<p>Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O₂ to H₂O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the <i>aa</i>₃-type oxidases, to which cytochrome <i>c</i> oxidase (Cyt<i>c</i>O) belongs, which uses energy released in the O₂-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO₂. Although as exergonic as O₂-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the <i>cbb</i>₃-type oxidases are the closest relatives to the NORs and as such provide a link between the <i>aa</i>₃ oxidases and the NORs. The <i>cbb</i>₃ oxidases have been shown to pump protons with nearly the same efficiency as the <i>aa</i>₃ oxidases, despite low sequence similarity.</p><p>This thesis is focused on measurements of membrane potential generating reactions during catalysis in the Cyt<i>c</i>O and the <i>cbb</i>₃ oxidase from <i>Rhodobacter sphaeroides</i>, and the NOR from <i>Paracoccus</i> <i>denitrificans</i>, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in Cyt<i>c</i>O showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of Cyt<i>c</i>O. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in <i>cbb</i>₃ oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O₂-reduction.</p>

Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O2 to H2O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the aa3-type oxidases, to which cytochrome c oxidase (CytcO) belongs, which uses energy released in the O2-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO2. Although as exergonic as O2-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the cbb3-type oxidases are the closest relatives to the NORs and as such provide a link between the aa3 oxidases and the NORs. The cbb3 oxidases have been shown to pump protons with nearly the same efficiency as the aa3 oxidases, despite low sequence similarity. This thesis is focused on measurements of membrane potential generating reactions during catalysis in the CytcO and the cbb3 oxidase from Rhodobacter sphaeroides, and the NOR from Paracoccus denitrificans, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in CytcO showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of CytcO. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in cbb3 oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O2-reduction.

Cytochrome c oxidase (COX) is complex IV of the electron transport system, and catalyzes the reduction of molecular oxygen to water. It possesses ten nuclear-encoded subunits, the largest of which is COX4. Bayesian analysis suggests the isoform pair for this subunit arose early in vertebrate evolution, and tissue distribution of the COX4 paralogs is similar in mammals and teleosts: COX4-1 is ubiquitously transcribed while COX4-2 is present in large amounts only in brain and respiratory tissue. This subunit is of interest due to its apparent sensitivity to oxygen. During hypoxia, transcription switches from COX4-1 to COX4-2 in some mammalian tissues. However, questions remain about the regulation of this response as well as its pervasiveness across vertebrates. I investigated these uncertainties by measuring the transcriptional response of the COX4 paralogs to hypoxia in a variety of vertebrate models, and assessing the hypoxic induction of putative oxygen-responsive elements (HRE1, HRE2, and ORE) from candidate vertebrate species in a transfection experiment. I also examined the conservation of key elements of the COX4-2 gene and polypeptide in vertebrates. It was found that the hypoxia-responsiveness of COX4-2 may not be vital to the cellular response to hypoxia. COX4-1 transcripts remained in excess during hypoxia in all of the vertebrate models used with the exception of western painted turtle (Chrysemys picta), where COX4-2 transcripts remained in excess during control and hypoxic treatments. Only the HRE2 element from human COX4-2 was activated with hypoxic exposure, yet this along with the other features of the gene and polypeptide were not well conserved across mammals, and nearly absent outside of this lineage. These results provide evidence that COX4-2 may respond to hypoxia in only select few mammalian tissues, or that the function of this gene is not related to the cellular hypoxic response. / Thesis (Master, Biology) -- Queen's University, 2012-11-25 20:51:59.419

The metabolic expense of producing and operating neural tissue required for adaptive behaviour is considered a significant selective force in brain evolution. In primates, brain size correlates positively with group size, presumably owing to the greater cognitive demands of complex social relationships in large societies. Social complexity in eusocial insects is also associated with large groups, as well as collective intelligence and division of labour among sterile workers. However, superorganism phenotypes may lower cognitive demands on behaviourally specialized workers resulting in selection for decreased brain size and/or energetic costs of brain metabolism. To test this hypothesis, we compared brain investment patterns and cytochrome oxidase (COX) activity, a proxy for ATP usage, in two ant species contrasting in social organization. Socially complex Oecophylla smaragdina workers had larger brain size and relative investment in the mushroom bodies (MBs)-higher order sensory processing compartments-than the more socially basic Formica subsericea workers. Oecophylla smaragdina workers, however, had reduced COX activity in the MBs. Our results suggest that as in primates, ant group size is associated with large brain size. The elevated costs of investment in metabolically expensive brain tissue in the socially complex O. smaragdina, however, appear to be offset by decreased energetic costs.

