Biocélulas a combustível (BFCs) são definidas como dispositivos bioeletroquímicos que utilizam componentes biológicos, como enzimas ou microrganismos, para converter energia química em energia elétrica. Neste estudo, reporta-se o desenvolvimento de um bioeletrodo composto por fibras flexíveis de carbono (FFC) modificadas com a enzima álcool desidrogenase (ADH), o qual foi utilizado juntamente com a levedura Saccharomyces cerevisiae , sendo enzima e microrganismos usados como biocatalisadores cooperativos para bioprodução e oxidação de etanol. A glicose é oxidada pelas células de levedura sob condições anaeróbias, e o etanol formado pela fermentação alcóolica é, em seguida, oxidado a acetaldeído pela enzima ADH. A oxidação de etanol pela ADH resulta ainda, na redução da molécula de nicotinamida adenina dinucleotídeo, NAD+ a NADH. Posteriormente, o NADH formado nessa reação é eletroquimicamente oxidado a NAD+ na superfície do bioeletrodo de FFC baseado em ADH (FFC-ADH). Avaliou-se a influência da temperatura e do pH na bioeletrocatálise de etanol pela ADH e a melhor resposta obtida foi em 40 ºC e pH 8,5. Além disso, obteve-se uma excelente correlação linear entre os valores de concentração de etanol e densidade de corrente, indicando que a resposta bioeletrocatalítica da ADH é diretamente proporcional à concentração de etanol produzido a partir da fermentação. O conceito de que microrganismos e enzimas podem trabalhar cooperativamente para produzir uma nova classe de bioeletrodos, foi introduzido nesse trabalho. Por fim, demonstrou-se, que o bioeletrodo cooperativo pode ser aplicado com sucesso em uma BFC, utilizando o biocátodo de difusão de gás contendo a enzima bilirrubina oxidase (BOx) imobilizada em sua superfície. / Biofuel cells (BFCs) are defined as bioelectrochemical devices that use biological components, such as enzymes or microorganisms, to convert chemical energy into electric energy. In this study, we report the development of a bioelectrode composed of flexible carbon fibers (FCF) modified with the enzyme alcohol dehydrogenase (ADH) together with Saccharomyces cerevisiae yeast, being enzyme and microorganisms used as cooperative biocatalysts for bioproduction and oxidation of ethanol. Glucose is oxidized by the yeast cells in anaerobic conditions, and ethanol is produced through alcoholic fermentation and then it is oxidized to acetaldehyde by the ADH enzyme. The ethanol oxidation by ADH also results in the reduction of the nicotinamide adenine dinucleotide molecule, NAD+ to NADH. Subsequently, the NADH produced in this reaction is electrochemically oxidized to NAD+ on the surface of the FCF bioelectrode based on ADH (FCF-ADH). The influence of temperature and pH on the bioelectrocatalysis of ethanol was evaluated and the best performance was found at 40 ºC and pH 8.5. Additionally, the results demonstrated an excellent linear correlation between the ethanol concentration and the current generated, which indicates that the bioelectrocatalytic response of ADH is directly proportional to concentration of ethanol produced from the fermentation. The present study has introduced the concept that microorganisms and enzymes can work cooperatively to produce a new class of bioelectrodes. Finally, it has been demonstrated that the cooperative bioelectrode can be applied successfully to BFC using a gas-diffusion biocathode containing the bilirubin oxidase enzyme (BOx) immobilized on its surface.

