituent at the C-24 position and in some cases, a double bond between C-22 and C-23 (Figure i). Furthermore, phytosterols are shown to have protective actions against colon, breast, and prostate cancer1; further investigation is required as their mode of action is unknown. Thus, reported herein is the design and synthetic implementation required to construct these naturally occurring compounds. Figure i : Cholesterol Construction towards a double bond flanked by two asymmetric carbon atoms, observed in the phytosterol side chain, will be synthesised using an asymmetric Horner-Wittig (H-W) reaction, involving a chiral α-substituted aldehyde and a chiral β-substituted phosphine oxide. In addition to the synthesis, the stereochemical outcomes of these H-W reactions were probed. The results demonstrated, that by varying the steric bulk, electronic nature, and aromatic properties of the groups β to the phosphorus and α to the aldehyde can control the cis/trans selectivity in alkene formation. Finally, to display the utility of this methodology, the phytosterol compounds will be synthesised and tested in MD-MBA-231 cancer cell lines, allowing further investigation into the phytosterol mechanism of action.

Phytosterols are major components of food and are structurally related to cholesterol, but differ from it by virtue of a carbon substituent at the C-24 position and in some cases, a double bond between C-22 and C-23 (Figure i). Furthermore, phytosterols are shown to have protective actions against colon, breast, and prostate cancer1; further investigation is required as their mode of action is unknown. Thus, reported herein is the design and synthetic implementation required to construct these naturally occurring compounds. Figure i : Cholesterol Construction towards a double bond flanked by two asymmetric carbon atoms, observed in the phytosterol side chain, will be synthesised using an asymmetric Horner-Wittig (H-W) reaction, involving a chiral α-substituted aldehyde and a chiral β-substituted phosphine oxide. In addition to the synthesis, the stereochemical outcomes of these H-W reactions were probed. The results demonstrated, that by varying the steric bulk, electronic nature, and aromatic properties of the groups β to the phosphorus and α to the aldehyde can control the cis/trans selectivity in alkene formation. Finally, to display the utility of this methodology, the phytosterol compounds will be synthesised and tested in MD-MBA-231 cancer cell lines, allowing further investigation into the phytosterol mechanism of action.

Le tocophérol, le phytostérol, le pourcentage de protéines des graines, l'huile et les teneurs en acides gras ont été mesurés dans une population de lignées recombinantes (RILS) de tournesol, cultivées sous conditions de sécheresse, irrigation et semis tardif. Une analyse génétique de QTL a été réalisée à partir de ces mesures, en utilisant une carte génétique basée sur des marques SSR et avec des gènes candidats (1) impliqués dans la voie métabolique de tocophérol et phytostérol, (2) des gènes codant des antioxydants enzymatiques, (3) des gènes liés à la sécheresse et (4) des gènes homologues à SEC14 chez Arabidopsis. Trois gènes candidats importants (VTE4, VTE2 et HPPD), qui codent pour des enzymes impliquées dans la biosynthèse du tocophérol, ont été cartographiés sur les groupes de liaison LG8 et LG14. Quatre SNPs sont identifiés pour PAT2, le gène homologue chez Arabidopsis SEC14, entre les deux parents (PAC2 et RHA266) et un SNP, identifié par alignement de séquences est converti en marqueur CAPS pour permettre l'analyse génotypique des RIL. Les gènes homologues à SFH3, HPPD, CAT et CYP51G1 ont été cartographiés grâce à la mise au point de marqueurs dominants, tandis que des marqueurs co-dominants ont permis la cartographie des gènes homologues à SEC14-1, VTE4, DROU1, POD, SEC14-2 et AQUA. Les gènes POD, CAT et GST, codant pour des antioxydants enzymatiques, ont également été cartographiés sur les groupes de liaison 17, 8 et 1, respectivement. Le QTL majeur pour la teneur en tocophérol a été identifié sur le groupe de liaison 8, qui explique 59,5% de la variation phénotypique (6.TTC.8). Il colocalsie également avec le QTL identifié pour la teneur en phytostérol (7.TPC.8). Sous condition de semis tardif, un QTL spécifique de la teneur en acide palmitique a été identifié sur le groupe de liaison 6 (PAC-LS.6). Il est situé entre les marqueurs ORS1233 et SSL66_1. Les QTLs pour le pourcentage d'huile de graines et la teneur en acide stéarique colocalisent sur les groupes de liaison 10 (PSO-PI.10 et SAC-WI.10) et 15 (PSO-PI.15 et SAC-LS.15). Sept QTLs associés à teneur en acides palmitique, stéarique, oléique et linoléique sont identifiés sur le groupe de