L’obésité, le diabète de type 2 et l’athérosclérose sont les principaux problèmes de santé publique affectant les pays développés. Bien que de nombreux traitements soient disponibles contre ces maladies, de nombreux progrès sont encore nécessaire pour le développement de composés plus actif et plus sûr. Mon laboratoire a montré que l’activation du récepteur aux acides biliaires TGR5 par ses ligands entrainait une augmentation de la dépense énergétique et réduisait le niveau des cytokines chez la souris, ce qui pourrait être une nouvelle voie vers le traitement de ces désordres métaboliques. Nous décrivons ici le développement d’un nouvel agoniste synthétique, spécifique et puissant pour TGR5. A partir d’une librairie de 20.000 composés, les composés 50980906, 13008574 et 37525283 ont été caractérisés comme les plus puissants et stables. Le composé 13008574 a montré une réduction significative sur la prise de poids de souris C57BL/6J après un régime alimentaire riche en graisses. Suite à l’activation de TGR5, nous avons observés une augmentation du niveau d'expression des gènes Ucp-1, Dio-2 et Cpt-1 dans le tissu adipeux brun et une augmentation de la clairance du glucose suite à une augmentation de la sécrétion de GLP-1 chez les souris traitées par le composé 13008574. Nous avons également montrés que le composé 13008574 n’a pas d’effet sur les souris TGR5-/- témoignant de sa spécificité. Enfin nous avons par ailleurs confirmé l'effet du composé 13008574 comme agent anti-inflammatoire, avec un effet protecteur face au développement de l'athérosclérose. Notre travail montre ainsi que le développement d’agonistes pour TGR5, puissant et sûr, est possible pour traiter le syndrome métabolique. / Obesity, type 2 diabetes and atherosclerosis, are amongst the main driving factors of a public health crisis that impacts developed countries. Although several drugs are available, there is still a large unmet medical need to find better and safer compounds to treat these diseases. In this context, my host laboratory discovered that activation of the membrane bile acid receptor TGR5 induces energy expenditure and reduces inflammation in mice, which would be beneficial to manage the above–mentioned disorders. INT-777, a semi-synthetic bile acid, is until now, one of the most specific TGR5 ligands. Here, we report the identification of a new synthetic, selective and potent TGR5 agonist. From a screen of 20,000 compounds as potential TGR5 activators, the compounds 50980906, 13008574 and 37525283 were the most potent and stable. In particular, 13008574 induced a significant reduction of body weight gain when C57BL/6J mice were exposed to a high fat diet, paralleled by an increase in the expression levels of Ucp-1, Dio-2 and Cpt-1 in brown adipose tissue. In addition, mice treated with 13008574 displayed improved glucose clearance, consequent to increased GLP-1 secretion. We showed furthermore that the effects of 13008574 were lost in TGR5-/- mice, testifying the specificity of the compound. In addition, 13008574 acts as an anti-inflammatory agent, with a protective effect on atherosclerosis development in LDLr-/- mice treated with a high cholesterol diet. Our work hence shows that potent, selective, and safe TGR5 agonists can be developed to cure the metabolic syndrome.

