Para a bioeletrocatálise de oxidação de etanol, a alteração da superfície eletródica e a otimização do processo de imobilização enzimática se fazem necessárias. Neste cenário, as fibras flexíveis de carbono (FFC) merecem destaque, pois além de sua superfície ser facilmente modificada devido à presença de carbono sp2, as mesmas possuem alta resistência mecânica e elasticidade, combinadas com a alta condutividade elétrica e térmica. Nesta tese de doutorado, apresenta-se como obter bioeletrodos de FFC modificadas com a enzima álcool desidrogenase (ADH) NAD-dependente, visando também aprimorar a oxidação da coenzima NADH (dinucleotídeo de nicotinamida e adenina). Os resultados mostram que quando as FCF são previamente submetidas a um tratamento oxidativo em meio ácido (KMnO4/H2SO4), obtém-se bioeletrodos estáveis, robustos e com alta área superficial. Além disso, observou-se que esses eletrodos possuem grupos funcionais contendo oxigênio que auxiliam na bioeletrocatálise de oxidação do etanol. Presume-se que presença de grupos quinonas seja responsável por facilitar a regeneração da coenzima, ou seja, estes grupos atuam decisivamente na oxidação do NADH. A alta qualidade dos bioeletrodos possibilitou manter a atividade catalítica da ADH por longo prazo, propriedade essa crucial para o estudo da oxidação do etanol acoplada à espectrometria de massas (DEMS). Devido a este estudo, foi possível observar concomitantemente a regeneração da coenzima (NADH -> NAD+) e a geração de acetaldeído como produto de bioeletroxidação do etanol, ambos em estado estacionário. Em suma, o estudo aqui apresentado introduz uma abordagem que combina não só o desenvolvimento de fibras de carbono tratadas quimicamente para aplicação em bioeletrocatálise, mas também um foco inédito no acoplamento entre a espectrometria de massas e a bioeletroquímica para a resolução de mecanismos enzimáticos. / Regarding the bioelectrocatalysis of the ethanol oxidation, the electrodic surface modification and the optimization of enzymatic immobilization are necessary. In this scenario, the flexible carbon fibers (FCF) are noteworthy, because besides their surface can be modified in an easy way due the presence of carbon sp2, they have high mechanical resistance and elasticity, combined with high electrical and thermal conductivity. In this doctoral thesis, it is presented how to obtain bioelectrodes of FFC modified with the enzyme alcohol dehydrogenase (ADH) NAD-dependent, as well as to improve the oxidation of the coenzyme NADH (nicotinamide adenine dinucleotide). The results show that when FCF is previously submitted to an oxidative treatment in acidic medium (KMnO4/H2SO4), stable, robust and high surface area bioelectrodes are obtained. In addition, it was observed that these electrodes have oxygen-containing functional groups that improve the bioelectrocatalysis of ethanol oxidation. There is proposed that the presence of quinone groups is responsible for facilitating the regeneration of the coenzyme, i. e., these groups act decisively in the oxidation of NADH. The high quality of the bioelectrodes allowed it to maintain the catalytic activity of the ADH for long term, property crucial for the study of the oxidation of ethanol coupled to mass spectrometry (DEMS). By using DEMS, there were possible to observe coenzyme regeneration and the generation of acetaldehyde as a bioelectrooxidation product of ethanol, both at steady state, which were simultaneously observed. In summary, the present study introduces an to an approach that combines not only the development of chemically treated carbon fibers for application in bioelectrocatalysis, but also an unprecedented focus on the coupling between mass spectrometry and bioelectrochemistry for the resolution of enzymatic mechanisms.

