The key defects characteristic of hyperglycemia in T2D include increased hepatic glucose production, a diminution of insulin secretion, and an absolute impairment in peripheral insulin action. The objective of my thesis was to investigate the molecular mechanisms leading to fatty acid induced β-cell dysfunction and determine the role of a novel transcriptional coregulator in the regulation of hepatic glucose homeostasis. The first part of my work focused on the chronic effects of fatty acids on human pancreatic β-cell function. Using microarray technology I established an important role for fatty acids in the pathogenesis of β-cell dysfunction. Accordingly chronic exposure of islets to oleate resulted in a significant reduction in glucose-stimulated insulin secretion and to an increase in the rate of reactive oxygen species generation. Additionally, pre-treatment of human islets with oleate led to a significant increase in the rate of oxidation of this fatty acid and to a significant decrease in glucose oxidation. My data indicate that chronic exposure of human islets to fatty acids activates inflammatory and metabolic pathways that lead to oxidative stress. In addition, the first part of my work demonstrated that fatty acids induce oxidative stress in vitro an effect that is preventable to a large extent by the use of antioxidants. In this setting and recapitulating the human islet data, fatty acids are causally linked to impaired insulin secretion, and the induction of oxidative stress. Our report demonstrated that oxidative stress plays a key role in the decrease in β-cell function induced by chronic lipotoxicity. My work also demonstrated that fatty acids are causally linked to the induction of endoplasmic reticulum stress in human islets. Finally, in the second part of my work I provide novel evidence for the role of PHIP in the regulation of hepatic gluconeogenesis. My work is the first to demonstrate that PHIP suppresses hepatic gluconeogenesis in vitro and in vivo. PHIP is amongst the few proteins that have ever been reported to suppress gluconeogenesis to date. PHIP thus represents a novel target for pharmaceutical intervention of diabetes and the suppression of hepatic glucose production.

The key defects characteristic of hyperglycemia in T2D include increased hepatic glucose production, a diminution of insulin secretion, and an absolute impairment in peripheral insulin action. The objective of my thesis was to investigate the molecular mechanisms leading to fatty acid induced β-cell dysfunction and determine the role of a novel transcriptional coregulator in the regulation of hepatic glucose homeostasis. The first part of my work focused on the chronic effects of fatty acids on human pancreatic β-cell function. Using microarray technology I established an important role for fatty acids in the pathogenesis of β-cell dysfunction. Accordingly chronic exposure of islets to oleate resulted in a significant reduction in glucose-stimulated insulin secretion and to an increase in the rate of reactive oxygen species generation. Additionally, pre-treatment of human islets with oleate led to a significant increase in the rate of oxidation of this fatty acid and to a significant decrease in glucose oxidation. My data indicate that chronic exposure of human islets to fatty acids activates inflammatory and metabolic pathways that lead to oxidative stress. In addition, the first part of my work demonstrated that fatty acids induce oxidative stress in vitro an effect that is preventable to a large extent by the use of antioxidants. In this setting and recapitulating the human islet data, fatty acids are causally linked to impaired insulin secretion, and the induction of oxidative stress. Our report demonstrated that oxidative stress plays a key role in the decrease in β-cell function induced by chronic lipotoxicity. My work also demonstrated that fatty acids are causally linked to the induction of endoplasmic reticulum stress in human islets. Finally, in the second part of my work I provide novel evidence for the role of PHIP in the regulation of hepatic gluconeogenesis. My work is the first to demonstrate that PHIP suppresses hepatic gluconeogenesis in vitro and in vivo. PHIP is amongst the few proteins that have ever been reported to suppress gluconeogenesis to date. PHIP thus represents a novel target for pharmaceutical intervention of diabetes and the suppression of hepatic glucose production.