Résumé : La prééclampsie (PE) est un désordre de la grossesse caractérisée par une dysfonction endothéliale faisant en sorte que l’endothélium devient moins sensible aux signaux de vasodilatation. La réponse provoquée par la liaison de la sérotonine au sous-type de récepteur S[indice inférieur 2] entraîne la libération de molécules aux propriétés vasoconstrictrices, qui, par une boucle de rétroaction positive, entraîne la libération de davantage de sérotonine par les plaquettes. Cette boucle amplifie la réponse et contribue ainsi à l’hypertension présente chez les femmes ayant une PE. Précédemment, il a été démontré par notre laboratoire que le Bisphénol A (BPA) s’accumulait davantage dans le placenta des femmes avec PE en comparaison aux femmes normotensives. Cette accumulation pourrait découler d’une perturbation de sa métabolisation qui impliquerait notamment la β-glucuronidase (GUSB). Des études chez les animaux ont quant à elles démontré que le BPA pouvait inhiber l’activité de la monoamine oxydase (MAO) à forte dose. Nous avons étudié l’effet du BPA à faible concentration (10 ng/ml) sur la MAO-A des cellules placentaires et démontré que le BPA inhibait la MAO-A de façon significative sans affecter son expression protéique. Afin d’expliquer l’accumulation particulière du BPA chez les femmes PE, nous avons comparé l’activité spécifique et l’expression protéique de la β-glucuronidase (GUSB) placentaire en utilisant un devis cas-témoins. Une tendance non significative suggère que la GUSB pourrait partiellement contribuer à l’accumulation du BPA chez les femmes PE. Nous avons étudié la relation entre la concentration sérique maternelle de BPA et la concentration à laquelle le fœtus est exposé par régression linéaire et corrélation de Spearman. Un tel modèle ne pourrait être utilisé pour déterminer de façon quantitative l’exposition fœtale. En revanche, en vue de la forte corrélation entre ces deux variables, une haute concentration sérique maternelle de BPA devrait se refléter par une haute exposition fœtale. Cette corrélation implique aussi que le métabolisme placentaire ne joue pas un rôle significatif dans la protection du fœtus. Le BPA pourrait ainsi contribuer à l’hypertension chez les femmes PE présentant une dysfonction endothéliale en inhibant la MAO-A et ainsi, favorisant la hausse de sérotonine circulante. Cette étude suggère les bases d’un mécanisme par lequel le BPA s’accumulerait davantage chez les femmes PE et affecterait ainsi la MAO-A placentaire et potentiellement, la MAO-A fœtale vu ses propriétés physico-chimiques. / Abstract : Preeclampsia (PE) is an hypertensive disorder of pregnancy characterized by a generalized endothelial dysfunction where the response to vasodilatation signals is compromised. The binding of serotonin to its S[subscript 2] receptor subtype 2 releases vasoconstrictor molecules which, by a positive retroaction loop, stimulates the release of more serotonin from platelets. This positive retroaction loop stimulates the vasoconstriction of blood vessels and contributes to the hypertension in women with PE. Previously, we showed that Bisphenol A (BPA) accumulates more in the placenta of women with PE than in normotensive women. This accumulation may be the result of an impaired metabolization due to the action of the β-Glucuronidase (GUSB). Animal studies showed that BPA at high dose could lower the activity of the monoamine oxidase A (MAO-A), an enzyme implicated in the metabolism of serotonin. We studied the impact of BPA at low dose (10 ng/ml) in trophoblastic primary cells and showed that even at low dose, BPA can lower its activity without affecting the protein expression. To determine if GUSB could be the cause of the BPA accumulation in women with PE, we studied its activity and protein expression in placental biopsies from women with and without PE. A nonsignificant tendency showed that the GUSB activity and protein expression were higher in women with PE. To study the impact of placental metabolism in the fetal exposure, we studied the relation between maternal and fetal concentrations of BPA with linear regression analysis and Spearman’s correlation. We showed that maternal BPA could not precisely predict the fetal exposure and that the placental metabolism is probably limited in light of the strong correlation between both variables. This strong correlation also implied that high maternal exposure would result in high fetal exposure. This study shows that the accumulation of BPA in preeclamptic women could contribute to maternal hypertension by interacting with serotonin levels. This accumulation could partially be attributed to a higher GUSB, but other factors are probably implicated. The strong correlation between maternal and fetal exposure implies that the placental metabolism of BPA is limited and does not protect the fetus significantly. This study suggests the basis of a mechanism explaining the abnormal accumulation of BPA in the placenta in women with PE and its impact on the placental MAO-A and potentially, the foetal MAO-A because of its physico-chemical properties.