liaison 14. Ils sont liés à l’homologue du gène HPPD. Par ailleurs, les caractères agronomiques tels que les jours du semis à la floraison, la hauteur des plantes, le rendement et la morphologie foliaire ont été étudiés. Des analyses association génétique ont permis d’identifier des QTLs intérêts sur les groupes de liaison 2, 10 et 13 pour les caractères étudiés, d’autres QTLs identifies sur les groupes de liaison 9 et 12 mettent en avant l'importance de ces régions génomiques pour les caractères de morphologie foliaire. Nous avons finalement identifié des marqueurs AFLP et quelques gènes candidats liés aux caractères impliqués dans la qualité des graines sous conditions irriguée et stress hydrique chez une population de mutants (M8). Deux lignées mutantes, M8-826-2-1 et M8-39-2-1, produisent un niveau significativement élevé d'acide oléique peuvent être utilisées dans les programmes de sélection en raison de la haute stabilité à l'oxydation et des propriétés cardiovasculaire apportés par l’acide oléique qu’elles produisent. L'augmentation du niveau de tocophérol dans les lignées mutantes, M8-862-1N1 et M8-641-2-1, est justifiée par le polymorphisme observé pour le gène, MCT, impliqué dans la voie métabolique du tocophérol. Le marqueur le plus important pour le contenu en tocophérol total est E33M50_16 qui explique 33,9% de la variation phénotypique. Un des gènes candidats les plus importants concernant la biosynthèse des acides gras, FAD2 (FAD2-1), est lié à la teneur en acides oléique et linoléique. Il explique plus de 52% de la variation phénotypique. / The genetic control of tocopherol, phytosterol, percentage of seed protein, oil and fatty acids content in a population of recombinant inbred lines (RILs) of sunflower under various conditions are studied through QTL analysis using genetic-linkage map based on SSR markers and introducing some important tocopherol and phytosterol pathway-related genes, enzymatic antioxidant-related genes, droughtresponsive family genes and Arabidopsis SEC14 homologue genes. Three important candidate genes (HPPD, VTE2 and VTE4), which encode enzymes involved in tocopherol biosynthesis, are mapped to linkage group 8(LG8) and LG14. One of the most important candidate genes coding for sterol methyltransferase II (SMT2) enzyme is anchored to LG17 by CAPS marker. Four SNPs are identified for PAT2, Arabidopsis Sec14 homologue gene, between two parents (PAC2 and RHA266). PAT2 is assigned to LG2 by CAPS marker. Squalene epoxidase (SQE1) is also assigned to LG15 by InDel marker. Through other candidate genes, POD, CAT and GST encoding enzymatic antioxidants are assigned to LG17, LG8 and LG1, respectively. The major QTL for total tocopherol content on linkage group 8 accounted for 59.5% of the phenotypic variation (6.TTC.8), which is overlapped with the QTL of total phytosterol content (7.TPC.8). Under late-sowing condition, a specific QTL of palmitic acid content on linkage group 6 (PAC-LS.6) is located between ORS1233 and SSL66_1 markers. Common chromosomic regions are observed for percentage of seed oil and stearic acid content on linkage group 10 (PSO-PI.10 and SACWI. 10) and 15 (PSO-PI.15 and SAC-LS.15). Overlapping occurs for QTLs of oleic and linoleic acids content on linkage groups 10, 11 and 16. Seven QTLs associated with palmitic, stearic, oleic and linoleic acids content are identified on linkage group 14. These common QTLs are linked to HPPD homologue, HuCL04260C001. QTLs controlling various traits such as days from sowing to flowering, plant height, yield and leaf-related traits are also identified under well-, partial-irrigated and late-sowing conditions in a population of recombinant inbred lines (RILs). The results do emphasis the importance of the role of linkage group 2, 10 and 13 for studied traits. Genomic regions on the linkage group 9 and 12 are important for QTLs of leaf-related traits in sunflower. We finally identified AFLP markers and some candidate genes linked to seed-quality traits under well-irrigated and water-stressed conditions in gammainduced mutants of sunflower. Two mutant lines, M8-826-2-1 and M8-39-2-1, with significant increased level of oleic acid can be used in breeding programs because of their high oxidative stability and hearthealthy properties. The significant increased level of tocopherol in mutant lines, M8-862-1N1 and M8- 641-2-1, is justified by observed polymorphism for tocopherol pathway-related gene; MCT. The most important marker for total tocopherol content is E33M50_16 which explains 33.9% of phenotypic variance. One of the most important candidate genes involving fatty acid biosynthesis, FAD2 (FAD2-1), is linked to oleic and linoleic acids content and explained more than 52% of phenotypic variance.