1-substituted 1,2,4-triazoles form a class of agricultural chemicals known as sterol-inhibiting fungicides (SIFs). These fungicides function through the inhibition of sterol synthesis, which ultimately affects cell membrane fluidity and permeability. Of the two main types of sterols in plants, free sterols (FSs) are thought to be incorporated into membranes while conjugated sterols such as steryl esters (SEs), hypothetically, regulate homeostasis by inserting or removing FSs from cell membranes under changing environmental conditions. Non-target algae species possess sterol synthesis pathways that are affected by a range of SIFs. One of the main objectives of the current study was to determine the reason for the observed sensitivity of C. fusca and the tolerance of C. kessleri to SIFs relative to total lipid, FS, SE and FFA composition. These parameters were measured using gas chromatography and mass spectroscopy techniques. Both quantitative and qualitative differences in sterol number and type were noted relative to the FS and SE composition of the two species of algae over time. Notably, SEs were detected in both species of algae, although presence and amount varied with the organism. While SEs were more abundant in C. kessleri, higher amounts of FSs were found in C. fusca. The FS/SE ratios were 64/36 and 88/12 percent of the total sterol in C. kessleri and C. fusca, respectively. Treatment of C. fusca with 2, 4, and 6 ppm and C. kessleri with 6,12, and 24 ppm propiconazole caused an accumulation of methylated precursor sterols, resulting in slightly more FSs in both algae. Only 3 of the FSs produced following treatment were different from the control in C. fusca while 9 new sterols were found in C. kessleri. Treatment also altered the SE fraction in both species, with fewer SEs produced compared to the control, but more novel sterols in C. kessleri, suggesting a possible inverse relationship between FSs and SEs in both organisms. Several studies have implicated lipid/sterol concentrations with the potential for cellular bioaccumulation of lipophilic xenobiotics as they relate to membrane permeability. Cell age and environmental parameters can also affect lipid composition of algae. Although cell age did not affect the qualitative sterol composition of C. fusca and C. kessleri, quantitative differences were observed. Plants exposed to chemical and other environmental stresses accumulate free fatty acids (FFAs), which may be linked to biophysical membrane changes. SIF sensitive C. fusca, had inherently higher levels of FFAs than C. kessleri. Qualitatively, C. fusca exhibited higher percentages of 18:1 and lower ratios of 18:2/18:3 FFAs than C. kessleri. In response to increasing SIF treatment, the ratio of 18:2/18:3 FFAs increased in C. kessleri and declined in C. fusca. The amount of total lipid produced in the cells of C. fusca was higher than in C. kessleri during all growth stages. Variations were observed in lipid measured as a percent dry weight compared to lipid/cell as the cultures age. Inherent differences in FS, SE, and lipid composition of C. fusca and C. kessleri as well as age related changes could account for the differences in the susceptibility of the two algae to propiconazole. / Master of Science

abstract: Synechocystis sp. PCC 6803 is a readily transformable cyanobacteria used to study cyanobacterial genetics, as well as production of biofuels, polyesters, and other industrial chemicals. Free fatty acids are precursors to biofuels which are used by Synechocystis cells as a means of energy storage. By genetically modifying the cyanobacteria to expel these chemicals, costs associated with retrieving the products will be reduced; concurrently, the bacteria will be able to produce the products at a higher concentration. This is achieved by adding genes encoding components of the Escherichia coli AcrAB-TolC efflux system, part of the resistance-nodulation-division (RND) transporter family, to Synechocystis sp. PCC 6803. AcrAB-TolC is a relatively promiscuous multidrug efflux pump that is noted for expelling a wide range of substrates including dyes, organic solvents, antibiotics, and free fatty acids. Adding components of the AcrAB-TolC multidrug efflux pump to a previously created high free fatty acid producing strain, SD277, allowed cells to move more free fatty acids to the extracellular environment than did the parent strain. Some of these modifications also improved tolerance to antibiotics and a dye, rhodamine 6G. To confirm the function of this exogenous