CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Diversos experimentos foram realizados com o objetivo de estudar a reaÃÃo de esterificaÃÃo enzimÃtica do Ãcido olÃico com a frutose em meio etanÃlico, visando a sÃntese de biosurfactantes biodegradÃveis. Para tanto, foi utizada a enzima CÃndida Antartica B na temperatura de 55 ÂC e em tempos de reaÃÃo variando entre 48, 72, 96 e 120 horas. De acordo com os resultados obtidos, constatou-se que a citada enzima catalisou preferencialmente o etanol presente no meio reacional para a formaÃÃo do Ãster oleato de etila. Este fato foi confirmado atravÃs do espectro de ressonÃncia magnÃtica nuclear (1H e 13C), bem como do espectro de infravermelho pela presenÃa de um pico de absorÃÃo em 1738,4 cm-1, caracterÃstico deste Ãster. Os resultados da reaÃÃo de formaÃÃo do oleato de etila indicam que o maior rendimento da reaÃÃo foi observado no tempo de 96 horas e que o tempo de 120 horas o rendimento foi inferior. Os experimentos realizados com o objetivo de se obter Ãsteres de frutose a partir do Ãcido olÃico em meio etanÃlico nÃo lograram Ãxito, apesar da literatura indicar a possibilidade de se realizar esta reaÃÃo em outros meios que utilizam solventes nÃo recomendados para o uso alimentÃcio. O oleato de etila apresenta carÃter lipofÃlico e na indÃstria de alimentos encontra aplicaÃÃo na desidrataÃÃo osmÃtica de tomates e pimentas do tipo âdedo de moÃaâ, facilitando a perda de Ãgua, ganho de aÃÃcar e cor mais luminosa. O uso do oleato de etila no processo de desidrataÃÃo diminui o tempo de secagem, aumenta sua eficiÃncia sem deixar nenhum tipo de resÃduo quÃmico. / Several experiments were done aiming at the study on the enzymatic esterification of the oleic acid with fructose in ethanolic medium, focusing the synthesis of biodegradable biosurfactants. For that purpose, it was utilized the enzyme, Candida antartica B., at temperature of 55 ÂC, in reacting time of 48, 72, 96 e 120 hours. Accordingly to the obtained results it was verified that the related enzyme catalyzed primarily the ethanol present in the reactional medium to form the ester: ethyl oleate. This fact was confirmed through magnetic nuclear resonance spectra (1H and 13C) as well as, through infrared spectrum, by the presence of absorption peak at 1738, 4 cm- 1, characteristic of that ester. The results of the reaction of ethyl oleate production indicate that the highest yield was observed in about 96 hours time, and for the 120 hours time it was observed also an inferior yield. The experiments accomplished for the obtention of fructose esters from oleic acids in ethanolic medium were not successful like in other solvents not recommended for food use. The ethyl oleate shows a lipophilic character and in the food industry it finds application in the osmotic dehydration of tomatoes and peppers âdedo de moÃaâ, improving water release, sugar yield and solar brightness. The use of ethyl oleate in the dehydration process decreases the drying time increases its efficiency without leaving any traces pf chemical residues

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-04-19T23:12:45Z No. of bitstreams: 1 Cintia_Kruger_2017.pdf: 1926572 bytes, checksum: 365416cbae7153ee1220106ae7f6204f (MD5) / Made available in DSpace on 2018-04-19T23:12:45Z (GMT). No. of bitstreams: 1 Cintia_Kruger_2017.pdf: 1926572 bytes, checksum: 365416cbae7153ee1220106ae7f6204f (MD5) Previous issue date: 2017-09-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The objective of this study was to produce the lipase of Penicillium sumatrense by solid-state fermentation (SSF), in this manner taking advantage of agroindustrial residues for the production of an enzyme that is useful in biocatalysis. It was studied as a substrate, the agroindustrial residue, crambe cake (Crambe abyssinica hochst), provided by MS Foundation, Mato Grosso do Sul. The enzymatic activity was determinated by the titrimetric method, using