This study focuses on the characterisation of three novel cytochrome P450-partner (P450-fusion) enzymes of unknown structure and function. Despite several well-established P450 functions, new structures of P450s are published frequently, with the P450-redox partner fusion systems being among the most discussed, due to their enhanced activity and biotechnological potential. Other, more intriguing, P450-fusions involve partners with functions distinct from electron transfer. Understanding why evolution drove the ‘partner’ proteins to evolve into a single unit is often unclear, but provides an important challenge for the understanding of the breadth of biochemical reactions mediated by P450s. The first P450-fusion analysed (Chapter 3) is CYP116B1 from a soil bacterium, Cupriavidus metallidurans, that displays important environmental implications. The enzyme was characterised as a functional fusion, composed of three domains: a P450 from the CYP116B family, and a phthalate dioxygenase reductase (PDOR)-like reductase binding FMN and a 2Fe-2S cluster. CYP116B1 is a stable, cytosolic enzyme but can undergo FMN cofactor loss. Studies included redox potentiometry of the intact fusion and its individual domains using spectro-electrochemical and EPR methods to enable the determination of midpoint redox potentials for individual cofactors. The CYP116B1 EPR signature was shown to be typical of P450s, and changed upon binding heme-coordinating inhibitors of the azole class. Extensive compound library screening did not reveal a substrate-like physiological “hit”. However, catalytic activity was detected towards selected thiocarbamate herbicides. GC-MS data revealed the enzymatic mechanism of herbicide degradation. The second system studied (Chapter 4) is P450-CAD, an atypical fusion of an uncharacterised soluble P450 and a cinnamyl alcohol dehydrogenase (ADH) module from Streptomyces ghanaensis; a member of the major antibiotic producing genus of bacteria. The CAD module appears unlikely to be a redox partner, but instead possibly mediates substrate/product exchange with the P450. The intact fusion was shown to aggregate during extraction. Genetic dissection of domains revealed that this was due to the highly insoluble ADH moiety. The heme domain (HD) was soluble and was characterised extensively. The enzyme displays an unusual spectrum when in the FeII-CO complex (Amax = 445 nm). The P450-CAD HD catalytic activity is supported by heterologous redox partners (E. coli flavodoxin reductase [FldR] and flavodoxin [FldA], and spinach ferredoxin reductase [FdR] and ferredoxin [Fdx]). The CAD-HD binds fatty acid substrates of carbon chain length C8-14, with the highest affinity for 12-methylmyristic acid (12M14C acid), the C12 lauric acid, its aldehyde and alcohol, indicating that the terminal methyl group is important for binding to the enzyme. Unusually, the CAD-HD also binds a range of detergent compounds. Further analysis included SEC-MALLS, thermostability and structural studies. The final enzyme studied (Chapter 5) is the P450-BDOR (a P450 linked to a benzoate dioxygenase reductase) redox-partner fusion. The unconventional trait of this enzyme is the inclusion of an FCD (a fatty acid metabolism regulator protein [FadR] C-terminal DNA-binding domain). From the point of view of P450s, DNA interaction would represent an unprecedented function, suggesting novel functions for a P450 enzyme. Thus, this enzyme requires extensive research with the expectations that new information will contribute to an expansion of knowledge of P450 diversity. This study provides initial analytical insights into the P450-BDOR system, supported with functional and kinetic data on the P450 and its reductase domain.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22502号 / 農博第2406号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5282(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 梅澤 俊明, 教授 矢﨑 一史, 教授 渡邊 隆司 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24671号 / 農博第2554号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5452(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 阪井 康能, 教授 白井 理 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

As biocélulas a combustível proporcionam meios de se obter energia de maneira mais sustentável, limpa e renovável e apresentam grande potencial para serem usadas como fontes de energia alternativas para dispositivos eletrônicos de baixa demanda energética. Esta dissertação investiga a bioeletro-oxidação de etanol pela enzima Álcool Desidrogenase (ADH) empregando-se bioânodos com polimerização simultânea do mediador (poli - verde de metileno) e polímero condutor (polipirrol). Uma vez preparado o bioânodo foram realizados estudos para verificar a oxidação do cofator (NADH), formação do produto da bio-oxidação de etanol, estabilidade e também estudos visando aumentar a eficiência energética gerada empregando nanotubos de carbono para aumento de área eletroquímicamente ativa e melhor comunicação com os sítios ativos das enzimas. Foi também preparada uma biocélula completa sem membrana polimérica visando diminuir resistência de transferência de prótons do sistema, com a configuração final constituída de ADH/NAD+, etanol, Lacase/O2 com e sem MWCNT. O bioânodo se mostrou relativamente