efflux pump, the genes encoding components of the AcrAB-TolC efflux pump were also added to Synechocystis sp. PCC 6803 and shown to grow on a greater concentration of various antibiotics and rhodamine 6G. Various endogenous efflux systems have been elucidated, but their usefulness in expelling products currently generated in Synechocystis is limited. Most of the elucidated pumps in the cyanobacteria are part of the ATP-binding cassette superfamily. The knowledge of the resistance-nodulation-division (RND) family transporters is limited. Two genes in Synechocystis sp. PCC 6803, slr2131 and sll0180 encoding homologs to the genes that encode acrB and acrA, respectively, were removed and the modifications resulted in changes in resistance to various antibiotics and a dye and also had an impact on free fatty acid secretion. Both of these deletions were complemented independently with the homologous E. coli gene and the resulting cyanobacteria strains had some of the inherent resistance restored to chloramphenicol and free fatty acid secretion was modified when compared to the wild-type and a high free fatty acid producing strain. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2018

High fibre diets are associated with reduced risk of type 2 diabetes mellitus (T2DM). This may be due to short-chain-fatty-acids (SCFA) influencing insulin resistance and secretion via changes in free-fatty-acids (FFA) and specific gut hormones. We aimed to determine the postprandial effects of inulin, a fermentable, soluble fibre in healthy subjects. Twelve fasted subjects were studied for 6 hours after either 80g high fructose corn syrup (HFCS), 56g HFCS plus 24g inulin, or 56g HFCS drinks using a randomized, cross-over design. SCFA were higher after inulin beginning at 4 hours. FFA were lower 4 hours after inulin than 56gHFCS. GLP-1 was higher 30 minutes after inulin than 56 and 80HFCS, while ghrelin was lower from 4-6 hours after inulin. The results support the hypothesis that inulin and SCFA generated from colonic fermentation of dietary fibre may improve insulin resistance and secretion via modulation of FFA and specific gut hormones.

High fibre diets are associated with reduced risk of type 2 diabetes mellitus (T2DM). This may be due to short-chain-fatty-acids (SCFA) influencing insulin resistance and secretion via changes in free-fatty-acids (FFA) and specific gut hormones. We aimed to determine the postprandial effects of inulin, a fermentable, soluble fibre in healthy subjects. Twelve fasted subjects were studied for 6 hours after either 80g high fructose corn syrup (HFCS), 56g HFCS plus 24g inulin, or 56g HFCS drinks using a randomized, cross-over design. SCFA were higher after inulin beginning at 4 hours. FFA were lower 4 hours after inulin than 56gHFCS. GLP-1 was higher 30 minutes after inulin than 56 and 80HFCS, while ghrelin was lower from 4-6 hours after inulin. The results support the hypothesis that inulin and SCFA generated from colonic fermentation of dietary fibre may improve insulin resistance and secretion via modulation of FFA and specific gut hormones.

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-26Bitstream added on 2014-06-13T18:56:51Z : No. of bitstreams: 1 felicidade_i_me_botfm.pdf: 442429 bytes, checksum: bbae716d0f4846cf367d5ed80870142c (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Em diversas partes do mundo, a prevalência da obesidade tem aumentado rapidamente. A Organização Mundial da Saúde estima que, em 2015, haverá 700 milhões de pessoas obesas (índice de massa corporal ≥30kg/m2) em todo o mundo. No Brasil, aproximadamente, 50% da população acima dos 20 anos apresentam sobrepeso (índice de massa corporal ≥25kg/m2) e 16% já demonstram quadro de obesidade. A obesidade é um dos fatores desencadeadores de diversas doenças crônicas, assim, é importante identificar características que predispõem indivíduos à obesidade, ou condições negativas que ocorrem durante o desenvolvimento da obesidade, as quais alteram o metabolismo e o estado de saúde de maneira negativa. O objetivo do presente estudo foi avaliar diferenças na expressão de genes-alvo do PPARα em células mononucleares periféricas do sangue (PBMCs) e na concentração plasmática de ácidos graxos livres no sangue, entre indivíduos obesos e não-obesos, após 24 horas de jejum. Este estudo foi o primeiro realizado na população brasileira. Em ambos os grupos (obeso e não-obeso), a concentração plasmática de ácidos graxos livres (AGL) elevou-se de forma significante após as 24horas de jejum, em