olive oil as substrate. In order to optimize the lipase production, was performed an experimental design 22, aiming to evaluate the effects of the variables involved in the process (substrate granulometry and moisture). The response variable chosen was the enzymatic activity in U (Total Units), of each fermented enviorement. Statistical analyzes were performed by the software Statistica 7.0. The results of the experimental design indicated that the enzymatic activity was maximum (243,0 U) when using a substrate moisture of 60 % (m/m, wet basis), substrate granulometry of 28 mesh, 29°C, after 96 h of fermentation. The dry fermented solid produced from the industrial waste crambe cake (24,3 U gss-1) was used in the synthesis studies of ethyl oleate in n-heptane studying the variables temperature and molar ratio (MR, acid:alcohol). The ester yield was evaluated by the method of LowryTinsley. The best conditions for ester production were 37C, MR of 1:3 and the amount of enzyme in the reaction of 60,0 U, obtaining a yield or conversion in ester of 100,0% after 30 min of reaction. It was also found that the time required to reach equilibrium was 40 min. The crambe cake was reused 10 times for the synthesis reaction of ethyl oleate. In the sixth reuse the enzyme reached half of the yield initially obtained (half life time). The results obtained in this work are inedited, since no papers were found in the literature using lipase of Penicillium sumatrense produced by solid-state fermentation with applications in biocatalysis and show the feasibility of using fermented solids directly in reactions of ester synthesis in organic solvents. Key-words: enzymes; SSF; ethyl oleate. / O objetivo deste trabalho foi estudar a produção da lipase de Penicillium sumatrense por Fermentação no Estado Sólido (FES), utilizando como substratos resíduos agroindustriais à produção de uma enzima com características especiais para utilização em biocatálise. Foi estudado como substrato, o resíduo agroindustrial, torta de crambe (Crambe abyssinica hochst), fornecida pela Fundação MS, Mato Grosso do Sul. A atividade enzimática foi determinada pelo método titulométrico utilizando-se o óleo de oliva como substrato. Para otimizar a produção de lipases foi realizado um planejamento experimental 22, visando avaliar os efeitos das variáveis envolvidas no processo (granulometria do substrato e umidade). A variável resposta escolhida foi a atividade enzimática em U (Unidades totais), de cada meio fermentado. As análises estatísticas foram realizadas pelo software Statistica 7.0. Os resultados do planejamento experimental indicaram que a atividade enzimática foi máxima (243,0 U) quando utilizamos uma umidade do substrato de 60 % (m/m, base úmida) granulometria do substrato de 28 mesh, 29°C, após 96 h de fermentação. O sólido fermentado seco produzido a partir do resíduo industrial torta de crambe (24,3 U gss-1) foi empregado nos estudos da síntese do oleato de etila em n-heptano estudando-se as variáveis temperatura e razão molar (RM, ácido:álcool). O rendimento em éster foi avaliado pelo método de LowryTinsley. As melhores condições para a produção do éster foram 37C, RM de 1:3 e a quantidade de enzima no meio reacional de 60,0 U, obtendo-se um rendimento ou conversão em éster de 100,0% após 30 min de reação. Verificou-se também que o tempo necessário para atingir o equilíbrio químico foi de 40 min. A torta de crambe foi reutilizada 10 vezes para a reação de síntese do oleato de etila. Na sexta reutilização a enzima atingiu a metade do rendimento obtido inicialmente (tempo de meia vida). Os resultados obtidos neste trabalho são inéditos, uma vez que não foram encontrados trabalhos na literatura utilizando-se a lipase de Penicillium sumatrense produzida por FES com aplicações em biocatálise e mostram a viabilidade de utilização de sólidos fermentados diretamente em reações de síntese de ésteres em solventes orgânicos.