estável, apresentando um decaimento médio de 36% do valor inicial da densidade de potência após 20 semanas de estocagem. Quando operado em condição contínua (tempo = 4h, E (V)= ½ PCA), o decaimento foi de 39% e de 66% em 12 horas de operação. Os valores de densidade de potência foram melhorados com a adição de MWCNTs sobre o suporte de C antes da eletropolimerização do filme simultâneo, em pH 8,9, obteve-se 275 + 12 ?W cm-2. A eletrólise para este sistema mostrou a formação de acetaldeído com conversão de 18% de etanol. Para a biocélula completa, os melhores resultados foram com a presença de MWCNTs no bioânodo, obtendo-se uma potência de 12,5 +0,9 ?W cm-2. Os resultados obtidos são bastante promissores comparado com a literatura atual e mostram que esse sistema pode ser empregado para construção de BFC. / Biofuel cells provide the means to obtain energy in a more sustainable, clean, and renewable way and have great potential as alternative energy sources for low-energy electronic devices. This dissertation investigates ethanol bioelectrooxidation by the enzyme Alcohol Dehydrogenase (ADH) at bioanodes with simultaneous polymerization of the mediator (polymethylene green) and conductive polymer (polypyrrole). After preparing the bioanode, we investigated cofactor (NADH) oxidation, the ethanol biooxidation product,and bioanode stability. We also conducted studies to increase the generated energy efficiency by using carbon nanotubes to augment the electrochemically active area and to improve communication with the enzyme active sites. We also prepared a complete biofuel cell without polymer membrane to decrease the system proton transfer resistance: the final configuration consisted of ADH / NAD +, ethanol, Laccase , and O2 with or without MWCNTs. The bioanode was relatively stable. The mean decay was 36% of the initial power density after 20 weeks of storage. When the bioanode was operated in continuous condition (time = 4h, E (V) = ½ PCA), the decay was 39% and 66% in 12 hours of operation. The power density values increased upon addition of MWCNTs to the C-support before simultaneous film electropolymerization at pH 8.9, to give 275 + 12 ?W cm-2. Under electrolysis conditions, this system produced acetaldehyde and converted 18% of ethanol. For the complete biofuel cell, the best results were with the presence of MWCNTs in the bioanode, which provided a power of 12.5 +0.9 ?W cm-2. The results obtained here are quite promising if compared to the current literature and show that this system can be used to construct a BFC.

Naudojant spektrofotometrinį metodą tirtas ADH IIG reaktingumas su skirtingais mediatoriais ir įvairiais alkoholiais. Nustatytos tariamųjų kinetinių parametrų KM ir Vmax reikšmės, bei paskaičiuotos katalitinės ir bimolekulinės alkoholių oksidacijos bei mediatorių redukcijos reaktingumo konstantos. Tirta FERO ir ABTS oksidacija katalizuojama L95 lakaze. Gautos pradinių reakcijos greičių priklausomybės nuo substratų koncentracijų. Rezultatų analizei pritaikius Michaelio-Menten lygtį, gautos tariamųjų KM ir Vmax reikšmės, iš kurių buvo paskaičiuotos katalitinės ir oksidacinės konstantos. Pamatuotos pradinio reakcijos greičio priklausomybės nuo buferinių tirpalų pH ir jų komponentų, bei nustatytos optimalios substratų oksidacijos reakcijos pH reikšmės. Naudojant deguoninį elektrodą buvo registruotas deguonies sunaudojimas vykstant L95 lakazės katalizuojamai FERO ir ABTS oksidacijai ir paskaičiuotas pradinis reakcijos greitis bei sunaudoto deguonies priklausomybės nuo substratų koncentracijų. Registruotas ir deguonies sunaudojimas vykstant ADH IIG katalizuojamai alkoholio oksidacijai ir lakazes katalizuojamai mediatoriaus (antrojo qADH substrato) regeneracijai. Darbą sudaro 6 dalys: įvadas, literatūros apžvalga, eksperimentinė dalis, rezultatai ir jų aptarimas, išvados, literatūros sąrašas. Darbo apimtis – 85 p. teksto, 50 paveikslų, 8 lentelės, 109 bibliografiniai šaltiniai. / Reactivity of ADH IIG with different mediators and various alcohols were investigated by spectrophotometric method. Values of apparent kinetic parameters KM and Vmax were determined, catalytic and bimolecular alcohols oxidation and mediators reduction reactivity constants were calculated. The FERO and ABTS oxidation catalysed by L95 laccase were investigated. The initial reaction rate dependencies on the substrates concentrations were obtained. The analysis of the results was performed by applying Michael-Menten equation and the values of apparent KM and Vmax, as well as catalytic and oxidation constants were calculated. The dependencies of the initial reaction rate on the buffer solutions pH and their components were measured also the optimal pH of the substrates oxidation reactions were determined. The oxygen consumption was registered L95 laccase catalysed oxidation of FERO and ABTS by oxygen electrode and initial reaction rate was calculated, as so as oxygen consumption dependences on substrate concentrations. The oxygen consumption ADH IIG catalysed oxidation of alcohols and laccases catalysed regeneration of mediators (second qADH substrate) were registered. Structure: introduction, 3 chapters, conclusions, references. Thesis consist of: 85 p. of text, 50 pictures, 8 tables, 109 bibliographical entries.