Os ácidos graxos (AG) podem influenciar o processo secretório de insulina induzido pela glicose. Os AG ω3 interferem em diversos processos fisiológicos, sendo que nas ilhotas pancreáticas, os AG ω3 colaboram para a diminuição da lipotoxicidade induzida pelo ácido palmítico. Ao ácido palmítico são atribuídos efeitos deletérios em diversos tecidos, assim como nas células β, onde ele promove a alteração da composição dos fosfolípides de membrana, do potencial elétrico da mesma e consequentemente do processo de extrusão dos grânulos de insulina. A exposição crônica das células β ao excesso de ácido palmítico é tóxica, provocando diminuição da resposta secretória de insulina, redução da oxidação e captação de glicose e aumento de espécies reativas de oxigênio (EROs) que, em quantidades suprafisiológicas, irão contribuir para a falência e morte da célula β. As EROs podem ser de origem mitocondrial, através do metabolismo dos nutrientes ou ainda proveniente da ativação do complexo enzimático NADPH oxidase, o qual é modulado pela glicose e pelos AG, incluindo o ácido palmítico. Em contrapartida, os AG ω3 exercem efeitos anti-inflamatórios e antioxidantes em diversos sistemas, contribuindo para melhora de perfil lipídico e resistência periférica à insulina. O objetivo deste trabalho foi verificar o possível efeito protetor dos AG ω3 contra os efeitos deletérios do ácido palmítico em células β pancreáticas. Nas células β, a partir dos resultados obtidos, a presença de AG ω3 mostrou-se eficaz para prevenir o dano secretório e o aumento de EROs, além de contribuir para manutenção da viabilidade celular e da captação de glicose nas ilhotas tratadas com ácido palmítico, desempenhando um importante papel protetor na célula β. / Fatty acids (FA) may influence the process of glucose-induced insulin. The ω3 FA interferes in several physiological processes, and in the pancreatic islets collaborate to decrease the lipotoxicity induced by palmitic acid. Palmitic acid induces deleterious effects in several tissues, as well as in β cells, where it promotes the alteration of the membrane phospholipid composition, the plasma membrane electric potential, and consequently, the process of the insulin granules extrusion. Chronic exposure of β cells to high concentration of palmitic acid is toxic, leading to decreased insulin secretory response, reduced oxidation and uptake of glucose, and an increase in reactive oxygen species (ROS) which, in supraphysiological amounts, will contribute to β-cell failure and death. ROS may be of mitochondrial origin, through the metabolism of nutrients or even from the activation of the enzymatic complex NADPH oxidase, which is modulated by glucose and FA, including palmitic acid. In contrast, ω3 FA exerts anti-inflammatory and antioxidant effects in several systems, contributing to the improvement of lipid profile and peripheral resistance to insulin. The aim of this study was verify the protective possible effect of AG ω3 against the deleterious effects of palmitic acid on pancreatic β cells. Our results shown that the presence of ω3 FA was effective in preventing secretory damage and increase of EROs, also contributing to the maintenance of cell viability and glucose uptake in the islets treated with palmitic acid, playing an important β-cell protective role.

A eletroquímica direta de enzimas redox depende da distância entre os sítios redox da proteína e a superfície do eletrodo e também da eficiência na imobilização dessas enzimas na superfície eletródica. Dessa forma, a obtenção de enzimas mais hidrofóbicas possibilita a melhora na interação entre elas e a superfície de eletrodos sólidos, como os de carbono. Neste estudo, foi desenvolvida uma rota para a obtenção da glicose oxidase oligomerizada (Ol-GOx) com o objetivo de melhorar a interação entre a enzima e a superfície de fibras de carbono, uma vez que enzimas oligomerizadas contêm suas porções hidrofóbicas expostas.Para tanto, diferentes ácidos de Brønsted foram utilizados, sendo que a enzima obtida a partir da reação com o ácido trifluorometanosulfônico (TFMS) foi a que se manteve ativa cataliticamente. A Ol-GOx se mostrou um biocatalisador promissor devido a sua hidrofobicidade e seu tamanho, os quais permitiram uma imobilização mais eficiente em superfícies de carbono. Após a caracterização estrutural, concluiu-se que a Ol-GOx é formada por um oligômero composto por 10 unidades de GOx nativa com raio hidrodinâmico de aproximadamente 96 nm. Por voltametria cíclica estudou-se a transferência direta de elétrons (TDE) entre o cofator dinucleotídeo de flavina e adenina (FAD) e a superfície das fibras de carbono, sendo observado um aumento de 7 vezes nas correntes faradaicas em relação ao obtido para a GOx nativa. Além disso, as propriedades bioeletrocatalíticas foram melhoradas em 30% quando analisada a oxidação da glicose. Concluiu-se ainda que quanto maior a quantidade de folhas-β presente na estrutura proteica, maior a TDE observada entre a enzima e a superfície das fibras de carbono. / The direct electrochemistry of redox enzymes is dependent on the distance between the active centers of the protein and the electrode surface, and also on the efficiency in the immobilization of these enzymes on the electrodic surface. Thus, the synthesis of more hydrophobic enzymes could lead to better interaction between the redox enzymes and the solid electrode surfaces, such as carbon electrodes. In this study, it was proposed a chemical route to obtain oligomerized glucose oxidase (Ol-GOx), aiming to improve the interaction between the enzyme and the surface of carbon fibers, since oligomerized proteins have their hydrophobic chains exposed. After structural characterization, it was concluded that Ol-GOx is formed by 10 dimeric units of native GOx with a hydrodynamic radius corresponding to approximately 96 nm. By cyclic voltammetry, it was studied the direct electron transfer (DET) between the flavin adenine dinucleotide (FAD) cofactor and the surface of carbon fibers, where it was observed an increase of 7-fold in the faradaic currents in comparison to that observed for native GOx. Besides, bioelectrocatalytic properties are 30% improved, when analyzed the glucose oxidation by cyclic voltammetry. It was also concluded that the greater the β-sheet content in protein structure, the higher the DET observed between the enzyme and the carbon fibers surface.

