In this study, two symbiotic fungi of Southern Pine Beetle (SPB), Entomocorticium peryii and Entomocorticium sp.A were evaluated in terms of polyphenol oxidase (PPO) production. The effect of different inhibitors, inducers and assay parameters such as temperature and pH on enzyme activity were investigated and maximum PPO activity was observed at 30&deg / C, pH 8.0 and when tannic acid was used as an inducer. Copper-chelator salicyl hydroxamic acid (SHAM) and pcoumaric acid, both indicated as inhibitors of tyrosinase and catechol oxidase significantly reduced the activity. For biochemical characterization studies, the enzyme was concentrated by ultrafiltration. To determine type of the enzyme, activity staining after Native-PAGE was carried out. Type of polyphenol oxidase produced by E. peryii and E. sp.A was determined as catechol oxidase by activity staining. However higher activity was observed on hydroquinone (p-diphenol) rather than catechol (o-diphenol). The enzyme obeys Michealis-Menten kinetics with Km and Vmaxvalues being 10.72 mM hydroquinone and 59.44 U/ml for E. peryii and 8.55 mM hydroquinone and 73.72 U/ml for E. sp.A respectively..

ABSTRACT BIOACTIVE SURFACE DESIGN BASED ON CONDUCTING POLYMERS AND APPLICATIONS TO BIOSENSORS Ekiz, Fulya M. Sc., Department of Biotechnology Supervisor: Prof. Dr. Levent Toppare Co-Supervisor: Prof. Dr. Suna Timur June 2012, 88 pages An underlying idea of joining the recognition features of biological macromolecules to the sensitivity of electrochemical devices has brought the concept of biosensors as remarkable analytical tools for monitoring desired analytes in different technological areas. Over other methods, biosensors have some advantages including high selectivity, sensitivity, simplicity and this leads to solutions for some problems met in the measurement of some analytes. In this context, conducting polymers are excellent alternatives with their biocompatibility and ease of applicability for an efficient immobilization of biomolecules in preparing biosensors. Using several materials and arranging the surface properties of the electrodes, more efficient and seminal designs can be achieved. In this thesis, it is aimed to create new direct biosensors systems for the detection of several analytes such as glucose and pesticides thought to be harmful to the environment. Recently synthesized conducting polymers (polyTBT) / (poly(2-dodecyl-4,7-di(thiophen-2-yl)-2H-benzo[ d][1,2,3]triazole) and (poly(TBT 6 -NH2 ) / poly(6-(4,7-di(thiophen-2-yl)-2H-benzo[d][1,2,3]triazol-2-yl)hexan-1-amine) were utilized as a matrices for biomolecule immobilization. After successful electrochemical deposition the polymers on the graphite electrode surfaces, immobilization of glucose oxidase (GOx) and choline oxidase (ChO) were carried out. Amperometric measurements were recorded by monitoring oxygen consumption in the presence of substrates at -0.7 V. The optimized biosensors showed a very good linearity with rapid response times and low detection limits (LOD) to glucose and choline. Also, kinetic parameters, operational and storage stabilities were determined. Finally, designed biosensor systems were applied for glucose and pesticide detection in different media.