L’homéostasie énergétique ou ‘balance énergétique’ est l’équilibre qui s’établit chez l’Homme et l’animal adulte entre la prise quotidienne de nutriments sous la forme de glucides, de lipides ou de protéines et leur oxydation pour ne produire que la quantité énergétique strictement nécessaire. Pour maintenir cette balance l’organisme doit recueillir en permanence des signaux nerveux, métaboliques ou hormonaux de la part de cellules spécifiques. Ces senseurs des besoins énergétiques transmettent alors à des centres régulateurs leurs informations qui en retour, par voie hormonale ou nerveuse, informent les organes effecteurs des mesures à prendre pour stocker, produire ou consommer l’énergie. Les trois principaux centres de cette balance sont 1/ le cerveau, centre intégrateur de l’information ; 2/ un groupe d’organes effecteurs parmi lesquels le foie, le tissu adipeux, les muscles squelettiques, le pancréas et 3/ un centre senseur de la qualité et de la quantité des aliments, le tractus gastrointestinal (Migrenne 2006). En plus d’être la source d’énergie nécessaire à la vie des cellules, les nutriments tels que les acides gras, le cholestérol ou encore le glucose sont aussi des molécules de signalisation cellulaire à la fois par leur fixation à des récepteurs membranaires mais aussi via des récepteurs nucléaires. Un déséquilibre dans l’homéostasie énergétique dû à une alimentation déséquilibrée, à un manque d’exercice physique ou à des facteurs génétiques est une caractéristique de l’obésité et de complications telles que le diabète de type 2 et les maladies cardiovasculaires (Hill, 2006). Au cours de ma thèse je me suis intéressé à l’intestin pour son rôle de régulateur de l’homéostasie énergétique dans un contexte physiologique ou physiopathologique d’obésité via sa capacité à sécréter l’incrétine Glucagon-Like Peptide-1 (GLP-1) en réponse au glucose et aux acides biliaires. J’ai étudié plus particulièrement le rôle du récepteur nucléaire en tant que senseurs moléculaires des acides biliaires FXR dans les cellules sécrétrices de cette incrétine car à l’heure actuelle rien n’était connu quant à son rôle ni même quant à son expression dans la cellule L. Pour cela, j’ai utilisé des lignées cellulaires murines et humaines où j’ai mis au point les conditions expérimentales pour répondre aux questions posées. Grâce à des ARN d’intestins issus de différents modèles de souris la relevance chez le rongeur a été testée. La relevance de ces résultats sur des biopsies intestinales humaine a également été testée. Grâce à ces outils, j'ai pu montré que FXR dans les cellules L était fonctionnel et que son activation interférait avec la voie de la glycolyse entrainant moins de synthèse et de sécrétion de GLP-1. Cela nous a permis de proposer un nouveau mécanisme moléculaire par lequel les séquestrants des acides biliaires exercent leur effets bénéfiques chez des patients atteints de diabète de type 2. / Originally identified as dietary lipid detergents, bile acids (BA) are now recognized as signaling molecules which bind to the transmembrane receptor TGR5 and the nuclear receptor FXR (Farnesoid X Receptor). Upon binding to TGR5 at the surface of enteroendocrine L cells, bile acids (BA) promote the secretion of the incretin GLP-1 which potentiates the glucose-induced insulin secretion by pancreatic beta-cells. More than 50% of the insulin secretion in response to glucose is mediated by GLP-1 and the other incretin Glucose-dependent Insulinotropic Polypeptide (GIP). Once secreted, GLP-1 is rapidly (2-3 minutes) degraded by the endothelial enzyme Dipeptydil Peptidase 4 (DPP4). GLP-1 analogues and DPP4 inhibitors are successfully used for the treatment of T2D. FXR is a ligand-activated nuclear receptor highly expressed in the liver and in the distal intestine. FXR controls BA, lipid and glucose metabolism. Whether FXR is expressed, functional in intestinal enteroendocrine L cells and in which extend its activation affects GLP-1 production are not yet reported. Encouraging data were obtained during my M2 training course. The aim of my thesis was thus to assess whether FXR in enteroendocrines cells could participate in the control of the deregulation of glucose homeostasis. Multiple in vitro, ex vivo and in vivo human and murine models allowed us to show that FXR is present and functional in L cells. FXR activation decreases GLP-1 production and secretion in L cells by inhibiting glycolysis pathway through an interference with the carbohydrate responsive transcription factor ChREBP. Finally, I identified an additional mechanism of action of the bile acid sequestrant Colesevelam, a molecule currently successfully used in USA for treating type 2 diabetic patients.