Neste projeto, demonstramos a modulação da enzima NAD(P)H oxidase pela glicose, palmitato e interleucina - 1? através da análise da expressão protéica do componente p47PHOX e pela atividade dessa enzima via produção de superóxido e peróxido de hidrogênio. Demonstramos também a participação da enzima NAD(P)H oxidase no processo de secreção de insulina induzido pela glicose pois a inibição da enzima pelo DPI e oligonucleotídeo anti p47PHOX promoveu uma diminuição da secreção do hormônio. A partir desse dado passamos a avaliar o mecanismo de ação da enzima no processo secretório e demonstramos que a inibição dessa enzima promove uma inibição de genes essenciais no processo de secreção de insulina como GLUT-2 e glicocinase.Assim podemos concluir que a enzima NAD(P)H oxidase é modulada pela glicose, palmitato e interleucina 1? e que essa enzima participa do processo de secreção insulina modulando genes essenciais para o processo secretório como GLUT-2 e glicocinase. / The expression and activity of the componenents of NAD(P)H oxidase in pancreatic islets were described for the first time in our laboratory (OLIVEIRA, HR et ai, 2003). It was shown the gene and protein expression of the components of this enzyme in Seta cells and that enzyme activation is mediated by glucose. Glucose induced insulin secretion was followed by increase in EROS generation and this increase was in part mediated by NAD(P)H oxidase activation (the same mechanism observed in phagocytes). In this study, the modulation of NAD(P)H oxidase activity by glucose, palmitate and interleukin 1ß as investigated through protein expression of p47phox vity of this enzyme through superoxide and hydrogen peroxide production. To determinate the role of NAD(P)H oxidase in the process of glucoseinduced insulin secretion the enzyme was inhibited by DPI and oligonucleotide anti p47phox, in the both cases the enzyme inhibition produced a decrease on insulin secretion. In order to investigated NAD(P)H oxidase mechanism of action in insulin secretion, we shown that the inhibition enzyme by DPI reduced the GLUT-2 and glucokinase gene expression. We can concluded hat NAD(P)H oxidase was modulated by glucose, palmitate and interleukin 1ß and that enzyme participed in process of glucoseinduced insulin secretion through modulation of GLUT-2 and glucokinase gene expression.

Cytochrome c oxidase (COX) is one of two known Cu-containing enzymes in mitochondria. Delivery and insertion of copper into COX are very complex processes that require multiple steps and involve a large number of assisting factors. One of the involved components is Cox11p, a copper binding protein in the inner mitochondrial membrane that is conserved from prokaryotes to eukaryotes. Cox11p is essential for respiratory growth and implicated in the assembly of the CuB site located in subunit Cox1p of COX. In the thesis the topology of Cox11p was determined and evidence for its association with the mitochondrial translation machinery is provided. The interaction of Cox11p with mitoribosomes is mediated by its single evolutionary conserved transmembrane segment and appears to be indirect and mediated by another conserved membrane protein(s). A model is proposed in which the CuB site is co-translationally formed by a transient interaction between Cox11p and the nascent Cox1p in the mitochondrial intermembrane space. In addition the genetic and biochemical characterization of S. pombe Cox11p homologue was performed. Two versions of cox11+ gene are detected in a haploid S. pombe genome. Cells lacking either of the cox11+ copies remain respiratory competent, whereas deletion of both S. pombe cox11+ alleles appears to result in either spore lethality or in severe decrease of spores viability. Thus, both versions of SpCox11p are functional and important. In S. pombe Cox11p exists as a tandem with the mitoribosomal protein Rsm22p. This precursor protein is cleaved during mitochondrial import into two mature protein species corresponding to Rsm22p- and Cox11p-like moieties.