Several epidemiological studies found that hypertensive patients have an increased risk to develop kidney cancer. Hyperaldosteronism frequently results in arterial hypertension and contributes to the development and progression of kidney injury, with reactive oxygen species (ROS) playing an important role. ROS are thought to be associated with many pathological conditions such as cancer and other disorders, like cardiovascular complications , which often go along with hypertension. The aim of the present work was to investigate whether the effects of elevated aldosterone concentrations might be involved in the increased cancer incidence of hypertensive individuals. First, the potential capacity of aldosterone to induce oxidative stress and DNA damage was investigated in vitro and in vivo. In LLC-PK1 porcine kidney cells and MDCK canine kidney cells the significant formation of ROS, and especially of superoxide (O2˙ˉ) was assessed. With two genotoxicity tests, the comet assay and the micronucleus frequency test, the DNA damaging potential of aldosterone was quantified. In both genotoxicity tests a dose-dependent increase in aldosterone-induced structural DNA damage was observed. Oxidative stress and DNA damage were prevented by antioxidants, suggesting ROS as a major cause of DNA damage. Furthermore, the oxidatively modified DNA lesion 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG), was found to be significantly elevated. In kidneys of rats with desoxycorticosterone acetate (DOCA)/salt-induced hypertension, which is a model of severe mineralocorticoid-dependent hypertension, elevated levels of ROS and superoxide were found, compared to kidneys of sham rats. Also DNA strand breaks, measured with the comet assay and double strand breaks, visualized with antibodies against the double strand break-marker gamma-H2AX were significantly elevated in kidneys of DOCA/salt-treated rats. In addition, significantly increased amounts of 8-oxodG were detected. Proliferation of kidney cells was found to be increased, which theoretically enables the DNA damage to manifest itself as mutations, since the cells divide. Second, the effects of aldosterone on the activation of transcription factors and signaling pathways were investigated. A significant activation of the potentially protective transcription factor Nrf2 was observed in LLC-PK1 cells. This activation was triggered by an increase of ROS or reactive nitrogen species (RNS). In response to oxidative stress, glutathione synthesis and detoxifying enzymes, such as the subunits of the glutathione-cysteine-ligase or heme oxygenase 1 were rapidly induced after 4 h. Nevertheless, after 24 h a decrease of glutathione levels was observed. Since ROS levels were still high after 24 h, but Nrf2 activation decreased, this adaptive survival response seems to be transient and quickly saturated and overwhelmed by ROS/RNS. Furthermore, Nrf2 activation was not sufficient to protect cells against oxidative DNA damage, because the amounts of double strand breaks and 8-oxodG lesions steadily rose up to 48 h of aldosterone treatment. The second transcription factor that was time- and dose-dependently activated by aldosterone in LLC-PK1 and MDCK cells was NF-kappaB. Furthermore, a significant cytosolic and nuclear activation of ERK was detected. Aldosterone induced the phosphorylation of the transcription factors CREB, STAT1 and STAT3 through ERK. Third, the underlying mechanisms of oxidant production, DNA damage and activation of transcription factors and signaling pathways were