Introdução: Os fitoesteróis são indicados no tratamento da hipercolesterolemia. Embora sua ação sobre a redução do colesterol já tenha sido exaustivamente estudada, não se encontra bem elucidada na literatura, a forma como atua sobre biomarcadores inflamatórios e endoteliais em indivíduos hipercolesterolêmicos. Objetivo: Avaliar o efeito do fitoesterol adicionado ao leite de soja sobre as concentrações plasmáticas de lípides e biomarcadores, além de alterações em vias transcricionais envolvidos na patogênese da aterosclerose em células linfomononucleares em indivíduos moderadamente hipercolesterolêmicos. Metodologia: Foram estudados 38 pacientes, recrutados no Ambulatório do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Inicialmente, os pacientes foram submetidos a um período basal de três semanas para avaliar a aderência e tolerabilidade ao leite de soja padrão (Placebo). Foram orientados a manter os hábitos alimentares e a atividade física. Receberam 400 mL/dia de leite de soja padrão ou enriquecido com fitoesteróis (1,6 g/dia) por dois períodos de quatro semanas em que o estudo foi randomizado, cego e cruzado. Resultados: O peso corporal foi mantido durante o estudo. O tratamento com fitoesterol reduziu o colesterol total em 5,5 % (P<0,001), a lipoproteína de baixa densidade (LDL) em 6,4 % e os triglicérides em 8,3 % (P<0,05) e aumentaram as concentrações de campesterol e sitosterol no plasma em 18,9% e 21,6%, respectivamente. Não houve alteração nos valores de lipoproteína de alta densidade (HDL). Com relação aos biomarcadores de aterosclerose, os fitoesteróis provocaram redução apenas na endotelina-1 em 11 % (P<0,05). Esta ação foi independente da redução do LDL-C. Não houve alteração no RNAm para 3-hydroxy-3-methylglutaryl coenzyme A redutase e receptor de LDL. Conclusão: Os fitoesteróis são eficientes na redução da concentração plasmática do colesterol total, triglicérides, LDL-C e da endotelina-1, sendo esta última independente da redução do colesterol / Background: Phytosterols are recommended in the treatment of moderate hypercholesterolemia. Although their mechanisms of action on cholesterol reduction has been exhaustively studied, other activities, such as their effects on inflammatory arterial markers and on endothelial function, have not yet been fully investigated. Objective: To evaluate the efficacy of phytosterols added to soy milk on the reduction of plasma lipids, on biomarkers involved in the pathogenesis of atherosclerosis and on transcriptional changes in 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase and low density lipoprotein receptor in moderately hypercholesterolemic individuals. Design: Moderately hypercholesterolemic patients (n=38, mean age 58 y) were initially followed over a 3-wk baseline period to evaluate their adherence to the program and their tolerance to the added milk. Patients received soy milk (400 mL/d) or soy milk + phytosterol (1.6 g/d) for 2 periods of 4-wk each. The study was randomized, blinded and crossed. Results: Body weight was maintained during the study. Phytosterol treatment reduced the total cholesterol concentration 5.5 % (P < 0.001), low density lipoprotein (LDL) 6.4 % and triglycerides 8.3 % (P<0.05), without modifying high density lipoprotein (HDL). Phytosterol reduced plasma endothelin-1 by 11 % (P<0.05), independently of LDL-C lowering activity. No effects on LDL receptor or on HMGCoA reductase mRNA expression in mononuclear blood cells were found. Sitosterol and campesterol plasma concentrations increased with phytosterol consumption by 18.9% and 21.6%, respectively. Conclusions: Phytosterol effectively lowered plasma total cholesterol, LDL-C, triglycerides and endothelin-1 concentration. The reduction in endothelin-1 concentration was independent of the decrease in the LDL-C concentration