Assembly of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the correct insertion of subunits, accessory proteins, cofactors and prosthetic groups. Most of the fundamental biological knowledge concerning mitochondrial copper homeostasis and insertion of copper into COX derives from investigations in the yeast Saccharomyces cerevisiae. In this organism, Cox17 was the first identified factor involved in this pathway. It is a low molecular weight protein containing highly conserved twin Cx9C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is essential for its function. So far, the role of Cox17 in the mammalian mitochondrial copper metabolism has not been well elucidated. Homozygous disruption of the mouse COX17 gene leads to COX deficiency followed by embryonic death, which implies an indispensable role for Cox17 in cell survival. In this thesis, the role of COX17 in the biogenesis of the respiratory chain in HeLa cells was explored by use of siRNA. The knockdown of COX17 results in a reduced steady-state concentration of the copper-bearing subunits of COX and affects growth of HeLa cells accompagnied by an accumulation of ROS and apoptotic cells. Furthermore, in accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper center of COX, COX17 siRNA knockdown affects COX-activity and -assembly. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so called supercomplexes. While the abundance of COX dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. Accumulation of a novel ~150 kDa complex containing Cox1, but not Cox2 could be observed. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. Data presented here suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. Instead an interdependent assembly scenario for the formation of supercomplexes is proposed that requires the coordinated synthesis and association of individual complexes.

No description available.

No description available.

O estresse oxidativo e a enzima NADPH oxidase-2 (NOX2) estão associados com a diminuição da massa funcional de células-β em pacientes com diabetes do tipo 2 (DT2). Neste estudo, testamos o papel da NOX2 sobre a glicotoxicidade em células-β. Ilhotas de camundongo C57BL/6J nocautes ou não para NOX2 (NOX2-KO e WT, respectivamente) foram isoladas e cultivadas por até 3 semanas em 10 ou 30 mmol/l de glucose (G10 e G30, respectivamente). A secreção de insulina foi maior nas ilhotas NOX2-KO vs. WT sem apresentar diferenças metabólicas ou do potencial redox da glutationa citosólica (EGSH). O cultivo de ilhotas em G30 aumenta a concentração de H2O2 e a oxidação de tióis no compartimento citosólico, seguido por aumento de apoptose de células-β, mas, preservando a reposta máxima secretória. Estas respostas foram quase idênticas em ambos os tipos de ilhotas. Em conclusão, a NOX2 regula negativamente a secreção de insulina em ilhotas de camundongos C57BL/6J, mas não é um componente crítico para a sobrevivência de células β em um modelo in vitro de glicotoxicidade. / Oxidative stress and NADPH oxidase-2 (NOX2) enzyme are associated to the decline of the functional β-cell mass in type 2 diabetes (T2D). Here, we tested the role of NOX2 on β-cell glucotoxicity. NOX2 knockout (NOX2 KO) and wild type (WT) C57BL/6J mice islets were isolated and cultured up to 3 weeks at 10 or 30 mmol/l glucose concentrations (G10 and G30, respectively). The insulin secretion was higher in NOX2-KO vs. WT islets despite similar metabolic and cytosolic glutathione-redox potential (EGSH) changes. The prolonged culture at G30 increases the H2O2 concentration and cytosolic thiol oxidation, followed by increased βcell apoptosis but preserving maximal secretory response. These responses were almost identical in both types of islets. In conclusion, NOX2 is a negative regulator of insulin secretion in C57BL/6J mouse islets, but is not a critical component for β-cell survival in a model of glucotoxicity in vitro.