studied. Aldosterone exclusively acted via the MR, which was proven by the MR antagonists eplerenone, spironolactone and BR-4628, whereas the glucocorticoid receptor (GR) antagonist mifepristone did not show any effect. Furthermore, aldosterone needed cytosolic calcium to exert its negative effects. Calcium from intracellular stores and the influx of calcium across the plasma membrane was involved in aldosterone signaling. The calcium signal activated on the one hand, the prooxidant enzyme complex NAD(P)H oxidase through PKC, which subsequently caused the generation of O2˙ˉ. On the other hand, nitric oxide synthase (NOS) was activated, which in turn produced NO. NO and O2˙ˉ can react to the highly reactive species ONOO- that can damage the DNA more severely than the less reactive O2˙ˉ. In the short term, the activation of transcription factors and signaling pathways could be a protective response against aldosterone-induced oxidative stress and DNA damage. However, a long-term NF-B and ERK/CREB/STAT activation by persistently high aldosterone levels could unfold the prosurvival activity of NF-kappaB and ERK/CREB/STAT in aldosterone-exposed cells. DNA damage caused by increased ROS might become persistent and could be inherited to daughter cells, probably initiating carcinogenesis. If these events also occur in patients with hyperaldosteronism, these results suggest that aldosterone could be involved in the increased cancer incidence of hypertensive individuals. / Mehrere epidemiologische Studien haben ein erhöhtes Nierenkrebsrisko bei Patienten mit Bluthochdruck aufgedeckt. Hyperaldosteronismus führt oft zu arteriellem Bluthochdruck und trägt zur Entwicklung und zum Fortschreiten von Nierenschäden bei, wobei reaktive Sauerstoffspezies (ROS) eine wichtige Rolle spielen. Immer häufiger werden ROS mit Krankheitsbildern wie Krebs und kardiovaskulären Erkrankungen, die mit Bluthochdruck einhergehen, in Verbindung gebracht. Das Ziel dieser Arbeit war es, zu untersuchen, ob erhöhte Aldosteronkonzentrationen an dem gesteigerten Krebsrisiko von hypertensiven Patienten beteiligt sein könnten. Zunächst wurde die potentielle Kapazität von Aldosteron, oxidativen Stress und DNA-Schaden in vitro und in vivo induzieren zu können, untersucht. In der Schweine-Nierenzelllinie LLC-PK1 und der Hunde-Nierenzelllinie MDCK wurde die Entstehung von ROS und speziell die Bildung von Superoxid (O2˙ˉ) nachgewiesen. Das gentoxische Potential von Aldosteron wurde mit zwei Genotoxizitätstests, dem Comet Assay und dem Mikrokernfrequenztest bestimmt. In beiden Genotoxizitätstests konnte ein dosis-abhängiger Anstieg des strukturellen DNA-Schadens beobachtet werden. Antioxidantien konnten den oxidativen Stress und die DNA-Schäden verringern, was annehmen lässt, dass ROS die Hauptursache für die Entstehung der DNA-Schäden sind. Darüberhinaus wurden signifikant erhöhte Mengen der oxidativ modifizierten DNA Läsion 8-Oxo-7,8-dihydro-2´-deoxyguanosin (8-oxodG) gefunden. In Nieren von Ratten mit Desoxycorticosteron-Acetat (DOCA) und Salz-induziertem Bluthochdruck, ein Modell für massiven Mineralocorticoid-induzierten Bluthochdruck, wurde ebenfalls eine erhöhte Bildung von ROS und O2˙ˉ in Nieren von DOCA/Salz-Ratten im Vergleich zu Sham-Ratten beobachtet. Auch im Comet Assay erfasste DNA-Strangbrüche und Doppelstrangbrüche, die mit Hilfe von Antikörpern gegen den Doppelstrangbruchmarker gamma-H2AX sichtbar gemacht wurden, waren in den Nieren der DOCA/Salz-behandelten Ratten signifikant erhöht. Weiterhin wurden erhöhte 8-oxodG-Spiegel in DOCA/Salz-Ratten