COX deficiency in human presents a plethora of phenotypes which is not surprising given the complexity of the enzyme structure and the multiple factors and many steps required for its assembly. A functional COX requires three mitochondrially encoded subunits (Cox1p, Cox2p and Cox3p), at least 10 nuclearly encoded subunits, some of which are tissue specific, and a yet unknown number of assembly factors. Mutations in four of these factors, hSco1p, hSco2p, hCox10p and hSurf1p, have been associated with lethal COX deficiency in patients. Sco proteins, conserved from prokaryotes to eukaryotes, are probably involved in the insertion of copper in COX. The role of hSco1p and hSco2p in this process was investigated in this work. Moreover the importance of some hSco mutations found in patients was analysed. Both in vitro and in vivo analyses show that the hSco proteins are localised in the mitochondria. Both proteins are per se unable to substitute for ySco1p. However, a chimeric construct consisting of the N-terminal portion, the TM and a part of the C-terminal portion of ySco1p and the remaining C-terminal part derived from hSco1p was able to complement a ysco1 null mutant strain. This construct was used to define the role of a point mutation (P174L) found in the hSCO1 gene of infants suffering from ketoacidotic coma. These mutation was shown to affect the COX activity and the levels of Cox1p and Cox2p. The fact that copper was able to suppress this mutation, strongly outlined the importance of Sco proteins in the copper insertion in COX. The C-terminal portions of recombinant hSco1p and hSco2p were purified from E. coli by affinity chromatography. The purified proteins were subjected to atomic emission and absorption analyses and were shown to specifically bind copper. A stoichiometry of 1:1 for hSco2p and of 0,6:1 for hSco1p was determined. To identify the Aa residues involved in copper binding, in vitro mutagenesis was performed. hSco1p and hSco2p, lacking the cysteines of the predicted metal binding site CxxxC, show a dramatic decrease in the ability to bind copper. A model for the structure of the metal binding site in hSco proteins is proposed. hSco proteins could bind copper with trigonal coordination, involving the two cysteines of the CxxxC motif and a conserved histidine. The purified recombinant proteins were also used in an enzymatic assay to test their ability to reduce disulfide bridges, similar to thioredoxin-like proteins involved in the assembly of bacterial COX. Both hSco proteins were not able to act as thioredoxins suggesting a role for the hSco proteins as copper chaperones. To define the pathway of the copper transfer to COX, hSco proteins were tested for their ability to interact with hCox17p, a mitochondrial copper chaperone, and with Cox2p, which contains two copper ions. An interaction between hSco1p and Cox2p was detected. Both hSco proteins were shown to homomerise and to form heterodimers one with each other. Two mutations found in hSCO2 patients suffering from hypertrophic cardiomyopathy, (E140K and S225F) were shown not to affect the copper binding properties, the intracellular localisation and the ability to form homomers. In accordance to these data, a model is proposed in which hSco2p dimers transfer copper to hSco1p dimers. hSco1p dimers interact with COX and insert copper in the binuclear centre of Cox2p.

No description available.

Heme-copper oxidases are transmembrane proteins that are found in aerobic and anaerobic respiratory chains. During aerobic respiration, these enzymes reduce dioxygen to water. The energy released in the reaction is used to transport protons across a biological membrane. Stored as proton electrochemical gradient, the energy can be used to regenerate ATP. It is known that aa3 oxidases, which are the most common oxidases, transport pumped protons and protons used for the catalytic reaction using two proton pathways. However, the molecular mechanism of pumping is still being debated. When oxygen is available in very small quantities, oxygen reductases with high affinity for oxygen are expressed by organisms like Thermus thermophilus. The proton pumping mechanism in the ba3 oxidase is slightly different from that of aa3 oxidases as this enzyme only uses a single proton uptake pathway. Here we analyzed the reaction mechanism of ba3 oxidase and found evidence that the first proton taken up by the four-electron reduced ba3 oxidase is transferred to a site distant from the catalytic site, the pump site, and that only every second proton taken up from solution is pumped. Data obtained from studies using site-directed mutagenesis and flow-flash spectroscopy suggest a probable location of the pump site. Under anaerobic conditions, some organisms are able to generate a proton- motive force using nitrate and nitrite as electron acceptors. In this process, the cytotoxic reaction intermediate nitric oxide is produced. Nitric oxide reductase (NOR), a deviant heme-copper oxidase that reduces NO to the rather harmless N2O, does not pump any protons. The catalytic mechanism of nitric oxide reduction by NOR is very poorly understood. Here we demonstrate that substrate inhibition, which occurs in NOR from Paracoccus denitrificans above 5 μM NO, can already be observed before the electrons from the low-spin hemes re-distribute to the active site. Furthermore, we found that a single specific proton pathway is used for proton-transfer leading from the periplasm to the active site.

ABSTRACT IMMOBILIZATION OF INVERTASE, POLYPHENOL OXIDASE AND GLUCOSE OXIDASE IN CONDUCTING COPOLYMERS OF THIOPHENE-CAPPED POLYTETRAHYDROFURAN AND PYRROLE B&ouml / y&uuml / kbayram, AySe Elif Ph.D., Department of Chemistry Supervisor: Prof. Dr. Levent Toppare January 2005, 123 pages Immobilization of invertase, polyphenol oxidase (PPO) and glucose oxidase (GOD) enzymes were performed in electrochemically synthesized two types of conducting copolymers. One end and two end thiophene-capped polytetrahydrofuran (TPTHF-1 and TPTHF-2) were copolymerized with pyrrole under conditions of constant potential electrolysis. The copolymers were characterized by thermal, spectroscopic and scanning electron microscopy analyses. Immobilization was carried out via entrapment of enzymes in two types of matrices during the copolymerization of pyrrole with the insulating polymers in the presence of sodium dodecyl sulphate (SDS). Kinetic parameters: Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes. Temperature optimization, pH optimization, operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase and glucose oxidase were used to determine the amount of their substrates in samples. Polyphenol oxidase converts mono and diphenols to quinone. Amount of phenolic compounds in two kinds of wines were determined by analyzing the quinone amount. Glucose oxidase converts &amp / #61538 / -D-glucose to D-glucono-1,5-lactone. Glucose amount was determined in two kind of factory-produced orange juices by analyzing D-glucono-1,5-lactone.