<p>The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism. </p><p>Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21. </p><p>Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme. </p><p>Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression.</p><p>The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.</p>

The present investigation is devoted to studies on porcine members of the cytochrome P450 4A (CYP4A) and CYP8B1 subfamilies, which are involved in bile acid biosynthesis and fatty acid metabolism. Hyocholic acid is considered to fulfil the requirements for trihydroxy bile acids in the domestic pig (Sus scrofa) in the absence of cholic acid. Hyocholic acid is a 6α-hydroxylated product of chenodeoxycholic acid and the enzyme catalyzing the 6α-hydroxylation was cloned and found to be an atypical member of the CYP4A subfamily. The primary structure of this porcine enzyme, designated CYP4A21, shows about 75% overall sequence identity to members of the CYP4A subfamily expressed in rabbit and man. Divergent amino acids in a “signature sequence” in the active site of all hitherto known CYP4A fatty acid hydroxylases, were found to be important determinants for the 6α-hydroxylase activity of CYP4A21. Two homologous CYP4A fatty acid hydroxylases, designated CYP4A24 and CYP4A25, expressed in pig liver and kidney were cloned. These two cDNAs encode proteins of 504 amino acids similar to CYP4A21. The overall identity between CYP4A24 and CYP4A25 is 97% compared to 94% identity to CYP4A21. Whereas CYP4A21 clearly deviates regarding structural features and catalytic activity it is more difficult to establish whether CYP4A24 and CYP4A25 are distinct enzymes or allelic variants of a single enzyme. Cloning of the CYP4A21 gene showed a conserved organization compared to CYP4A genes in other species. A segment of the CYP4A24 gene was also cloned and comparison with the CYP4A21 gene revealed an extensive sequence identity also within introns as well as within the proximal promoter regions. This indicates that CYP4A21 and CYP4A fatty acid hydroxylases have a common origin and evolved by gene duplication. The CYP4A21 and CYP4A fatty acid hydroxylases, however, show distinct patterns of expression. The key enzyme in cholic acid biosynthesis, CYP8B1, was markedly expressed in fetal pig liver compared to livers from young pigs. The opposite was shown for the expression of CYP4A21. An apparently conserved pig CYP8B1 gene was cloned and was intronless, similar to CYP8B1 genes from other species. The pig gene encoded a protein of 501 amino acids with 81% identity to CYP8B1 expressed in rabbit and man. Unlike other CYP8B1 genes, the pig promoter lacked a TATA-box. This might offer one explanation for the unusual expression pattern, which appears to be restricted to pig fetal life.

La préparation de polymères à base d’acides biliaires, molécules biologiques, a attiré l'attention des chercheurs en raison des applications potentielles dans les domaines biomédicaux et pharmaceutiques. L’objectif de ce travail est de synthétiser de nouveaux biopolymères dont la chaîne principale est constituée d’unités d’acides biliaires. La polymérisation par étapes a été adoptée dans ce projet afin de préparer les deux principales classes de polymères utilisés en fibres textiles: les polyamides et les polyesters. Des monomères hétéro-fonctionnels à base d’acides biliaires ont été synthétisés et utilisés afin de surmonter le déséquilibre stoechiométrique lors de la polymérisation par étapes. Le dérivé de l’acide lithocholique modifié par une fonction amine et un groupement carboxylique protégé a été polymérisé en masse à températures élevées. Les polyamides obtenus sont très peu solubles dans les solvants organiques. Des polyamides et des polyesters solubles en milieu organique ont pu être obtenus dans des conditions modérées en utilisant l’acide cholique modifié par des groupements azide et alcyne. La polymérisation a été réalisée par cycloaddition azoture-alcyne catalysée par l'intermédiaire du cuivre(Ι) avec deux systèmes catalytiques différents, le bromure de cuivre(I) et le sulfate de