Disponibiliza??o do trabalho em conte?do parcial, conforme Termo de Autoriza??o. / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-03-27T19:34:05Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) fernanda_souza_paula_parcial.pdf: 515579 bytes, checksum: f72e2289acf5068c063e2bd00f0fefc3 (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-04-24T16:35:34Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) fernanda_souza_paula_parcial.pdf: 515579 bytes, checksum: f72e2289acf5068c063e2bd00f0fefc3 (MD5) / Made available in DSpace on 2017-04-24T16:35:34Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) fernanda_souza_paula_parcial.pdf: 515579 bytes, checksum: f72e2289acf5068c063e2bd00f0fefc3 (MD5) Previous issue date: 2017 / O trabalho investiga a utiliza??o de plataformas eletroqu?micas contendo filmes polim?ricos derivados do ?cido 4-aminosalicico (4-AMS) para imobiliza??o da enzima Urato oxidase (UOx) visando aplica??o na quantifica??o de ?cido ?rico (AU) em amostras de urina. Investigou-se a eletrodeposi??o do 4-AMS pelas t?cnicas de voltametria c?clica (VC) e cronoamperometria (CA) sobre eletrodos de grafite (EG). Por VC foram realizados 100 ciclos de potencial na faixa de -0,25 a 1,25 V ? 50 mV/s em solu??o 2,50 mM do mon?mero preparado em H2SO4 0,50 M. Utilizando a CA, a eletropolimeriza??o foi realizada a potencial constante de +0,928 V durante 5600s no mesmo meio reacional utilizado na VC. O poli(4-AMS) obtido por VC e CA mostrou dois pares redox, os quais est?o relacionados a eletroatividade do filme polim?rico, na regi?o de potencial de +0,50/+0,40 V. Contudo, maiores valores de Ipa e Ipc foram obtidos para os eletrodos modificados por VC, sugerindo que estes filmes s?o mais eletroativos. A deposi??o do azul da Pr?ssia (AP), mediador da rea??o de per?xido, foi investigada sobre os EG, com posterior modifica??o com poli(4-AMS). Notou-se que a presen?a do AP n?o altera o perfil voltam?trico da eletropolimeriza??o do 4-AMS. Contudo, quando comparada com a eletropolimeriza??o somente nos EG, obteve-se filmes mais resistivos e com menor eletroatividade. Analisando as propriedades eletroqu?micas e morfol?gicas, por VC conseguiuse filmes mais uniformes, com maior quantidade de material depositado e maior eletroatividade. A eletropolimeriza??o foi realizada tamb?m sobre eletrodos impressos de grafite contendo azul da Pr?ssia (EI/AP), onde posteriormente imobilizou-se 5 U da UOx, e o biossensor foi acoplado a uma c?lula de fluxo num sistema de an?lise por inje??o em fluxo (FIA) de linha ?nica. A vaz?o e o volume da al?a de amostragem foram otimizados em 2,10 mL/min e 200 ?L, repectivamente. O valor de pH da solu??o do analito foi otimizado em 8,27. Medidas de reprodutibilidade mostraram desvio padr?o de 2,15% (n=10). O biossensor respondeu linearmente para AU na faixa de 1,0 x 10-5 a 2,0 x 10-4 M, com limite de detec??o de 3,0 ?M. Amostras de urina foram dilu?das (1:10) e injetadas diretamente no biossensor. A reposta foi reprodut?vel mostrando baixo desvio padr?o para as medidas, e valores encontrados dentro da faixa esperada para o analito em amostras de urina. Testes de adi??o e recupera??o mostraram valores de 97,35% (?2,43). O biossensor mostrou-se bastante promissor para a proposta do trabalho, apresentando resultados muito satisfat?rios para as an?lises e par?metros investigados. / Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / This work investigates the use of electrochemical platforms containing polymeric films derived from 4-aminosalicylic acid (4-ASA) modified with the enzyme Urate oxidase (UOx) for quantification of uric acid (UA) in urine samples. The electrodeposition of 4-ASA was investigated through Cyclic Voltammetry (CV) and Chronoamperometry (CA) on graphite electrodes (GE). 100 cycles were performed in the range of -0.25 to 1.25 V at 50 mV/s in 2.50 mM monomer solution prepared in 0.50 M H2SO4. Using CA, the deposition was performed at a potential of +0.928 V for 5600 s in the same CV reaction medium. The poly(4-ASA) showed two redox pairs related to the electroactivity of the polymeric film in the potential range of + 0.50 /+ 0.40 V. However, higher values of Ipa and Ipc were obtained for the electrodes modified through CV, suggesting that these films are more electroactive. The deposition of Prussian blue (PB), mediator of the peroxide reaction, was investigated on the GE with subsequent modification with poly (4-ASA). It was observed that the presence of PB does not alter the voltammetric profile of 4-ASA electropolymerization. However, when compared with the electropolymerization in bare GE, more resistive films were obtained with lower electroactivity. Analyzing the electrochemical and morphological properties through CV, more uniform films were obtained, with more material deposited and greater electroactivity. The electropolymerization of poly(4-ASA) was also conducted on screen printed electrodes containing Prussian Blue (SPE/PB), with subsequent immobilization of 5U of Uox. This biosensor was coupled to a flow cell in a Flow Injection Analysis (FIA) system of single line. The flow rate and the sampling loop volume were optimized at 2.10 mL/min and 200 ?L, respectively. The pH value of the analyte solution was optimized at 8.27. Reproducibility measures showed a standard deviation of 2.15% (n = 10). The biosensor responded linearly to UA in the range of 1.0 x 10-5 to 2.0 x 10-4 M, with a detection limit of 3.0 ?M. Urine samples were diluted (1:10) and directly injected over the biosensor. The response was reproducible with low standard deviation and values found within the range expected for the analyte in urine samples. Addition and recovery tests showed values of 97.35% (?2.43). The biosensor is very promising for the work proposal, presenting very satisfactory results for the analyzes and investigated parameters.