Despite advances in healthcare, cardiovascular disease (CVD) remains the leading cause of death in the United States. Elevated levels of plasma cholesterol are highly predictive of CVD and stroke and are the principal driver of atherosclerosis. Unfortunately, current cholesterol lowering agents, such as statins, are not known to reverse atherosclerotic disease once it has been established. In preclinical models, agonists of nuclear receptor, LXR, have been shown to reduce and reverse atherosclerosis. Phytosterols are bioactive non-cholesterol sterols that act as LXR agonists and regulate cholesterol metabolism and transport. We hypothesize that stigmasterol would act as an LXR agonist and alter intestinal cholesterol secretion to promote cholesterol elimination. Mice were fed a control diet, or a diet supplemented with stigmasterol (0.3% w/w) or T0901317 (0.015% w/w), a known LXR agonist. In this experiment we analyzed the sterol content of bile, intestinal perfusate, plasma, and feces. Additionally, the liver and small intestine were analyzed for relative levels of transcripts known to be regulated by LXR. We observed that T0901317 robustly promoted cholesterol elimination and acted as a strong LXR agonist. Stigmasterol also promoted cholesterol elimination but did not alter LXR-dependent gene expression. Stigmasterol promoted transintestinal cholesterol secretion through an LXR-independent pathway.

Introdução: Os fitoesteróis são indicados no tratamento da hipercolesterolemia. Embora sua ação sobre a redução do colesterol já tenha sido exaustivamente estudada, não se encontra bem elucidada na literatura, a forma como atua sobre biomarcadores inflamatórios e endoteliais em indivíduos hipercolesterolêmicos. Objetivo: Avaliar o efeito do fitoesterol adicionado ao leite de soja sobre as concentrações plasmáticas de lípides e biomarcadores, além de alterações em vias transcricionais envolvidos na patogênese da aterosclerose em células linfomononucleares em indivíduos moderadamente hipercolesterolêmicos. Metodologia: Foram estudados 38 pacientes, recrutados no Ambulatório do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Inicialmente, os pacientes foram submetidos a um período basal de três semanas para avaliar a aderência e tolerabilidade ao leite de soja padrão (Placebo). Foram orientados a manter os hábitos alimentares e a atividade física. Receberam 400 mL/dia de leite de soja padrão ou enriquecido com fitoesteróis (1,6 g/dia) por dois períodos de quatro semanas em que o estudo foi randomizado, cego e cruzado. Resultados: O peso corporal foi mantido durante o estudo. O tratamento com fitoesterol reduziu o colesterol total em 5,5 % (P<0,001), a lipoproteína de baixa densidade (LDL) em 6,4 % e os triglicérides em 8,3 % (P<0,05) e aumentaram as concentrações de campesterol e sitosterol no plasma em 18,9% e 21,6%, respectivamente. Não houve alteração nos valores de lipoproteína de alta densidade (HDL). Com relação aos biomarcadores de aterosclerose, os fitoesteróis provocaram redução apenas na endotelina-1 em 11 % (P<0,05). Esta ação foi independente da redução do LDL-C. Não houve alteração no RNAm para 3-hydroxy-3-methylglutaryl coenzyme A redutase e receptor de LDL. Conclusão: Os fitoesteróis são eficientes na redução da concentração plasmática do colesterol total, triglicérides, LDL-C e da endotelina-1, sendo esta última independente da redução do colesterol / Background: Phytosterols are recommended in the treatment of moderate hypercholesterolemia. Although their mechanisms of action on cholesterol reduction has been exhaustively studied, other