especial, no grupo não-obeso, onde a média da concentração elevou-se 3 vezes se comparada com a média da concentração no tempo 0h. A média da concentração plasmática basal (0h) de AGL revelou-se 2 vezes maior no grupo obeso quando comparada com a média do grupo não-obeso. No entanto essa diferença não foi observada após as 24h de jejum. Os genes estudados participam diretamente na β-oxidação mitocondrial hepática e musculoesquelética e são ativados por PPARα. Após o jejum, todos os genes estudados evidenciaram um incremento na expressão (fold change) quando comparados com o... / The prevalence of obesity has increased rapidly in many parts of the world. The World Health Organization estimates that in 2015, there should be 700 million obese people (body mass index ≥ 30kg / m²) around the world. In Brazil, approximately 50% of the population aged 20 years and older are overweight (body mass index ≥ 25kg / m²) and 16% already meet the criterion for obesity. Obesity is considered a trigger factor for several chronic diseases, so it is important to identify traits that predispose individuals to obesity or negative conditions that occur during the development of obesity, affecting metabolism and health negatively. The aim of this study was to evaluate differences in the expression of PPAR-alpha target genes in peripheral blood mononuclear cells (PBMCs) and in plasma free fatty acid concentration in the blood of obese and non-obese subjects, after 24 hours of fasting. This was the first study conducted in the Brazilian population. In both groups (obese and non-obese), plasma free fatty acid (FFA) concentration increased significantly after 24 hours of fasting, especially in the non-obese group, in which the mean concentration rose 3 times compared with the mean concentration at time 0h. The baseline (0h) mean of plasma FFA concentration was 2 times higher in the obese group compared with the mean from the non-obese group. This difference, however, was not observed after 24h of fasting. The studied genes participate directly in hepatic and musculoskeletal mitochondrial beta-oxidation and are activated by PPAR-alpha. After fasting, all the studied genes had an increase in fold change compared with time 0h, in both groups. By identifying the preferential beta-oxidation during fasting, the CPT1A gene presented an increase in fold change compared with the CPT1B gene, indicating thus that beta-oxidation... (Complete abstract click electronic access below)

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Para uma produção eficiente e economicamente viável de ésteres é necessária a utilização de catalisadores durante o processo, uma vez que a transesterificação e a esterificação são reações de equilíbrio, sendo necessário deslocar o equilíbrio de reação na direção desejada. Nesse trabalho, ésteres metílicos e etílicos de ácidos graxos de cadeia longa foram produzidos usando cloretos de cobre como catalisadores. Para definir a melhor condição reacional, foram estudadas três diferentes razões em massa de AGLs de dendê/ metanol (ou etanol) / catalisador. Definida a melhor condição reacional, foram produzidos monoésteres metílicos e etílicos a partir de ácidos graxos livres do destilado da soja assim como das borras do refino correspondente do óleo de soja e de dendê. Os ésteres metílicos e etílicos foram caracterizados através das técnicas de cromatografia gasosa acoplada à espectrometria de massas (CG/MS) e ressonância magnética nuclear de prótons (RMN 1H). Observou-se que as reações empregando cloreto de cobre como catalisador reagem preferencialmente com AGLs em relação aos triésteres. Dentre esses ácidos graxos livres, os de cadeia insaturada, são os que reagem mais facilmente com o catalisador. Em relação ao álcool empregado, observou-se que as reações em etanol são mais rápidas que as realizadas em metanol, devido a facilidade de solubilização do catalisador e matéria prima lipídica no etanol. A reação com metanol, por sua vez ocorre de forma mais lenta, devido a solubilidade parcial entre os reagentes no meio e consequentemente, com separação instantânea de duas fases ao término da agitação, facilitando a extração do produto. A ampliação da estequiometria de reação em 160 e 80 vezes, para o catalisador anidro e hidratado, respectivamente, em relação a massa de AGLs de dendê foi obtida com sucesso em ambos os catalisadores, mostrando a viabilidade do emprego do cloreto de cobre como catalisador em escala industrial e a possibilidade de ser estendido a outras matérias primas. Desse modo, a