cuivre(II). Seul le bromure de cuivre(Ι) s’est avéré être un catalyseur efficace pour le système, permettant la préparation des polymères avec un degré de polymérisation égale à 50 et une distribution monomodale de masse moléculaire (PDI ˂ 1.7). Les polymères synthétisés à base d'acide cholique sont thermiquement stables (307 °C ≤ Td ≤ 372 °C) avec des températures de transition vitreuse élevées (137 °C ≤ Tg ≤ 167 °C) et modules de Young au-dessus de 280 MPa, dépendamment de la nature chimique du lien. / Bile acids have drawn attention in the synthesis of polymers for biomedical and pharmaceutical applications due to their natural origin. The objective of this work is to synthesize main-chain bile acid-based polymers. The step-growth polymerization was used to prepare two important classes of polymers used in textile fibers, polyamides and polyesters. Heterofunctional bile acid-based monomers were synthesized and used in order to overcome stoichiometric imbalances during step-growth polymerization. The lithocholic acid derivative bearing amine and protected carboxylic functional groups was polymerized in bulk at high temperatures, yielding polyamides that were poorly soluble in common organic solvents. Soluble triazole-linked polyamides and polyesters were obtained when the cholic acid derivative bearing azide and alkyne functional groups was polymerized under mild conditions via copper(Ι)-catalyzed azide-alkyne cycloaddition. Two different catalytic systems, copper(Ι) bromide and copper(ΙΙ) sulfate, were tested. Only copper(Ι) bromide proved to be an effective catalyst for the system, allowing the synthesis of the polymers with a degree of polymerization of ca. 50 and an unimodal molecular weight distribution(PDI ˂ 1.7). The main-chain cholic acid-based polymers are thermally stable (307 °C ≤ Td ≤ 372 °C) with high glass transition temperatures (137 °C ≤ Tg ≤ 167 °C) and Young’s moduli in excess of 280 MPa, depending on the chemical structure of the linker.

Es wurden 44 Hunde mit einem kongenitalen Portosystemischen Shunt präoperativ hinsichtlich ihrer Leberzellintegrität (ALT, AP, GLDH, GGT) und ihrer hepatischen Synthese- (Harnstoff, Cholesterin, Albumin) bzw. Metabolisierungsrate (Ammoniak) untersucht. Die hepatische Durchblutungs- und Resorptionsrate wurde anhand des Verlaufs der Serumgallensäuren im Gallensäuren-Stimulationstest mit Ceruletid (0,3µg/kg KM i.v.)vor und 30 min nach Stimulation (FSBA; PSBA) beurteilt. Eine Verlaufsuntersuchung erfolgte bei den Hunden, bei denen das Shuntgefäß in zwei Operationen verschlossen worden war. Die Untersuchungen wurden jeweils prae operationem, am zweiten, vierten und siebten Tag post operationem sowie abschließend mindestens 120 Tage nach vollständigem Verschluss des Shuntgefäßes durchgeführt. Es wurden die Ergebnisse der einzelnen Untersuchungstage untereinander mit Hilfe des gepaarten t-Tests sowie mit einer Kontrollgruppe (63 lebergesunde Hunde)unter Anwendung des unpaaren t-Tests verglichen. / The hepatic enzymes: alanine aminotransferase (ALT), alkaline phosphatase (AP), glutamat dehydrogenase (GLDH), gamma-glutamyl transferase (GGT); the hepatic synthetic (urea, cholesterol, albumin) and metabolic activity (ammonia) and the hepatic blood flow (serum bile acid stimulation test) were determined in 44 dogs with congeintal portosystemic shunt and in 63 healthy dogs. After determination of fasting serum bile acids (FSBA), the gallbladder contraction was induced by administration of 0,3µg/kg iv ceruletide (Takus). Blood samples of the poststimulatin serum bile acids (PSBA) were taken 30 minutes post administration. The portosystemic shunt was first attenuated (surgery 1) and 4 weeks later completely ligated (surgery 2). All dogs treated with this surgical procedure were examined with the described laboratory design before surgery, the second, the fourth, the seventh day after surgery and approxiamtely 120 days after complete ligation in a follow up study. To compare the developmentof the biochemical and hepatic alterations the paired and unpaired t-test were used.