Alzheimer’s disease (AD) is the leading cause of dementia but despite being identified over a century ago, current treatments remain limited. To date, no disease-modifying therapies are available. Soluble, intracellular forms of β-amyloid (Aβ), a protein associated with AD, have been identified and intracellular targets of Aβ are being investigated as potential targets for new drugs. Amyloid binding alcohol dehydrogenase (ABAD) was previously identified as a mitochondrial target of Aβ and is known to be up-regulated in AD. This interaction results in production of reactive oxygen species and cell death. Using a small peptide, known as the “decoy peptide”, disruption of this interaction has been shown to reverse biochemical and behavioural symptoms in an AD mouse model. The work reported in this thesis describes the approaches taken to develop methods for in vitro and ex vivo study of the interaction between ABAD and Aβ. A fluorogenic assay for measuring the intracellular activity of ABAD in living cells was developed and using this technique, the intracellular inhibition of ABAD by Aβ was observed for the first time. Surface plasmon resonance was used to measure binding between ABAD and Aβ and also showed the first quantitative analysis of direct binding of the decoy peptide to Aβ42. In order to synthesise small molecule inhibitors of ABAD activity with the aim of developing a molecular probe of the enzyme’s activity, compounds were identified by screening a fragment-based library. Subsequent optimisation of the compound structure led to a 10-fold improvement in the IC50 and has resulted in a lead compound for future development. A similar screening strategy was employed to identify potential small molecule inhibitors of the ABAD-Aβ interaction. This research has resulted in a range of tools and methods for studying ABAD activity and interactions, which will greatly benefit future work on developing compounds that inhibit the ABAD-Aβ interaction to provide a novel method for treating Alzheimer’s disease.

Made available in DSpace on 2014-08-20T13:25:41Z (GMT). No. of bitstreams: 1 Tese_Juliana_Severo_Castelo_Branco.pdf: 7339681 bytes, checksum: 5c86995be26b0fd8b4d1fda6177461ec (MD5) Previous issue date: 2008-10-01 / Among the cereals, wheat (Triticum aestivum L.) is the most important crop reaching the third largest grain production in the world. Wheat is cultivated under many different environmental conditions, presenting high grain yield capacity, nutritional quality and high degree of adaptability. In the State of Rio Grande do Sul, Brazil, approximately 5.4 million hectares are considered hydromorphic soils and are mostly used for rice farming and cattle grazing, due to its low drainage capability. Annually, only one million hectares are cultivated with rice, leaving large amounts of land without cropping or cultivated with forage crops. Therefore, there is a strong challenge for breeding programs to develop genotypes capable of tolerating this environment, with a satisfactory cost/benefit ratio for farmers. In order to suppress the energy deficit provoked by hypoxic conditions, several species redirect their metabolic pathways in order to garantee the extra ATP production resulting from an increase in fermentation rates. In those species, a group of proteins that cathalyse glycolisis and phosphate sugar metabolism such as the enzyme alcohol dehydrogenase (ADH), is selectively synthesized. The objective of this work was to verify the possibility of selecting wheat genotypes tolerant to flooding stress, in the greenhouse, by using different periods of flooding conditions alterned with draining periods. Also, to estimate the degree of conservation of the ADH1 gene in some plant species. The results indicate that it is possible to detect genetic variability for the character flooding tolerance in greenhouse. The condition of eight days of flooding revealed that the genotypes BRS 120 and BRS 208 were the most tolerant and the genotypes CD 111 and BRS 194 were the most sensitive. The molecular phylogenetic analysis obtained for the alcohol dehydrogenase (ADH1), indicates that Poaceae members present a high degree of conservation. The closest species were Saccharum oficinarum and Zea mays, presenting higher nucleotide identity. The model species (Oryza sativa) presented a higher dissimilarity regarding the remaining species of the Poaceae, suggesting that this enzyme has acquired novel functions and or mutations that were selected under the flooding