utilização do cloreto de cobre como catalisador na reação de ácidos graxos livres de cadeia longa ou de óleos vegetais se constitui em uma metodologia simples e prática, sendo uma opção viável para a produção de monoalquil esteres de cadeia longa em condições brandas de reação quando comparada aos métodos já estabelecidos e utilizados inclusive em escala industrial. / For an economically feasible and efficient production of esters is necessary to use catalysts in the esterification and transesterification reactions, which are reversible. It is necessary to shift the reaction equilibrium in the desired direction. In this work we developed a novel reaction to produce methyl and ethyl esters of long chain fatty acids using copper chloride as catalysts. To establish optimal reaction conditions, we studied three different mass ratios of FFAs palm (or soybean) / methanol (or ethanol) / catalyst. Employing the best reaction conditions, methyl and ethyl mono-esters were produced from free fatty acid of the soybean oil deodorization distillate (DDOS) as well as the corresponding refined soybean and palm oil. The methyl and ethyl esters were characterized by gas chromatography coupled to mass spectrometry (GC / MS) and proton nuclear magnetic resonance (1H NMR). It was observed that reactions using copper chloride as catalyst are faster with free fatty acids than with neutrals refined oils. Among these, free fatty acids of unsaturated chain react faster than the saturated ones. Regarding the alcohol employed, it was observed that the reactions in ethanol are faster than those performed in methanol, due to higher of solubility of the catalyst and feedstock lipid in ethanol. The reaction with methanol, in turn occurs more slowly, due to partial solubility of the reactants in the solvent that results in, instantaneous phases separation. The scale up of reaction at 80 and 160 times for both, anhydrous and hydrated catalyst, was successfully performed showing the feasibility of this novel process use in industrial scale. Thus, the use of copper chloride as the catalyst in the reaction of long chain free fatty acids constitutes a simple and practical methodology, being a feasible option for the production of long chain monoalkyl esters under mild conditions compared to methods already established and also used in industrial scale.

O presente estudo teve como objetivo selecionar procedimentos de purificação de óleos vegetais, com enfoque na desacidificação, comparando a eficiência de métodos de refino alternativos (adsorção e extração liquido-liquido) com o convencional (alcalino). Para tanto, foram utilizados óleos com baixo impacto na cadeia alimentícia, como óleo de andíroba e óleo de macaúba, visando contribuir para a ampliação de matérias-primas lipídicas na produção de biodiesel. A triagem do método de purificação mais adequado foi baseada na redução de ácidos graxos livres, na quantidade recuperada de matéria-prima após o refino e na qualidade do biodiesel gerado. Adotando óleos com diferentes teores de ácidos graxos livres, óleo de andíroba (17,7%) e óleo de macaúba (7,9%) a desacidificação por adsorção, empregando diferentes adsorventes foi excluída com base nos baixos valores de desacidificação (<34,1%) e de recuperação em massa de óleo (22,2-43,1%). A acidez mais elevada do óleo de andíroba se mostrou como um fator limitante adicional, portanto, o uso deste óleo foi eliminado para as etapas posteriores de triagem. Em contrapartida, o refino por extração liquido-liquido (ELL) usando etanol como solvente forneceu valores mais elevados de recuperação mássica (>75,9%) e de redução de ácidos graxos livres (>48,8%). Buscou-se por meio da utilização de um método estatístico determinar as condições adequadas de refino por ELL usando etanol hidratado. Esta etapa foi realizada por meio de uma matriz de planejamento fatorial composto de face centrada 22, avaliando simultaneamente a influência da porcentagem mássica de agua no solvente alcoólico (2, 4 e 6%) e da razão mássica solvente/óleo (1:1, 1,5:1, 2:1). De acordo com a análise estatística foi possível estabelecer que a adição de 5,3% de agua no etanol e razão mássica etanol/óleo de 1,79:1 maximizaram a remoção de ácidos graxos livres (64,4%) sem ocasionar elevadas perdas de óleo refinado (93,9%). Adotando as condições otimizadas de ELL, o óleo de macaúba foi refinado em único e duplo estagio. Constatou-se que o teor de ácidos graxos livres em relação ao óleo bruto (7,9%) foi reduzido expressivamente em ambos os casos para valores de 2,9% (refino