Host metabolism is profoundly altered during bacterial infection, both as a consequence of immune activation and secondary to virulence strategies of invading pathogens. As a result, the metabolic pathways that regulate nutrient acquisition, energy storage, and resource allocation in host cells must adapt to pathogen stress in order to meet the physiological demands of the host during infection. In this work, we uncover that the synthesis of the monounsaturated fatty acid (MUFA) oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Oleate is also important for proper lipid storage and abundance. We found that exposure to pathogenic bacteria drives rapid somatic depletion of lipid stores in C. elegans. Activating the p38/MAPK immune signaling pathway in the absence of pathogens was also sufficient to drive loss of somatic fat. In addition, we found that transcriptional suppression of MUFA synthesis occurs during P. aeruginosa infection, in a manner dependent on pathogen virulence. Finally, we showed that the host compensates for the pathogen-induced depletion of fatty acids by promoting the redistribution of oleate from non-intestinal tissues to support immune function in the intestine. Together, these data add to the known health-promoting effects of MUFAs, and suggest an ancient link between nutrient stores, metabolism, and host responses to bacterial infection.

No description available.

Un déséquilibre de la balance énergétique constitue la principale cause du développement des pathologies métaboliques telles que l’obésité et le diabète de type 2. Au sein du cerveau, l’hypothalamus joue un rôle primordial dans le contrôle de la prise alimentaire et du métabolisme périphérique via le système nerveux autonome. Ce contrôle, repose sur l’existence de différentes populations neuronales au sein de l’hypothalamus médio-basal (MBH), neurones à neuropeptide Y (NPY)/Agouti-related peptide (AgRP), et neurones a proopiomelanocortine (POMC), dont l’activité est directement modulée par les variations des taux circulants des nutriments tels que le glucose et les acides gras (FA). Alors que les mécanismes de détection et le métabolisme intracellulaire du glucose ont été largement étudiés, l’implication du métabolisme intracellulaire des FA dans leurs effets centraux, est très peu comprise. De plus, on ignore si le glucose, module le métabolisme intracellulaire des acides gras à longue chaine (LCFA) dans le MBH. Le but de notre première étude est, de déterminer l'impact du glucose sur le métabolisme des LCFA, le rôle de l’AMP-activated protein kinase (AMPK), kinase détectrice du statut énergétique cellulaire, et d'établir s’il y a des changements dans le métabolisme des LCFA en fonction de leur structure, du type cellulaire et de la région cérébrale. Nos résultats montrent que le glucose inhibe l'oxydation du palmitate via l’AMPK dans les neurones et les astrocytes primaires hypothalamiques, in vitro, ainsi que dans les explants du MBH, ex vivo, mais pas dans les astrocytes et les explants corticaux. De plus, le glucose augmente l'estérification du palmitate et non de l’oléate dans les neurones et les explants du MBH, mais pas dans les astrocytes hypothalamiques. Ces résultats décrivent le devenir métabolique de différents LCFA dans le MBH, ainsi que, la régulation AMPK - dépendante de leur métabolisme par le glucose dans les astrocytes et les neurones, et démontrent pour la première fois que le métabolisme du glucose et des LCFA est couplé spécifiquement dans les noyaux du MBH, dont le rôle est critique pour le contrôle de l'équilibre énergétique. Le deuxième volet de cette thèse s’est intéressé à déterminer les mécanismes intracellulaires impliqués dans le rôle de la protéine de liaison ACBP dans le métabolisme central des FA. Nous avons démontré que le métabolisme de l’oléate et non celui du palmitate est dépendant de la protéine ACBP, dans les astrocytes hypothalamiques ainsi que dans les explants du MBH. Ainsi, nos résultats démontrent qu’ACBP, protéine identifiée originellement au niveau central, comme un modulateur allostérique des récepteurs GABA, agit comme un régulateur du métabolisme intracellulaire des FA. Ces résultats ouvrent de nouvelles pistes de recherche liées à la régulation du métabolisme des acides gras au niveau central, ainsi que, la nouvelle fonction de la protéine ACBP dans la régulation du métabolisme des FA au niveau du système nerveux central. Ceci aiderait à identifier des cibles moléculaires pouvant contribuer au développement de nouvelles approches thérapeutiques de pathologies telles que l’obésité