beobachtet. Auch eine erhöhte Proliferationsrate in DOCA/Salz-behandelten Ratten konnte festgestellt werden, was theoretisch dazu führen könnte, dass sich die DNA-Schäden als Mutationen manifestieren, da sich die Zellen teilen. Im zweiten Teil der Arbeit wurde der Einfluss von Aldosteron auf die Aktivierung von Transkriptionsfaktoren und Signalwegen untersucht. Zunächst konnte die Aktivierung des potentiell schützenden Transkriptionsfaktors Nrf2 in LLC-PK1 Zellen mittels electrophoretic mobility shift assay (EMSA) beobachtet werden. Diese Aktivierung wurde durch den Anstieg an ROS und reaktiven Stickstoffspezies (RNS) ausgelöst. Als Antwort auf den oxidativen Stress, wurde die Glutathion-Synthese und detoxifizierende Enzyme, wie die Untereinheiten der Glutathion-Cystein-Ligase oder Hämoxygenase 1, nach 4 Stunden rasch hochreguliert. Nichtsdestotrotz konnte nach 24 Stunden eine Abnahme des Glutathionspiegels festgestellt werden. Da die Konzentration an ROS nach 24 Stunden immer noch signifikant erhöht war, die Aktivierung von Nrf2 allerdings stark zurückgegangen ist, scheint diese adaptive Überlebensstrategie nur kurzfristig, und somit schnell durch ROS/RNS gesättigt zu sein. Weiterhin war die Aktivierung von Nrf2 nicht ausreichend, um die Zellen vor dem durch Aldosteron-induzierten DNA-Schaden zu schützen, da Doppelstrangbrüche, sowie 8-oxodG-Läsionen bei bis zu 48-stündiger Inkubation mit Aldosteron stetig anstiegen. Der zweite Transkriptionsfaktor, der zeit- und dosisabhängig durch Aldosteron aktiviert wurde, war NF-kappaB. Ausserdem wurde die cytosolische und nukleäre Aktivierung von ERK nachgewiesen. Aldosteron induzierte weiterhin die Phosphorylierung der Transkriptionsfaktoren CREB, STAT1 und STAT3 durch ERK. Im dritten Teil dieser Arbeit wurden die zugrundeliegenden Mechanismen der Entstehung von ROS/RNS, des DNA-Schadens und der Aktivierung von Transkriptionsfaktoren untersucht. Aldosteron wirkte ausschließlich über den MR, bewiesen durch Einsatz der MR-Antagonisten Eplerenon, Spironolakton und BR-4628. Der Glucocorticoid-Rezeptor-Antagonist Mifepriston zeigte dagegen keinen Effekt. Weiterhin benötigte Aldosteron cytosolisches Calcium, um seine negativen Effekte auszuüben. Es waren intrazelluäres Calcium, sowie ein Calciuminflux über die Plasmamembran am Aldosteronsignal beteiligt. Einerseits wurde der prooxidative Enzymkomplex NAD(P)H-Oxidase von Calcium durch die Proteinkinase C (PKC) aktiviert, was wiederum zur Bildung von O2˙ˉ führte. Andererseits kam es durch erhöhtes cytosolisches Calcium zur Aktivierung der NO-Synthase (NOS), welche daraufhin Stickoxid (NO) produzierte. NO und O2˙ˉ können zu dem hochreaktiven Peroxynitrit (ONOO-) reagieren, welches die DNA mehr schädigen kann als das etwas weniger reaktive O2˙ˉ. Kurzfristig könnte die Aktivierung der Transkriptionsfaktoren und Signalwege eine schützende Wirkung gegen den durch Aldosteron-induzierten oxidativen Stress und DNA-Schaden in den Zellen haben. Allerdings kann eine länger anhaltende Aktivierung von NF-kappaB und ERK/CREB/STAT durch permanent hohe Aldosteronspiegel zur Induktion einer Überlebensstrategie durch NF-kappaB und ERK/CREB/STAT in Aldosteron-exponierten Zellen führen. Der DNA-Schaden, der durch erhöhte ROS-Spiegel entsteht, könnte persistent und somit an Tochterzellen weitervererbt werden, was eventuell zur Entstehung von Krebs beitragen könnte. Falls diese Effekte auch in Patienten mit Hyperaldosteronismus gefunden werden können, dann könnte Aldosteron an der erhöhten Krebsinzidenz bei Bluthochdruck beteiligt sein.