em único estagio) e de 1,0% (refino em duplo estagio), com a vantagem de não promover oxidação do material lipídico. Entretanto, a recuperação mássica de óleo refinado obtida na ELL em duplo estagio foi menor, da ordem de 78,5%. Para validar a metodologia de purificação, foram realizadas reações de etanólise das amostras de óleo refinado por ELL em único e duplo estagio, óleo bruto e óleo refinado por via alcalina utilizando como catalisador oxido nióbio impregnado com sódio. Enquanto a acidez do óleo bruto inibiu o catalisador, os produtos originados pelas amostras de óleo refinadas por ELL em único e duplo estagio contiveram teores em ésteres etílicos (97,4% e 97,8%, respectivamente) que atenderam as especificações recomendadas pela Agencia Nacional de Petróleo, Gás Natural e Biocombustível para uso como combustível. Esses valores foram similares a conversão alcançada na reação conduzida com o óleo refinado por via alcalina (99,0%), que teve desvantagens como recuperação mássica de 80,1% e aumento do índice de peroxido, indicando que a extração liquido-liquido tem potencial para desenvolvimento e aplicação em processos de refino de óleos vegetais para posterior utilização na síntese de biodiesel. / This study aimed at comparing the purification of non-edible vegetable oils by two different types of refining (adsorption and liquid-liquid extraction) with conventional alkali refining, contributing to the expansion of lipid feedstocks for biodiesel production. The use of non-edible vegetable oils is related to the final cost of biodiesel by increasing product competitiveness. The screening of the purification methods for vegetable oils (andiroba oil and macaw palm oil) was based on the reduction of free fatty acids content, oil mass recovery after the refining and the quality of biodiesel. Adopting oils with different levels of free fatty acids, andiroba oil (17.7%) and macaw palm oil (7.9%), the deacidification by adsorption, employing different adsorbents was excluded on the basis of the low deacidification values (<34.1%) and the oil mass recovered (22.2-43.1%). The higher acidity of andiroba oil was found to be an addition drawback and therefore, this oil was disqualified for further testing. On the other hand, the purification by liquid-liquid extraction (LLE) of macaw palm oil using ethanol as solvent gave higher values for oil mass recovery (>75.9%) and reduction of free fatty acids (>48.8). To determine the suitable conditions for oil refining by LLE using hydrated ethanol as solvent a statistical method was proposed. For this a 22 central composite face-centered experimental design was adopted to simultaneously evaluate the influence of the water percentage in the alcoholic solvent (2, 4, and 6%), and mass ratio of solvent-to-oil (1:1, 1.5:1 and 2:1). According to the statistical analysis it was possible to establish that the addition of 5.3% of water in the ethanol and mass ratio of ethanol to oil of 1.79:1 maximizes the removal of free fatty acids (64.4%) resulting in high refined oil recovered (93.9%). Adopting the optimized LLE conditions, the macaw palm oil was refined in single and double-stage. It was noted that the level of free fatty acids in relation to crude oil (7.9%) was significantly reduced in both cases for values of 2.9% (refining in single stage) and 1.0% (refining in double-stage), with the advantage of not promoting lipid oxidation. However, the percentage of the recovered refined oil obtained in LLE double-stage was slightly lower, in the order of 78.5%. To validate the purification methodology, ethanolysis reactions catalyzed by niobium oxide impregnated with sodium were performed using samples refined by LLE (single and double-stage), oil refined by alkali neutralization and crude oil. The obtained dataset demonstrated that the catalyst was inhibited by the high acidity level of the crude oil, while refined oil by LLE (single-stage and double-stage) gave levels of ethyl esters contents of 97.4% and 97.8%, respectively that meet the criteria established by the Brazilian National Agency of Petroleum, Natural Gas and Biofuels to be used as a fuel. These values were similar to that attained in the reaction using refined oil by alkaline protocol (99.0%), which had disadvantages such as oil mass recovered of 80.1% and oil oxidation (increased peroxide value), indicating that the liquid-liquid extraction has the potential for development and application in vegetable oils refining processes for further using as feedstocks in the biodiesel synthesis.