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / This paper describes obtaining eight lithocholic acid derivatives, as well as inhibitory activity. Interest in the use of lithocholic acid as starting material for the preparation of derivatives arose because of it being widely studied in order to discover new biological activities and such studies have shown good results. In this work it was used for the synthesis of a series of derivatives with modifications at the C-3 and/or C-24 of the steroid skeleton. The series was prepared using simple chemical and showed good yields. Aiming to investigate the antibacterial activity of the same and its derivatives, aiming to structure-activity relationships, tests were performed with bacteria Escherichia coli, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa, try some of them presented significant results. And lithocholic acid derivatives were characterized by spectroscopic IR, 1H NMR, 13C-BB NMR and 13C-DEPT NMR and mass spectrometry as well as comparison with data in the literature and described constitute the body of the dissertation. / Esse trabalho descreve a obtenÃÃo de oito derivados do Ãcido litocÃlico, bem como, suas atividades antimicrobianas. O interesse pela utilizaÃÃo do Ãcido litocÃlico como material de partida na preparaÃÃo de derivados surgiu pelo fato do mesmo sendo bastante estudado com o objetivo de descobrir novas atividades biolÃgicas e tais estudos terem apresentado bons resultados. Nesse trabalho, o Ãcido litocÃlico foi submetido a modificaÃÃes moleculares nas posiÃÃes C-3 e/ou C-24 do esqueleto esteroidal. A sÃrie foi preparada utilizando quÃmica convencional e apresentando bons rendimentos. Com o objetivo de investigar a atividade antimicrobiana dos compostos, foram realizados testes com as bactÃrias Escherichia coli, Staphylococcus aureus, Bacillus cereus e Pseudomonas aeruginosa, tento alguns deles apresentado resultados bastante significativos. O Ãcido litocÃlico e seus derivados foram caracterizados por mÃtodos espectroscÃpicos de IV, RMN 1H, RMN 13C-BB e RMN 13C-DEPT e por espectrometria de massa, bem como comparaÃÃo com dados descritos na literatura.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-03-28T11:47:33Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-03-28T17:39:03Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-03-28T17:39:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-02-20 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As leishmanioses são um conjunto de doenças infecciosas causadas por protozoários do gênero Leishmania sp, responsáveis por um grande impacto socioeconômico mundial. Os tratamentos disponíveis para leishmanioses são limitados por apresentarem alta toxicidade, administração parenteral prolongada, alto custo e efeitos colaterais. Os esteroides constituem um grupo de compostos bastante diversificados com variada aplicabilidade na farmacologia. No entanto, a atividade antileishmanial desta classe ainda é pouco explorada. No presente trabalho, investigamos os efeitos antileishmanial de derivados sintéticos do colesterol e dos ácidos biliares cólico e desoxicólico. Além disso, determinamos, in vitro, possíveis alvos/alterações celulares importantes na atividade biológica do composto mais promissor, bem como analisamos in silico sua biodisponibilidade oral e propriedades farmacocinéticas. A atividade antipromastigota em Leishmania sp e citotoxicidade em macrófagos peritoneais foram determinadas pelo método colorimétrico MTT. A atividade antiamastigota foi avaliada em macrófagos infectados com L. amazonensis RFP após 72 horas de tratamento através de fluorimetria. Os efeitos do melhor composto em alvos celulares foram realizados utilizando marcação fluorescentes em promastigotas tratadas, e as análises in silico foram realizadas através de uma simulação computacional. Em uma análise geral dos resultados obtidos pelos testes in vitro, observa-se que entre os derivados esteroidais testados neste trabalho, os derivados do ácido desoxicólico apresentaram o melhor efeito antileishmanial para as formas promastigotas e amastigotas in vitro. Dentre esta série de compostos, o composto 11d (CI50 = 15,04 μM) é o