condition. / Entre os cereais, o trigo (Triticum aestivum L.) é a mais importante cultura, alcançando a terceira maior produção de grãos no mundo sobrepujado apenas pelo milho e arroz. O trigo é cultivado sob diversas condições ambientais, apresentando alta capacidade de produção de grãos, qualidade nutricional e alto grau de adaptabilidade. No Estado do Rio Grande do Sul, aproximadamente 5,4 milhões de hectares são considerados solos hidromórficos e são principalmente utilizados para cultivo de arroz e pastagem, devido a sua baixa capacidade de drenagem. Anualmente, apenas um milhão de hectares está sendo utilizado para o cultivo de arroz, deixando grandes quantidades de terras para o cultivo de culturas forrageiras. Portanto, existe um forte desafio para programas de melhoramento no desenvolvimento de genótipos capazes de tolerar esse ambiente, com uma boa relação custo/benefício para os agricultores. A fim de suprimir o déficit energético provocado pelas condições de hipoxia, várias espécies redirecionam suas vias metabólicas, a fim de garantir a produção extra ATP resultantes do aumento das taxas de fermentação. Nessas espécies, um grupo de proteínas que catalisam a glicólise e metabolismo de açúcares, como a enzima álcool desidrogenase (ADH), é sintetizada seletivamente. O objetivo deste trabalho foi verificar a possibilidade de seleção de genótipos de trigo tolerantes ao estresse por alagamento, em casa de vegetação, utilizando diferentes períodos de alagamento intercalados com drenagem. Além disso, estimar o grau de conservação do gene ADH1 em algumas espécies de plantas. Os resultados indicam que é possível a detecção de variabilidade genética para o caráter tolerância ao alagamento em casa de vegetação. A condição de oito dias de alagamento revelou que os genótipos BRS 120 e BRS 208 foram as mais tolerantes e os genótipos CD 111 e BRS 194 foram as mais sensíveis. A análise filogenética molecular obtida para o álcool desidrogenase (ADH1), indica que os membros Poaceae apresentaram um elevado grau de conservação. As espécies mais próximas foram Saccharum oficinarum e Zea mays, apresentando maior identidade de nucleotídeos. A espécie modelo para plantas monocotiledôneas (Oryza sativa) apresenta uma maior dissimilaridade quanto ao restante das espécies de Poaceae, sugerindo que esta enzima tenha adquirido níveis de funções ou mutações que foram selecionadas sob a condição alagamento.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol dehydrogenase (ADH) genes encode proteins that metabolize ethanol to acetaldehyde. Humans have seven ADH genes in a cluster. The hypothesis of this study was that by controlling the levels of ADH enzymes, cis-regulatory regions could affect the risk for alcoholism. The goal was thus to identify distal regulatory regions of ADHs. To achieve this, sequence conservation across 220 kb of the ADH cluster was examined. An enhancer (4E) was identified upstream of ADH4. In HepG2 human hepatoma cells, 4E increased the activity of an ADH4 basal promoter by 50-fold. 4E was cell specific, as no enhancer activity was detected in a human lung cell line, H1299. The enhancer activity was located in a 565 bp region (4E3). Four FOXA and one HNF-1A protein binding sites were shown to be functional in the 4E3 region. To test if this region could affect the risk for alcoholism, the effect of variations in 4E3 on enhancer activity was tested. Two variations had a significant effect on enhancer activity, decreasing the activity to 0.6-fold. A third variation had a small but significant effect. The effect of variations in the ADH1B proximal promoter was also tested. At SNP rs1229982, the C allele had 30% lower activity than the A allele. In addition to studying the regulatory regions of ADH genes, the effects of alcohol on liver-derived cells (HepG2) were also explored. Liver is the primary site of alcohol metabolism, and is highly vulnerable to injuries due to chronic alcohol abuse. To identify the effects of long term ethanol exposure on global gene expression and alternative splicing, HepG2 cells were cultured in 75 mM ethanol for nine days. Global gene expression changes and alternative splicing were measured using Affymetrix GeneChip® Human Exon 1.0 ST Arrays. At the level of gene expression, genes involved in stress response pathways, metabolic pathways (including carbohydrate and lipid metabolism) and chromatin regulation were affected. Alcohol effects were also observed on alternative transcript isoforms of some genes.