In this work, we have analysed the temporal dynamics of gut-dwelling bacteria and Borrelia in the gut of the deer tick Ixodes ricinus. Using quantitative PCR, we have shown that levels of the tick intestinal microflora are profoundly decreased at later stages of feeding on whole blood but not on serum. Even though we noted that host complement system manages to interfere with Borrelia viability in vitro, we did not see any effect of host complement on Borrelia acquisition in adult ticks in vivo. However, we revealed that host hemoglobin is essential for Borrelia proliferation in the tick gut. All together, these data imply that, during feeding, levels of gut-dwelling bacteria and Borrelia are determined by the host. While hemoglobin seems to be detrimental for gut-dwelling bacteria, Borrelia require it in order to proliferate. During off-host stage, we showed that levels of gut-dwelling microflora are regulated by an intestinal transmembrane enzyme Dual oxidase. In conclusion, we aimed, and mostly succeeded, to perform pilot experiments describing the biology of a complex process of regulating gut microflora in the vector Ixodes ricinus and extend it by its impact on Borrelia acquisition

A diabetes é um distúrbio endócrino do metabolismo dos carboidratos clinicamente caracterizado por hiperglicemia, resultante da incapacidade do organismo em secretar insulina, defeitos na sua ação ou ambos. Recentemente, as conseqüências neurológicas da diabetes no sistema nervoso central têm recebido maior atenção, entretanto os mecanismos pelos quais a hiperglicemia é capaz de danificar o tecido nervoso ainda permanecem pouco esclarecidos. Estudos recentes têm demonstrado que a hiperglicemia é capaz de induzir dano oxidativo em cérebro de ratos. Portanto, o presente trabalho objetivou produzir um modelo de hiperglicemia neonatal em ratos e investigar o papel do estresse oxidativo (EO) na neurotoxicidade da hiperglicemia neonatal. Para a indução do modelo de hiperglicemia neonatal foram utilizados ratos Wistar de 5 dias de vida que foram submetidos a administração intraperitoneal de 100 mg/Kg de peso corporal de estreptozotocina (STZ), sendo que 5 dias após a administração de STZ os animais foram sacrificados e a média glicêmica do grupo diabético (222 mg/dL) durante todo tratamento é 82% maior do que a média do grupo controle (121 mg/dL). Os efeitos da hiperglicemia neonatal induzida por STZ foram estudados sobre os seguintes parâmetros de EO em cérebro de ratos: as atividades das enzimas glicose-6-fosfatodesidrogenase (G6PD), 6-fosfogluconato-desidrogenase (6PGD) e NADPH oxidase (Nox); o conteúdo de ânion superóxido (O2 -); as atividades das principais enzimas antioxidantes superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GSHPx) e as substâncias reativas ao ácido tiobarbitúrico (TBA-RS). Os ratos submetidos ao modelo de hiperglicemia neonatal apresentaram alto conteúdo de O2•- através da ativação da NADPH oxidase, possivelmente esta ativação dependa do NADPH derivado das enzimas G6PD e 6PGD. Além disso, o aumento dos níveis de O2 - pode ter promovido um efeito rebote de aumento das atividades das principais enzimas antioxidantes (SOD, CAT e GSHPx) e ter induzido a lipoperoxidação em cérebro de ratos. Portanto, esses resultados sugerem que o EO pode representar um mecanismo envolvido nos efeitos da hiperglicemia no sistema nervoso central de ratos neonatos. No entanto, outros estudos parecem ser necessários a fim de melhor caracterizar o papel das espécies reativas na neurotoxicidade da hiperglicemia neonatal. / Diabetes is an endocrine disorder of carbohydrate metabolism characterized by hyperglycemia and is the result of body’s inability to secret insulin or a defect of insulin action or both. The neurological consequences of diabetes on the central nervous system have most recently been received greater attention, but the mechanisms by which hyperglycemia can cause brain damage remain poorly understood. Recent studies have shown that hyperglycemia induces oxidative damage in rat brain. Therefore, this study aimed to produce a model neonatal hyperglycemia and investigate the role of oxidative stress (OS) in the neurotoxicity of neonatal hyperglycemia. The neonatal hyperglycemia was induced by one intraperitoneal administration of 100 mg/ kg body weight of streptozotocin (STZ), 5 days after the STZ administration, the animals were killed and the glucose diabetic group mean (222 mg/dL) during all treatment was 82% higher than the control group mean (121 mg/dL). So, the effects of streptozotocin-induced neonatal hyperglycemia were studied on the following oxidative stress parameters from rat brain: the activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD) and NADPH oxidase (Nox), the content of superoxide anion (O2•-), the activities of the main antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSHPx), and thiobarbituric acid-reactive substances (TBA-RS). Rats subjected to a model of neonatal hyperglycemia presented high content of O2•- through activation of NADPH oxidase and it is possible that this activation was dependent of G6PD- and 6PGD-derived NADPH. Also, increased levels on O2•- may have promoted a rebout effect, through to enhanced antioxidant enzymes activities (SOD, CAT and GSHPx) and led to lipid peroxidation in the brain. So, these results suggest that OS could represent a mechanism to understand the harmful effect of hyperglycemia on the central nervous system. However, further studies appear to be worthwhile in order to better characterize the role of reactive species in the neurotoxicity of neonatal hiperglycemia.