mais seletivo para amastigotas de L. amazonensis (IS >9,78) e o mais específico para a forma intracelular desta espécie. Este composto tem como provável alvo celular a mitocôndria, aumentando a produção de EROs e diminuindo o potencial de membrana mitocondrial. Além do mais, o composto mostrou causar danos no conteúdo de DNA do parasito. Estudos das propriedades físico-químicas e farmacocinéticas do composto 11d mostrou que ele possui capacidade de permear por membranas biológicas sendo possível a administração por via oral. / Leishmaniasis is a group of infectious diseases caused by protozoa of the genus Leishmania sp, responsible for a great socioeconomic impact worldwide. The available treatments for leishmaniasis are limited because they present high toxicity, parenteral administration, high cost and side effects. Steroids are a diversified group of compounds with varied applicability in pharmacology. However, the antileishmanial activity of this class is still little explored. In the present work, we investigated the antileishmanial effects of groups of steroidal derivatives: cholesterol; colic and deoxycholic acid. In addition, we determined, in vitro, possible important cellular targets/alterations in the biological activity of the most promising compound and in silico analysis of its oral bioavailability and pharmacokinetics. The antipromastigote activity in Leishmania sp and cytotoxicity in peritoneal macrophages were determined by the colorimetric method MTT. The antiamastigote activity was evaluated in with L. amazonensis-infected macrophages after 72 hours of treatment. The effects of the best compound on promastigote targets were performed using fluorescent labeling on treated promastigotes, and in silico analyzes were performed using computational tools. In a general analysis of the results obtained by the in vitro tests, it is observed that among the steroid derivatives tested in this work, the derivatives of deoxycholic acid presented the best antileishmanial effect for the promastigote and amastigote forms in vitro. Among this series of compounds, compound 11d (IC50 = 15.34 μM) is the most selective for L. amazonensis (SI>9.78) and the most specific for the intracellular form of this species. This compound has as probable cellular target the mitochondria, increasing the ROS production and decreasing the ΔΨm. Moreover, the compound has been shown to cause damage to the DNA content of the parasite. In silico studies of the physicochemical and pharmacokinetic properties of compound 11d showed that it has the ability to permeate through biological membranes and oral administration is possible.

Orientador: Jaime Amaya Farfan / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-22T23:27:05Z (GMT). No. of bitstreams: 1 Jackix_ElisadeAlmeida_D.pdf: 1472562 bytes, checksum: 8830780df24de0113cef22e1e18df981 (MD5) Previous issue date: 2013 / Resumo: Diversos estudos têm sugerido que a ingestão frequente de vegetais reduz o risco de doenças crônicas não transmissíveis em função da presença de compostos como a fibra alimentar (FA) e demais substâncias bioativas associadas, os quais produzem efeitos positivos sobre a saúde humana. A FA inclui polissacarídeos não amiláceos, oligossacarídeos resistentes à digestão e lignina. Essas substâncias podem passar inalteradas pelo trato gastrintestinal ou serem fermentadas pela microbiota colônica, resultando em efeitos fisiológicos benéficos. A taioba (Xanthosoma sagittifolium) é uma arácea comestível originária da América tropical e possui considerável valor nutritivo. A porção tuberosa é consumida por diversas populações em todo mundo, no entanto, o consumo de suas folhas é menos difundido. O objetivo deste estudo foi caracterizar parcialmente a fração fibrosa da folha de taioba liofilizada (FTL), e avaliar possíveis efeitos fisiológicos in vivo em ratos Wistar machos alimentados com dieta AIN 93G, ou hiperlipídica. As folhas de taioba foram cozidas em água, liofilizadas e quimicamente caracterizadas. Por meio de ensaio biológico foram avaliados os efeitos da FTL sobre a produção de ácidos graxos de cadeia curta, sobre