activities, such as their effects on inflammatory arterial markers and on endothelial function, have not yet been fully investigated. Objective: To evaluate the efficacy of phytosterols added to soy milk on the reduction of plasma lipids, on biomarkers involved in the pathogenesis of atherosclerosis and on transcriptional changes in 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase and low density lipoprotein receptor in moderately hypercholesterolemic individuals. Design: Moderately hypercholesterolemic patients (n=38, mean age 58 y) were initially followed over a 3-wk baseline period to evaluate their adherence to the program and their tolerance to the added milk. Patients received soy milk (400 mL/d) or soy milk + phytosterol (1.6 g/d) for 2 periods of 4-wk each. The study was randomized, blinded and crossed. Results: Body weight was maintained during the study. Phytosterol treatment reduced the total cholesterol concentration 5.5 % (P < 0.001), low density lipoprotein (LDL) 6.4 % and triglycerides 8.3 % (P<0.05), without modifying high density lipoprotein (HDL). Phytosterol reduced plasma endothelin-1 by 11 % (P<0.05), independently of LDL-C lowering activity. No effects on LDL receptor or on HMGCoA reductase mRNA expression in mononuclear blood cells were found. Sitosterol and campesterol plasma concentrations increased with phytosterol consumption by 18.9% and 21.6%, respectively. Conclusions: Phytosterol effectively lowered plasma total cholesterol, LDL-C, triglycerides and endothelin-1 concentration. The reduction in endothelin-1 concentration was independent of the decrease in the LDL-C concentration

A aterosclerose é um processo inflamatório que se inicia na infância e progride com a idade, sendo o principal processo patológico que culmina nas doenças cardiovasculares. Ácidos graxos ômega 3 (N-3 FA) reduzem triacilgliceróis plasmáticos e inflamação, enquanto fitosteróis possuem ação hipocolesterolêmica. Portanto, o objetivo deste estudo foi de aplicar a combinação dessas duas classes de lipídios bioativos na infância, visando reduzir a aterosclerose na fase adulta. O estudo foi dividido em duas etapas. Inicialmente, o efeito de três ingredientes contendo N-3 FA foi avaliado em termos de perfil lipídico e biomarcadores inflamatórios. Camundongos adultos LDLr Knockout receberam uma dieta hiperlipídica e foram simultaneamente suplementados com água (CON), óleo de algas (ALG), óleo de peixe (FIS) e óleo de Echium (ECH) através de gavagem durante 4 semanas. Os animais suplementados com óleo de Echium apresentaram os maiores índices de redução de triacilgliceróis e VLDL, além de inibição da esteatose causada pelo dieta hiperlipídica. Entretanto, diferente dos óleos marinhos, tais efeitos não envolveram ativação de fatores de transcrição envolvidos no metabolismo lipídico, como PPAR&#945; e LXR&#945; hepáticos. Na segunda etapa deste estudo, camundongos Knockout LDLr recém-desmamados foram suplementados durante 2 meses com emulsões preparadas com óleo de soja (CON), óleo de de Echium (ECH), óleo de alga (ALG), fitosteróis isolados (PHY), óleo de alga + fitosteróis (ALG+PHY) e óleo de Echium + fitosteróis (ECH+PHY). A seguir, dislipidemia e estresse oxidativo foram induzidos através de uma dieta hiperlipídica por mais 2 meses. Todos os animais apresentaram estrias gordurosas na aorta, sendo que a área de lesão foi maior naqueles suplementados com fitosteróis isoladamente (PHY). Entretanto, esse efeito negativo foi totalmente revertido pela co-suplementação com N-3 FA. Observou-se que os fitosteróis isolados agiram como agonistas de LXR&#945;, e que a reversão proporcionada pelos N-3 FA envolveu aumento da expressão do fator de transcrição PPAR&#945; e redução do