Made available in DSpace on 2016-06-02T19:19:58Z (GMT). No. of bitstreams: 1 SOUZA_Tamera_2014.pdf: 3828061 bytes, checksum: 5a203182c573895f9074200bb9448449 (MD5) Previous issue date: 2014-02-24 / Financiadora de Estudos e Projetos / The layer-by-layer technique is one of the most used in the immobilization of molecules of biological interest, being largely employed in the fabrication of modified electrodes for biosensors. One of the main challenges in the development of biosensors is to eliminate interfering electroactive species within the detection system, therefore, this problem can be solved with the introduction of inorganic or organic electron mediators. Here, we carried out systematic studies for the application of hemoglobin acting as an electron mediator, and the self-assembly technique was used to immobilize haemoglobin in nanostructured thin films. The LbL films were tested with two polyelectrolytes, obtaining uniform films with activity kept during 15 days, without responses to possible interfering phospholipids. The layer-bylayer technique was also used to immobilize the enzyme cholesterol oxidase, alternated with different polyelectrolytes. The thin films showed a good performance as an amperometric biosensor for the detection of cholesterol at low concentrations (10-5 M), with the functioning of the biosensor attributed to the use of hemoglobin in mediating the effects of excluding interfering ascorbic acid and uric acid. Some tests were also performed in real samples using egg yolk, with good results obtained. The high sensitivity and selectivity of the biosensor have been attributed to the nanostructured thin nature of the films, despite recognition capability of the biomolecules used, with a strong potential to contribute for new device technologies for food analysis. / A técnica de automontagem ou layer-by-layer é uma das mais utilizadas na imobilização de moléculas de interesse biológico. Essa técnica é empregada na fabricação de eletrodos modificados utilizados, como por exemplo, os biossensores. Um dos desafios no desenvolvimento de biossensores é eliminar interferentes de espécies eletroativas dentro do sistema de detecção. Esse problema pode ser solucionado com a introdução de mediadores de elétrons inorgânicos ou orgânicos. Nessa dissertação foram realizados estudos sistemáticos para a aplicação da hemoglobina atuando como mediadora de elétrons. A técnica de automontagem foi utilizada para a imobilização da hemoglobina, testada com dois polieletrólitos, obtendo filmes uniformes, mantendo sua atividade por 15 dias e sem respostas aos possíveis interferentes fosfolipídios. A automontagem também foi utilizada para imobilizar a enzima colesterol oxidase, alternada com diferentes polieletrólitos. Os filmes finos apresentaram um bom desempenho como biossensor amperométrico na detecção de colesterol em baixas concentrações tais como 10-5 M. O bom funcionamento do biossensor deve- se ao emprego da hemoglobina com mediador excluindo os efeitos dos interferentes acido úrico e acido ascórbico. Também foram realizados testes com amostras reais utilizando a gema de ovo obtendo bons resultados. A alta sensibilidade e seletividade dos biossensores foram atribuídas à natureza do filme automontado e à capacidade de reconhecimento das biomoléculas. Este estudo abre caminho para novas tecnologias de dispositivos para análises de alimentos.