o metabolismo de ácidos biliares (AB) e indicadores bioquímicos. A FTL apresentou teores de fibra alimentar total, solúvel e insolúvel de 35,23 ± 0,26%, 6,82 ± 0,06% e 28,04 ± 0,21%, respectivamente. A análise da composição monossacarídica mostrou que a glicose é o monômero mais abundante (61%), sugerindo a presença de celulose em grandes quantidades. Os animais alimentados com dieta AIN 93G contendo taioba apresentaram maior peso e excreção de gordura fecal, e melhores perfis de ABs, por diminuir a proporção de ABs secundários. Nos ratos alimentados com dieta hiperlipídica a adição de FTL resultou em menor ganho de peso corporal e gordura hepática, e maior peso e conteúdo de gordura nas fezes; além disso, observaram-se maiores concentrações de ácidos graxos de cadeia curta e ABs fecais em comparação ao grupo controle, que recebeu baixos teores de fibra; também se observou menores concentrações de colesterol sérico e maior conteúdo de cinzas corporais nos grupos recebendo a FTL. Os resultados sugeriram que os componentes da folha de taioba possuem elevada capacidade de ligação com os ABs e alta fermentabilidade, indicando que a folha possui potencial para reduzir o risco de doenças cardiovasculares e câncer de cólon / Abstract: Several studies have suggested that frequent vegetable intake reduces the risk of coronary heart disease, diabetes, cancer and obesity due to the presence of compounds such as dietary fiber (DF) and other associated bioactive compounds, which exert positive effects on human health. DF includes non-starch polysaccharides, resistant oligosaccharides and lignin. These substances can pass unaltered through the gastrointestinal tract or be fermented by colonic microbiota, resulting in positive physiological effects. Taioba (Xanthosoma sagittifolium) is an edible aroid originated from tropical America that exhibits significant nutritive value. The tuberous portions are consumed by several populations worldwide, whereas the leafy portions of Taioba are generally discarded. The objective of this research was to partly characterize the DF of lyophilized taioba leaf (LTL), and to assess the possible in vivo physiological effects when fed to male Wistar rats receiving a normal AIN 93G diet, or a high-fat diet. Taioba leaves were boiled in water, lyophilized and chemically characterized. Biological evaluation was conducted to assess the effect of LTL on the intestinal short-chain fatty acid production, bile acid (BA) metabolism, and biochemical parameters. Total, soluble and insoluble dietary fiber contents in LTL were 35.23 ± 0.26%, 6.82 ± 0.06% and 28.04 ± 0.21%, respectively. Monosaccharide composition showed glucose as the main component (61%), suggesting the presence of cellulose. Animals fed the AIN 93G diets containing taioba leaf had increased fecal mass and fat excretion, in addition to improved BA profiles by diminishing the proportion of the secondary acids. In the rats fed the high-fat diet, the addition of LTL resulted in lower weight gain and liver fat concomitantly with higher fecal mass and lipids. Furthermore, these rats exhibited a higher fecal short chain fatty acid and bile salt concentration in the feces than the control group, which had a lower fiber intake. Additionally, lower serum cholesterol and higher body ash content were found in the groups receiving the leaf. The results suggested that taioba leaves have compounds with high BA binding capacity and high fermentability. These properties indicate that the leaf has the potential of lowering the risk of colon cancer and cardiovascular disease / Doutorado / Nutrição Experimental e Aplicada à Tecnologia de Alimentos / Mestre em Alimentos e Nutrição