aumento de LXR&#945;. Além disso, o óleo de Echium reduziu o estresse oxidativo no fígado através de mecanismos associados à modulação da atividade e expressão de enzimas antioxidantes. Nossos resultados sugerem que a melhor alternativa no desenvolvimento de alimentos funcionais para crianças, visando prevenção de aterosclerose na fase adulta, foi conferida pelos N-3 FA provenientes do óleo de Echium. / Atherosclerosis is an inflammatory process that begins in childhood and progresses with age, being the main pathological process that culminates in cardiovascular disease. Omega 3 fatty acids (n-3 FA) reduces triacylglycerol (TG) and inflammation, while phytosterols present hypocholesterolemic action. Therefore, the aim of this study was to apply the combination of these two classes of bioactive lipids in childhood to reduce atherosclerosis in adulthood. The study was divided into two steps. Initially, the effect of three ingredients containing n-3 FA was evaluated in terms of lipid and inflammatory biomarkers. LDLr knockout adult mice received a high-fat diet and were simultaneously supplemented with water (CON), algae oil (ALG), fish oil (FIS) and Echium oil (ECH) by gavage for 4 weeks. The animals supplemented with Echium oil presented higher reduction of TG and VLDL. Besides, Echium oil inhibited hepatic steatosis caused by high-fat diet. However, unlike of marine oils, this hypotriglyceridemic effect did not involve activation of transcription factors associated to lipid metabolism, such as PPAR&#945; and LXR&#945;. At the second step of this study, weaning LDLr knockout mice were supplemented with emulsions prepared with soybean oil (CON), Echium oil (ECH), algae oil (ALG), isolated phytosterols (PHY), algae oil + phytosterols (ALG + PHY) and Echium oil + phytosterols (PHY + ECH) for 2 months. Thereafter, dyslipidemia and oxidative stress were induced by a high fat diet for 2 months more. All animals showed fatty streaks in the aorta artery. The lesion area was greatest in mice supplemented with isolated phytosterol (PHY). However, this negative effect was completely reversed by co-supplementation with n-3 FA. It was observed that isolated phytosterols acted as LXR&#945; agonists, and that the reversal provided by the N-3 FA involved the increase of PPAR&#945; and reduction of LXR&#945; expression. Furthermore, the Echium oil reduced oxidative stress in liver through mechanisms associated with modulation of antioxidant enzymes activity and expression. Our results suggest that the best alternative to develop functional foods for children, aimed at preventing atherosclerosis in adulthood, was conferred by the N-3 FA from Echium oil.

A aterosclerose é um processo inflamatório que se inicia na infância e progride com a idade, sendo o principal processo patológico que culmina nas doenças cardiovasculares. Ácidos graxos ômega 3 (N-3 FA) reduzem triacilgliceróis plasmáticos e inflamação, enquanto fitosteróis possuem ação hipocolesterolêmica. Portanto, o objetivo deste estudo foi de aplicar a combinação dessas duas classes de lipídios bioativos na infância, visando reduzir a aterosclerose na fase adulta. O estudo foi dividido em duas etapas. Inicialmente, o efeito de três ingredientes contendo N-3 FA foi avaliado em termos de perfil lipídico e biomarcadores inflamatórios. Camundongos adultos LDLr Knockout receberam uma dieta hiperlipídica e foram simultaneamente suplementados com água (CON), óleo de algas (ALG), óleo de peixe (FIS) e óleo de Echium (ECH) através de gavagem durante 4 semanas. Os animais suplementados com óleo de Echium apresentaram os maiores índices de redução de triacilgliceróis e VLDL, além de inibição da esteatose causada pelo dieta