A espécie Garcinia brasiliensis, conhecida como "bacupari", é nativa da Amazônia e cultivada em todo o território brasileiro. Na medicina popular, suas folhas são utilizadas para tratar tumores, inflamações das vias urinárias e artrite, bem como para aliviar dores. Este trabalho foi conduzido para avaliar a atividade analgésica e antiinflamatória do extrato etanólico foliar (GbEE) e da 7-epiclusianona, uma benzofenona natural poliprenilada isolada de suas folhas, em modelos animais in vivo. A ação deste benzofenona também foi investigada em um ensaio antiinflamatório ex vivo, enfocando o “burst” respiratório de neutrófilos e as vias bioquímicas envolvidas. Foi constatada atividade atividade antiinflamatória tanto para GbEE v.o. ( 30, 100 3 300 mg /kg) como para 7-epiclusianona v.o.(5, 10 e 15mg/Kg) por reduzir o edema de pata induzido por carragenina e inibir o recrutamento de leucócitos na cavidade peritoneal. Além disso, através do modelo de indução de inflamação crônica foi observado uma redução estatisticamente significativa de formação do tecido granulomatoso. Por outro lado, efeito antinociceptivo periférico foi atribuído ao GbEE sugerindo ser mais eficaz contra dores inflamatórias, enquanto que para a benzofenona foi constatada tanto atividade antinociceptiva periférica como central, através de diferentes mecanismos. O composto 7-epiclusianona (10 a 100µg) também foi capaz de reduzir e até mesmo suprimir a liberação de ânion superóxido por fagócitos inflamatórios através de um mecanismo controlado pela fosforilação da proteína tirosina e pela estimulação direta da proteína quinase C, sugerindo novas abordagens terapêuticas para o tratamento de processos inflamatórios e desenvolvimento de novos fármacos. Os resultados mostraram atividades antiinflamatória e analgésica das folhas de G. brasiliensis e da benzofenona isolada, confirmando o uso tradicional dessa espécie para tratamento de dores e inflamações. / The species Garcinia brasiliensis, known as “bacupari”, is native to the Amazon and cultivated throughout Brazil. Their leaves are used in folk medicine to treat tumors, inflammation of the urinary tract and arthritis as well as to relief pain. This study was conducted to evaluate the anti-inflammatory and analgesic effects of the ethanolic extract of leaves (GbEE) and the 7-epiclusianone, a natural polyisoprenilated benzophenone isolated from G. brasiliensis, in several animal models. The action of the benzophenone was also investigated ex vivo throughout an anti-inflammatory assay, focusing neutrophil respiratory burst and the biochemical pathway. Antiinflammatory activity was found for both GbEE (30, 100 and 300 mg /kg) p.o. and for 7-epiclusianone (5, 10 and 15mg/kg) p.o. to reduce the paw edema induced by carrageenan and to inhibit leukocyte recruitment into the peritoneal cavity. In addition, through the induction model of chronic inflammation, was observed a statistically significant reduction of granulomatous tissue formation. Moreover, peripheral antinociceptive effect was attributed to GbEE suggesting that this extract could be more effective against inflammatory pain, whereas for benzophenone was verified both peripheral and central antinociceptive activity through different mechanisms.The compound 7-epiclusianone (10 to 100 μg) was also able to reduce and even suppress the release of superoxide anion by inflammatory phagocytes through a mechanism controlled by protein tyrosine phosphorylation and by direct stimulation of protein kinase C, suggesting new therapeutic approaches for the treatment of inflammatory processes and development of new drugs. So, it was demonstrated the anti-inflammatory and antinociceptive activities of the leaves of G. brasiliensis and the benzophenone isolated which supports previous claims of the traditional use of this species against inflammation and pain. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES

by Kwok-Ping Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1980. / Bibliography: leaves 111-118.