et le diabète de type 2. / An imbalance of energy balance is the main cause of the development of metabolic diseases such as obesity and type 2 diabetes. Within the brain, the hypothalamus plays an important role in the control of food intake and peripheral metabolism, via the autonomic nervous system. This control relies on the existence of different neuronal populations in the medio-basal hypothalamus (MBH), including neuropeptide Y (NPY), agouti-related peptide (AgRP) and proopiomelanocortin (POMC) neurons, the activity of which, is directly modulated by changes in the circulating levels of nutrients such as glucose and fatty acids (FA). While mechanisms governing the detection and the intracellular metabolism of glucose have been extensively studied, the involvement of FA intracellular metabolism, in their central effects is poorly understood. It is currently unknown if glucose regulates long chain fatty acids (LCFA) metabolism in the MBH. The aim of our first study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), a sensor of cellular energy status, and to establish if changes in LCFA metabolism, and its regulation by glucose, vary as a function of LCFA type, cell type and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures and MBH slices, ex vivo, but not in cortical astrocytes and slice preparations. In addition, our results show that glucose increases palmitate but not oleate esterification into neutral lipids, in neurons and MBH slices, but not in hypothalamic astrocytes. These findings reveal the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons and established for the first time the metabolic coupling of glucose and LCFA as a specific feature of the MBH, whose role is critical for the control of energy balance. During the second part of this thesis, we were interested to determine the intracellular mechanisms involved in the role of Acyl-CoA binding protein (ACBP), in the central metabolism of FA. We have shown that the metabolism of oleate but not palmitate is ACBP -dependent in hypothalamic astrocytes and MBH slices. Thus, our results demonstrate That ACBP, a protein originally identified as an allosteric modulator of GABA receptor peptide, acts as a regulator of intracellular metabolism of FA. These results open a new avenues of research related to the central regulation of fatty acid metabolism and the new function of ACBP protein in the regulation of FA metabolism in the central nervous system, which could help to identify molecular targets that may contribute to the development of new therapeutic approaches of diseases such as obesity and type 2 diabetes.

Surfactants are molecules that contain a water-soluble and afat-soluble part. They have important functions in productssuch as detergents, cosmetics, pharmaceuticals and foods aswell as in many industrial processes. Surfactants are used onvery large scale, which makes it important to decrease theirimpact on the environment. This can be done by starting withnatural materials, by improving the synthetic methods and byreducing the use of limited resources such as energy andorganic solvents. This thesis focuses on lipase-catalyzed synthesis ofsurfactants based on natural products. It also includesfunctional studies of the produced surfactants; as antioxidantsin oils, or as surfactants to solubilize pharmaceuticals. Unsaturated fatty acid esters of ascorbic acid weresynthesized with catalysis by Candida antarctica lipase B int-amyl alcohol and in ionic liquids. High yields ofascorbyl oleate were obtained in an ionic liquid that wasdesigned to improve the solubility of the fatty acid, when thereaction was performed under vacuum. Ascorbyl oleate wasamorphous and was a better antioxidant than ascorbyl palmitatein rapeseed oil. Polyethylene glycol (PEG) stearate, PEG 12-hydroxystearateand a series of PEG 12-acyloxy-stearates were synthesized in avacuum-driven, solvent-free system usingC. antarcticalipase B as catalyst. Critical micelleconcentration and solubilization capacity were determined forthe PEG 12-acyloxy-stearates. Their effects on living cellswere evaluated in studies of hemolysis and transepithelialelectrical resistance. Several PEG1500 12-acyloxy-stearateswere excellent solubilizers for pharmaceutical use and hadnegligible negative effects on living cells even at highconcentrations. Enzymatic and chemo-enzymatic methods offer uniquepossibilities to synthesize surfactants of high purity. Pureand well-defined surfactants enable new applications and areimportant for the understanding of surfactantstructure-function relationships.