hiperlipídica. Entretanto, diferente dos óleos marinhos, tais efeitos não envolveram ativação de fatores de transcrição envolvidos no metabolismo lipídico, como PPAR&#945; e LXR&#945; hepáticos. Na segunda etapa deste estudo, camundongos Knockout LDLr recém-desmamados foram suplementados durante 2 meses com emulsões preparadas com óleo de soja (CON), óleo de de Echium (ECH), óleo de alga (ALG), fitosteróis isolados (PHY), óleo de alga + fitosteróis (ALG+PHY) e óleo de Echium + fitosteróis (ECH+PHY). A seguir, dislipidemia e estresse oxidativo foram induzidos através de uma dieta hiperlipídica por mais 2 meses. Todos os animais apresentaram estrias gordurosas na aorta, sendo que a área de lesão foi maior naqueles suplementados com fitosteróis isoladamente (PHY). Entretanto, esse efeito negativo foi totalmente revertido pela co-suplementação com N-3 FA. Observou-se que os fitosteróis isolados agiram como agonistas de LXR&#945;, e que a reversão proporcionada pelos N-3 FA envolveu aumento da expressão do fator de transcrição PPAR&#945; e redução do aumento de LXR&#945;. Além disso, o óleo de Echium reduziu o estresse oxidativo no fígado através de mecanismos associados à modulação da atividade e expressão de enzimas antioxidantes. Nossos resultados sugerem que a melhor alternativa no desenvolvimento de alimentos funcionais para crianças, visando prevenção de aterosclerose na fase adulta, foi conferida pelos N-3 FA provenientes do óleo de Echium. / Atherosclerosis is an inflammatory process that begins in childhood and progresses with age, being the main pathological process that culminates in cardiovascular disease. Omega 3 fatty acids (n-3 FA) reduces triacylglycerol (TG) and inflammation, while phytosterols present hypocholesterolemic action. Therefore, the aim of this study was to apply the combination of these two classes of bioactive lipids in childhood to reduce atherosclerosis in adulthood. The study was divided into two steps. Initially, the effect of three ingredients containing n-3 FA was evaluated in terms of lipid and inflammatory biomarkers. LDLr knockout adult mice received a high-fat diet and were simultaneously supplemented with water (CON), algae oil (ALG), fish oil (FIS) and Echium oil (ECH) by gavage for 4 weeks. The animals supplemented with Echium oil presented higher reduction of TG and VLDL. Besides, Echium oil inhibited hepatic steatosis caused by high-fat diet. However, unlike of marine oils, this hypotriglyceridemic effect did not involve activation of transcription factors associated to lipid metabolism, such as PPAR&#945; and LXR&#945;. At the second step of this study, weaning LDLr knockout mice were supplemented with emulsions prepared with soybean oil (CON), Echium oil (ECH), algae oil (ALG), isolated phytosterols (PHY), algae oil + phytosterols (ALG + PHY) and Echium oil + phytosterols (PHY + ECH) for 2 months. Thereafter, dyslipidemia and oxidative stress were induced by a high fat diet for 2 months more. All animals showed fatty streaks in the aorta artery. The lesion area was greatest in mice supplemented with isolated phytosterol (PHY). However, this negative effect was completely reversed by co-supplementation with n-3 FA. It was observed that isolated phytosterols acted as LXR&#945; agonists, and that the reversal provided by the N-3 FA involved the increase of PPAR&#945; and reduction of LXR&#945; expression. Furthermore, the Echium oil reduced oxidative stress in liver through mechanisms associated with modulation of antioxidant enzymes activity and expression. Our results suggest that the best alternative to develop functional foods for children, aimed at preventing atherosclerosis in adulthood, was conferred by the N-3 FA from Echium oil.