A sulfito oxidase (SO) é uma enzima que catalisa a última reação na rota de degradação de aminoácidos sulfurados, a oxidação de sulfito a sulfato. A deficiência da SO é um erro inato do metabolismo que pode ser causado tanto pela deficiência isolada da enzima como por defeitos na síntese do seu cofator molibdênio, cuja principal característica bioquímica é o acúmulo tecidual e a excreção urinária aumentada de sulfito, tiossulfato e S-sulfocisteína. Os pacientes acometidos pela doença têm sintomatologia predominantemente neurológica, e exames de imagem evidenciam encefalomalácia cística, atrofia cerebral e edema, perda neuronal e astrogliose, os quais se concentram na região cortical. Pouco se sabe sobre os mecanismos envolvidos nos danos encontrados na deficiência da SO, porém dados apontam para uma ação tóxica dos metabólitos acumulados. Sendo assim, o objetivo deste estudo foi o de investigar os efeitos in vitro do sulfito e do tiossulfato sobre a neurotransmissão glutamatérgica e parâmetros de estresse oxidativo em fatias de córtex cerebral de ratos. As fatias foram expostas ao sulfito ou tiossulfato (10 – 500 μM) durante 1 ou 3 h para a realização dos experimentos, nos quais medimos a captação de glutamato dependente de sódio, a atividade da enzima glutamina sintetase, os níveis de substâncias reativas ao ácido tiobarbitúrico (TBA-RS), o conteúdo de glutationa (GSH) e de sulfidrilas, a formação de carbonilas e as atividades das enzimas antioxidantes glutationa peroxidase (GPx), glutationa redutase (GR), glutationa S-transferase (GST) e glicose-6-fosfato desidrogenase (G6PDH). Também avaliamos a viabilidade celular através dos testes liberação da lactato desidrogenase e a redução do MTT. Foi observado que o sulfito diminui a captação de glutamato e que o tiossulfato diminui a atividade da glutamina sintetase. Uma tendência quase significativa de que o sulfito diminui a atividade da glutamina sintetase também foi verificada. Quanto à homeostase redox, verificamos que o sulfito, na concentração de 10 μM, aumentou os níveis de TBA-RS e diminuiu as concentrações de GSH, sem alterar a formação de carbonilas. Já o tiossulfato não teve nenhum efeito significativo sobre esses parâmetros. Ainda verificamos que 500 μM de sulfito aumentaram o conteúdo de grupamentos sulfidril em córtex cerebral de ratos e o conteúdo de GSH em um meio sem amostra biológica, o que pode ser explicado pela capacidade do sulfito em reduzir pontes dissulfeto a grupos sulfidril. Ao medir as atividades das enzimas antioxidantes GPx, GR, GST e G6PDH, não houve diferença com qualquer dos metabólitos durante 1 h de incubação, porém, ao realizarmos os mesmos experimentos com amostras incubadas por 3 h com sulfito, observamos inibição das atividades da GPx, da GST e da G6PDH. Finalmente, observamos que o sulfito não alterou a redução do MTT e a liberação de lactato desidrogenase, indicando que os resultados encontrados não são devidos à morte celular. Pode ser concluído que um prejuízo na neurotransmissão glutamatérgica e estresse oxidativo causados pelos metabólitos acumulados na deficiência da SO estão envolvidos, pelo menos parcialmente, na disfunção neurológica observada nessa doença. / Sulfite oxidase (SO) is the enzyme that catalyzes the oxidation of sulfite to sulfate, which is the last step in the pathway of degradation of sulfur-containing amino acids. SO deficiency is an inborn error of metabolism caused either by isolated deficiency in this enzyme, or by defects in the synthesis of its molybdenum cofactor. The main biochemical characteristic of this disorder is the tissue accumulation and high urinary excretion of sulfite, thiosulfate and cysteine-S-sulfate. Patients present predominantly neurological symptoms and brain abnormalities, such as cystic encephalomalacia, brain atrophy and swelling and neuronal loss, which prevail in the cortical region. Although available data point towards a toxic mechanism of the accumulating metabolites, little is known about the exact pathomechanisms exerted by these compounds. Therefore, our objective in this study was to investigate the in vitro effects of sulfite and thiosulfate on glutamatergic neurotransmission and oxidative stress parameters in rat cerebral cortex slices, a system with preserved integrity. Slices were exposed to sulfite or thiosulfate (10 – 500 μM) for 1 or 3 h. After the incubation, we measured sodium-dependent glutamate uptake, glutamine synthetase activity, thiobarbituric acid-reactive substances (TBA-RS) levels, glutathione (GSH) and sulfhydryl content, carbonyl formation and the activities of the antioxidant enzymes glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST) and glucose-6-phosphate dehydrogenase (G6PDH). Lactate dehydrogenase and MTT reduction were also evaluated. We verified that sulfite reduced the glutamate uptake and that thiosulfate inhibited glutamine synthetase activity. A pronounced trend toward glutamine synthetase inhibition caused by sulfite was also seen. Regarding redox homeostasis, 10 μM sulfite increased TBA-RS levels and decreased GSH concentrations, without altering protein carbonyl formation. Moreover, thiosulfate had no effect on these parameters. Five hundred micromolar sulfite also increased sulfhydryl content in rat cerebral cortex slices and increased GSH content in a medium devoid of biological samples, which can be explained by the fact that sulfite is able to directly reduce disulfide bonds to thiol groups. We further verified that sulfite did not alter the activities of the enzymes GPx, GR, GST and G6PDH when cortical slices were incubated in the presence of sulfite during 1 h. However, after an incubation of 3 h, sulfite decreased the activities of GPx, GST and G6PDH. Finally, sulfite did not change MTT reduction and lactate dehydrogenase release, suggesting that the effects observed were not due to cell death. Therefore, it is concluded that glutamatergic neurotransmission impairment and oxidative stress induced by the accumulating metabolites in SO deficiency may contribute to the neurological dysfunction observed in this disorder.