<p>Surfactants are molecules that contain a water-soluble and afat-soluble part. They have important functions in productssuch as detergents, cosmetics, pharmaceuticals and foods aswell as in many industrial processes. Surfactants are used onvery large scale, which makes it important to decrease theirimpact on the environment. This can be done by starting withnatural materials, by improving the synthetic methods and byreducing the use of limited resources such as energy andorganic solvents.</p><p>This thesis focuses on lipase-catalyzed synthesis ofsurfactants based on natural products. It also includesfunctional studies of the produced surfactants; as antioxidantsin oils, or as surfactants to solubilize pharmaceuticals.</p><p>Unsaturated fatty acid esters of ascorbic acid weresynthesized with catalysis by Candida antarctica lipase B in<i>t</i>-amyl alcohol and in ionic liquids. High yields ofascorbyl oleate were obtained in an ionic liquid that wasdesigned to improve the solubility of the fatty acid, when thereaction was performed under vacuum. Ascorbyl oleate wasamorphous and was a better antioxidant than ascorbyl palmitatein rapeseed oil.</p><p>Polyethylene glycol (PEG) stearate, PEG 12-hydroxystearateand a series of PEG 12-acyloxy-stearates were synthesized in avacuum-driven, solvent-free system using<i>C. antarctica</i>lipase B as catalyst. Critical micelleconcentration and solubilization capacity were determined forthe PEG 12-acyloxy-stearates. Their effects on living cellswere evaluated in studies of hemolysis and transepithelialelectrical resistance. Several PEG1500 12-acyloxy-stearateswere excellent solubilizers for pharmaceutical use and hadnegligible negative effects on living cells even at highconcentrations.</p><p>Enzymatic and chemo-enzymatic methods offer uniquepossibilities to synthesize surfactants of high purity. Pureand well-defined surfactants enable new applications and areimportant for the understanding of surfactantstructure-function relationships.</p>

In this project I sought to analyse the effects of different free fatty acids (FFAs) on INS-1E β-cells. The saturated fatty acid palmitate is considered toxic whereas the monounsaturated fatty acid oleate is harmless. In my working hypothesis I assumed an additional protective effect of oleate when used in combination with palmitate. Furthermore I aimed to explore in detail the possible causes and signalling pathways responsible for apoptosis or sustained cell survival. I examined the Endoplasmic Reticulum (ER) stress response, called unfolded protein response (UPR), as one essential criterion deciding about cell death or life. Analysis of viability and apoptosis confirmed the deleterious effect of palmitate on INS-1E β-cells after 24h of incubation. Oleate proved not to be harmful and even reversed the toxicity of palmitate. When the main components of the UPR were assessed using Western blot analyses and quantitative PCR was performed I found positive proof that palmitate activated the UPR and ultimately led to apoptosis. By contrast, oleate completely prevented UPR signalling. I conclude that oleate rescues INS-1E β-cells by inhibiting ER stress and its signalling.

In case of metalworking operations, the purpose of lubrication is served by a complex mixture of two or more phases, these mixtures are known as metalworking fluids (MWFs). For many decades oil-in-water emulsions have been used as metalworking fluids. The particular advantage of using oil-in-water emulsion in metalworking operations is that it combines the cooling property of water and the lubrication property of the oil. To explain the lubrication mechanism for oil-in-water emulsions as metalworking fluids a variety of models and theories has been proposed. To understand the lubrication mechanism, the role of each ingredient in the tribological process needs to be studied. In the present study a model for lubrication which determines force and proximity regimes of droplets based on the droplet size distribution is proposed. Dynamic light scattering (DLS) is used to characterize the emulsions. The small droplets are found to be the ones which enhance lubricity. DLVO (Derjaguin-Landau-Verwey-Overbeek) theory is used to validate the results. The concentration and type of surfactant is found to be the performance controlling parameter. A further analysis of the three interfacial energetics; oil/water, oil/substrate, water/substrate, is studied in the presence and absence of surfactants with the help of a Goniometer, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM). Such energetics reflects the rate at which the excess surfactant molecules accumulate at the water/oil interface and desorb into the phases. The tribological response is recorded using AFM and the nanotribometer (NTR). Frictional response of the chemisorbed self-assembled monolayer of surfactant (sodium oleate) on the steel substrate reflects that a tribofilm helps in lubricating the contact under boundary lubrication by creating a low shear strength material. Water being the continuous phase in oil/water emulsion a thin water layer adjacent to steel substrate is always present. This thin layer on the solid substrate acts as a barrier to the lubricating oil droplets to reach the metal surface. The focus of the present work is also to investigate conditions which permit the disjoining of the water film to allow the oil to lubricate the metal substrate. AFM is used to study the interaction force between an oil droplet and the steel substrate through water. An oil encapsulated SiO2 colloidal probe used to simulate the oil droplet. The charge regulatory status of the substrates and interfaces are found to be critical in mapping the force characteristics when DLVO interaction is considered. The condition for activation of non-DLVO (hydration, hydrophobic, capillary) forces are also identified and found to be dependent on the physical states of surfaces. Disjoining of the thin film can be controlled by selecting surfactants based on interfacial energetics and attractive force characteristic can be achieved to facilitate lubrication.