A diabetes é um distúrbio endócrino do metabolismo dos carboidratos clinicamente caracterizado por hiperglicemia, resultante da incapacidade do organismo em secretar insulina, defeitos na sua ação ou ambos. Recentemente, as conseqüências neurológicas da diabetes no sistema nervoso central têm recebido maior atenção, entretanto os mecanismos pelos quais a hiperglicemia é capaz de danificar o tecido nervoso ainda permanecem pouco esclarecidos. Estudos recentes têm demonstrado que a hiperglicemia é capaz de induzir dano oxidativo em cérebro de ratos. Portanto, o presente trabalho objetivou produzir um modelo de hiperglicemia neonatal em ratos e investigar o papel do estresse oxidativo (EO) na neurotoxicidade da hiperglicemia neonatal. Para a indução do modelo de hiperglicemia neonatal foram utilizados ratos Wistar de 5 dias de vida que foram submetidos a administração intraperitoneal de 100 mg/Kg de peso corporal de estreptozotocina (STZ), sendo que 5 dias após a administração de STZ os animais foram sacrificados e a média glicêmica do grupo diabético (222 mg/dL) durante todo tratamento é 82% maior do que a média do grupo controle (121 mg/dL). Os efeitos da hiperglicemia neonatal induzida por STZ foram estudados sobre os seguintes parâmetros de EO em cérebro de ratos: as atividades das enzimas glicose-6-fosfatodesidrogenase (G6PD), 6-fosfogluconato-desidrogenase (6PGD) e NADPH oxidase (Nox); o conteúdo de ânion superóxido (O2 -); as atividades das principais enzimas antioxidantes superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GSHPx) e as substâncias reativas ao ácido tiobarbitúrico (TBA-RS). Os ratos submetidos ao modelo de hiperglicemia neonatal apresentaram alto conteúdo de O2•- através da ativação da NADPH oxidase, possivelmente esta ativação dependa do NADPH derivado das enzimas G6PD e 6PGD. Além disso, o aumento dos níveis de O2 - pode ter promovido um efeito rebote de aumento das atividades das principais enzimas antioxidantes (SOD, CAT e GSHPx) e ter induzido a lipoperoxidação em cérebro de ratos. Portanto, esses resultados sugerem que o EO pode representar um mecanismo envolvido nos efeitos da hiperglicemia no sistema nervoso central de ratos neonatos. No entanto, outros estudos parecem ser necessários a fim de melhor caracterizar o papel das espécies reativas na neurotoxicidade da hiperglicemia neonatal. / Diabetes is an endocrine disorder of carbohydrate metabolism characterized by hyperglycemia and is the result of body’s inability to secret insulin or a defect of insulin action or both. The neurological consequences of diabetes on the central nervous system have most recently been received greater attention, but the mechanisms by which hyperglycemia can cause brain damage remain poorly understood. Recent studies have shown that hyperglycemia induces oxidative damage in rat brain. Therefore, this study aimed to produce a model neonatal hyperglycemia and investigate the role of oxidative stress (OS) in the neurotoxicity of neonatal hyperglycemia. The neonatal hyperglycemia was induced by one intraperitoneal administration of 100 mg/ kg body weight of streptozotocin (STZ), 5 days after the STZ administration, the animals were killed and the glucose diabetic group mean (222 mg/dL) during all treatment was 82% higher than the control group mean (121 mg/dL). So, the effects of streptozotocin-induced neonatal hyperglycemia were studied on the following oxidative stress parameters from rat brain: the activities of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD) and NADPH oxidase (Nox), the content of superoxide anion (O2•-), the activities of the main antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSHPx), and thiobarbituric acid-reactive substances (TBA-RS). Rats subjected to a model of neonatal hyperglycemia presented high content of O2•- through activation of NADPH oxidase and it is possible that this activation was dependent of G6PD- and 6PGD-derived NADPH. Also, increased levels on O2•- may have promoted a rebout effect, through to enhanced antioxidant enzymes activities (SOD, CAT and GSHPx) and led to lipid peroxidation in the brain. So, these results suggest that OS could represent a mechanism to understand the harmful effect of hyperglycemia on the central nervous system. However, further studies appear to be worthwhile in order to better characterize the role of reactive species in the neurotoxicity of neonatal hiperglycemia.