Abstract α-Methylacyl-CoA racemase (Amacr) is an auxiliary enzyme of β-oxidation and participates in the elimination of methyl-branched fatty acids in peroxisomes and in mitochondria and in the synthesis of bile acids in peroxisomes. Amacr catalyzes in reversible manner the isomerization of fatty acyl-CoA esters with a methyl group in the R-configuration to the corresponding S-configuration, which allows them to serve as substrates for the next reaction in their metabolism. The substrates of Amacr include the acyl-CoA esters of 2R-pristanic acid, a metabolite derived from phytol, and 25R-THCA and 25R-DHCA (tri- and dihydroxycholestanoic acid), the bile acid intermediates derived from cholesterol. AMACR-deficiency in humans results in the accumulation of R-isoforms of its substrates. Patients with adult onset AMACR-deficiency suffer from neurological disorders. The more severe infantile form of the deficiency is characterized by liver disease. Amacr-deficient mice show a bile acid pattern similar to that of human patients with accumulation of bile acid intermediates in their body. In contrast to humans, Amacr-deficient mice are clinically symptomless on a regular laboratory chow diet. Supplementation of phytol in their diet triggers the disease state with liver abnormalities. In this study it was shown that in spite of the disruption of a major metabolic pathway, Amacr-deficient mice are able to readjust their cholesterol and bile acid metabolism to a new balanced level allowing them to live a normal life span. A double knockout mouse model deficient in Amacr and MFE-1 (peroxisomal multifunctional enzyme type 1) was generated in this work. Characterization of this mouse line showed that MFE-1 can contribute to peroxisomal side-chain shortening of C27 bile acid intermediates in both Amacr-dependent and Amacr-independent pathways. In addition, this work confirmed that Amacr-deficient mice are unable to thrive when phytol is supplemented in their chow. The main cause of death was liver failure accompanied by kidney and brain abnormalities. The detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways and Amacr-deficient mice are not able to respond adequately. The results of this study emphasize the indispensable role of Amacr in detoxification of α-methyl branched fatty acids. / Tiivistelmä α-Metyyliasyyli-koentsyymi-A-rasemaasi (Amacr) osallistuu metyyli-haarautuvien rasvahappojen eliminointiin peroksisomeissa ja mitokondrioissa ja sappihappojen synteesiin kolesterolista peroksisomeissa. Amacr katalysoi käänteisesti rasvahappojen asyyli-koentsyymi-A-estereiden isomerisaatio-reaktiota, jossa stereokemiallisesti R-asemassa oleva metyyliryhmä siirtyy S-asemaan. Tämä on edellytys eliminointiketjun seuraavan reaktion tapahtumiselle. Amacr-entsyymin substraatteja ovat fytolin aineenvaihdunnassa syntyvän 2R-pristaanihapon ja kolesterolista sappihapposynteesireitin välituotteina syntyvien 25R-trihydroksikolestaanihapon ja 25R-dihydroksikolestaanihapon (25R-THCA ja 25R-DHCA) asyyli-koentsyymi-A-esterit. Ihmisellä Amacr-entsyymin puutos johtaa R-muodossa olevien substraattien kertymiseen, joka aiheuttaa neurologisia oireita aikuisiässä alkavassa sairauden muodossa. Lapsuusiässä alkavassa tautimuodossa potilaille kehittyy vakava maksasairaus. Tutkimuksen tulokset osoittivat, että Amacr-poistogeenisten hiirten elinikä ei lyhene huolimatta yhden tärkeän aineenvaihduntareitin estymisestä. Tämä on hyvä esimerkki siitä, kuinka nisäkäs pystyy mukauttamaan kolesteroli- ja sappihappoaineenvaihduntaansa vastaamaan muuttunutta tilannetta aineenvaihdunnassa. Tässä työssä tuotettiin myös kaksoispoistogeeninen hiirimalli, jonka Amacr- ja peroksisomaalinen monitoiminnallinen entsyymi tyyppi 1- (MFE-1) entsyymit ovat toimimattomat. Tämä hiirimalli paljasti, että MFE-1 pystyy osallistumaan 27:ää hiiltä sisältävien sappihappovälituotteiden sivuketjun lyhentämiseen sekä Amacr entsyymin kanssa että ilman sitä. Työn tulokset myös osoittivat, että Amacr-poistogeeniset hiiret eivät ole elinkykyisiä, jos niiden ravinto sisältää fytolia. Maksan toiminnanvajaus oli näiden hiirten tärkein kuolinsyy, mutta hiirten munuaisten ja aivojen kudosrakenteissa oli myös muutoksia. Maksassa fytolin metaboliittien vaarattomaksi tekeminen aiheuttaa villityypin hiirillä useamman aineenvaihduntareitin aktivoitumisen, mutta Amacr-poistogeeniset hiiret eivät pysty reagoimaan tähän samalla tavalla. Tämä työ osoittaa, että Amacr-entsyymin elintärkeä tehtävä on osallistua ravinnon mukana elimistöön joutuvien α-metyylihaarautuvien rasvahappojen eliminaatioon.