Les composés hydrophobes sont connus comme des sources de carbone qui peuvent être utilisées par les levures comme Yarrowia lipolytica pour de multiples applications. Ces composés causent parfois des perturbations aux levures mais sont aussi rapportés comme conférant aux cellules une certaine résistance contre les stress environnementaux. Dans le cadre de cette thèse, nous avons étudié le rôle de l'oléate de méthyle comme source de carbone sur la résistance de la levure Y. lipolytica en réponse au choc d’un composé amphiphile, la -dodécalactone, et au stress thermique. Les résultats obtenus montrent que les cellules ayant poussé sur oléate sont beaucoup plus résistantes au choc lactone ainsi qu’au stress thermique que les cellules ayant poussé sur glucose. L’action de la lactone se trouve au niveau de la membrane où elle cause une fluidification membranaire et une déplétion de stérols qui sont considérés comme la cause de la mort cellulaire. Ce travail met en évidence le rôle des corps lipidiques dans la réponse cellulaire qui se manifeste de différentes manières en réponse à ces stress. Une accumulation des corps lipidiques est importante pour la résistance de la cellule aux stress. Les cellules ayant poussé sur glucose transforment leur stérol libre en esters de stéryle pour former les corps lipidiques en réponse au choc lactone, ce qui augmente leur sensibilité. Tandis que les cellules ayant poussé sur oléate qui ont accumulé des corps lipidiques pendant leur croissance ont tendance à convertir leurs esters de stéryle en stérol libre pour compenser la déplétion de stérol membranaire causée par la lactone ce qui diminue leur sensibilité. L'homéostasie de l'ergostérol, liée à la présence de corps lipidiques, semble donc jouer un rôle clé dans la résistance cellulaire à ces stress. Ce travail relève aussi que la présence de lipides modifie le processus de mort cellulaire programmée de Y. lipolytica en réponse à un stress thermique. / Hydrophobe compounds are known as carbon source which could be used by yeast like Yarrowia lipolytica for multi purposes. These compounds may cause disturbance in yeast but also reported as confer some resistance to cells towards environmental stress. Here, we study the role of methyl oleate as carbon source on resistance of Y. lipolytica in response to stress of an amphiphilic compound, -dodecalactone, and to heat shock. Results show that cells grown in oleate are more resistant to these stresses than cells grown in glucose. This work reveals the role of the lipids bodies in cells response to stress and that cells manifest in different ways in response to these stresses. An accumulation of lipids bodies is required for the resistance of cells towards stress as glucose grown cells transform their free sterol into steryl esters to form the lipids bodies in response to lactone shock which increases their sensibility towards lactone. In the case of oleate grown cells which accumulated the lipids bodies during their growth, these cells have tendency to convert their steryl esters into free sterol in order to compensate sterol depletion causing by lactone shock and decrease their sensibility. Homeostasis of ergosterol, linked with presence of lipids bodies, seems to be the key for cellular resistance to stresses. This work reveals also that the presence of lipids bodies modifies the processes of programmed cells death in response to heat shock.