Orientador: Mário Roberto Maróstica Júnior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T22:33:18Z (GMT). No. of bitstreams: 1 Furlan_CibelePriscilaBusch_M.pdf: 1142828 bytes, checksum: b13310e35dd9859ae5548ba7a4d91a00 (MD5) Previous issue date: 2013 / Resumo: Obesidade se tornou o maior problema de saúde pública ao redor no mundo e é associado, geralmente, com doenças crônicas não transmissíveis como a diabetes. Alguns componentes dos alimentos estão relacionados com benefícios à saúde como modulação do estresse oxidativo, hormônios e inflamação. O ácido linoléico conjugado (CLA) é um ácido graxo que pode exercer papel antioxidante, possivelmente pela influencia na auto oxidação lipídica. Outros lipídios, esteroidais das plantas, os fitosteróis são considerados também compostos bioativos, uma vez que eles são responsáveis pela redução da circulação do LDL colesterol, bem como pela redução do peso corporal. Em adição, esses compostos são anti-inflamatórios, e apresentam importante potencial para prevenir efeitos adversos da obesidade. Nessas condições, o objetivo do presente trabalho foi investigar os efeitos da suplementação do CLA e fitosterol, sozinhos e em combinação, em relação aos hormônios da saciedade, glicose, resistência a insulina, perda de peso, consumo e os efeitos no fígado e sua influencia no sistema antioxidante de defesa em ratos machos Sprague-Dawley alimentados com dieta hiperlipídica.Quarenta ratos Sprague-Dawley recém-desmamados foram divididos em cinco grupos (n=8) aleatoriamente: grupo Padrão, grupo Hiperlipídico Padrão, e três grupos hiperlipídicos com adição de 2% de CLA, 2% de fitosterol e 2% de CLA mais 2% de fitosterol. Todas as dietas tiveram como base a AIN-93G, sendo as dietas experimentais high-fat adicionadas de 35% de banha animal e 2% de cada suplemento. O tempo de experimento foi de 65 dias. O grau de sensibilidade à insulina foi calculado nas últimas semanas experimentais, por meio dos métodos Teste de Tolerância à Glicose e Teste de Tolerância à Insulina. O acompanhamento do ganho de peso e consumo foi feito por pesagem. As análises das enzimas do estresse oxidativo, 8-F2-Isoprostana e hormônios foram determinadas utilizando kits comerciais, apenas as análises de malondialdeído foram feitas pelo método de TBARS. A dieta suplementada com CLA, fitosterol ou a combinação de ambos, por 65 dias foi efetiva na redução da massa gorda, dos tecidos adiposos e do consumo alimentar. Ademais, CLA mas não o fitosterol modulou a ação da leptina na obesidade. No entanto, os suplementos apresentaram efeito na modulação das enzimas do estresse oxidativo indicando possível dano mitocontrial e citotoxicidade, além de não reverterem o processo de intolerância a glicose e resistência a insulina. Por outro lado, os suplementos reduziram as gotas de gordura no fígados dos animais suplementados / Abstract: Obesity is one of the most serious public health problems worldwide and is generally associated with chronic diseases such as diabetes. Some food compounds are linked to benefits in health as they modulate oxidative stress, hormones and inflammation. Conjugated linoleic acid (CLA) is a fatty acid that could exert an antioxidant role, possibly by its influence on lipid auto-oxidation. Other lipids, steroidal plant, the phytosterols are considered also bioactive compounds, since they are responsible for the reduction of LDL cholesterol circulation, as well as reduction in body weight. In addition, these compounds are considered to play anti-inflammatory roles, and they are potentially important in preventing the adverse effects of obesity. Thus, the aim of the present study was to investigate the effect of CLA and phytosterol supplementation, alone or in combination, in relation to satiety hormones, glucose and insulin resistance, weight loss and adipose tissue, and feed consumption and liver effects and their influence in the modulation of antioxidant system on male Sprague-Dawley rats fed a high-fat diet. Forty rats of the Sprague-Dawley specie, males and healthy were divided into five groups (n=8) randomly: standad group, High-Fatstandad group, and three groups that received the standard experimental high-fat diet with addition of 2% CLA, with addition of 2% phytosterol and with addition of 2% CLA plus 2% phytosterols. All diets were based on the AIN- 93G, and experimental diet high-fat add of 35% lipids, and 2% each supplement. The period of experiment was 65 days. The insulin sensitivity was calculated in the last week experimental by the methods Tolerance Test Glucose and Insulin Tolerance Test. The monitoring of consumption and weight gain was made by weighing. The analysis of the enzymes of oxidative stress, 8-F2- isoprostane and hormones were determined using commercial kits, only the analyzes of malondialdehyde were made by the method of TBARS. The diet supplementations with CLA, phytosterols or their combination, for 65 days were effective in reducing body fat, adipose tissue and feed consumption. Furthermore, CLA, but not phytosterols, modulated the action of leptin in obesity. However, the supplementation had an effect in modulating the enzymes of oxidative stress indicating possible damage and cytotoxicity mitocontrial, moreover both supplements did not reverse the process of glucose intolerance and insulin. On the other hand, the supplementation reduced the liver fat droplets of the animals / Mestrado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestra em Alimentos e Nutrição