A β-galactosidase (E.C 3.2.1.23) é uma das enzimas mais empregadas na indústria de alimentos sendo utilizada na hidrólise da lactose. Neste trabalho foram utilizadas duas metodologias para imobilização desta enzima. Na primeira delas foi empregado como suporte um material híbrido à base de sílica que possui um grupo orgânico catiônico covalentemente ligado. A adsorção da enzima a este material apresentou eficiência que variou de 74 a 53% com o aumento da quantidade de enzima aplicada ao suporte. A baixa estabilidade térmica da enzima imobilizada obtida e as prováveis fracas interações envolvidas na sua adsorção a este suporte podem explicar o decréscimo de atividade observada durante as sucessivas bateladas de hidrólise da lactose. Na primeira batelada o grau de hidrólise foi de 90,9% e no final da última batelada (4ª), a enzima foi capaz de converter apenas 13% do substrato. A segunda metodologia utilizada foi imobilização covalente da enzima em um filme de celulose/líquido iônico modificado com uma poliamina e ativado com glutaraldeído. A presença da poliamina foi confirmada por análises de infravermelho. Após a imobilização, a enzima reteve 60% de sua atividade inicial. Bons resultados de hidrólise da lactose em batelada foram obtidos tanto a 7ºC como a 35ºC e foi possível reutilizar a enzima imobilizada por 16 ciclos consecutivos, a 7ºC, sem mudanças significativas na atividade enzimática. O valor de Km para a enzima imobilizada no material híbrido à base de sílica foi de 9,17 mM e para a enzima imobilizada nos filmes de celulose foi de 11,22 mM, ambos apresentaram um acréscimo quando comparados ao Km enzima livre (1,25 mM), devido à dificuldade de acesso do substrato ao sítio ativo da enzima. Não houve mudança no pH e temperatura ótimos da enzima imobilizada em relação à enzima livre em nenhum dos métodos testados. / β-galactosidase (E.C 3.2.1.23) is the most widely used enzymes in the food industry and its employed in the lactose hydrolysis process. In this study, two methodologies were used to test their immobilization. In the first, the enzyme was immobilized by adsorption in one silica based hybrid material that contains a cationic organic group covalently linked. The efficiency of immobilization showed a decrease of 74 to 53% by increasing the protein load applied to the support. The low thermo stability of the immobilized enzyme and the probable weak interactions involved in their adsorption, could explain the decrease in enzyme activity observed in the successive batch hydrolysis of lactose. In the first run, the degree of lactose hydrolysis was 90.9% and, at the end of the last run (4th), the enzyme was able to convert only 13% of the substrate. The second methodology used was the covalent immobilization of the enzyme on a cellulose/ionic liquid film, modified with a polyamine and activated using glutaraldehyde. The presence of a polyamine was confirmed by infrared analysis. After immobilization, the enzyme retained 60% of its initial activity. Highly efficient lactose conversion was achieved in a batch process at 7ºC and 35ºC and was possible to reuse the immobilized enzyme in 16 repeated cycles, at 7ºC, without any drastic decrease in enzyme activity. Km value for the immobilized enzyme in silica based hybrid material was 9.17 mM and for the enzyme immobilized in the film of cellulose/ionic liquid was 11.22 mM, both showing an increase compared with the Km value for free enzyme (1.25 mM), due to the difficulty of access of the substrate to the active sites of the enzyme. The immobilized enzyme did not show any changes in the optimal pH and temperature when compared to the free enzyme in both methods tested.