Abstract The metabolism of reactive oxygen species (ROS) in cancer cells is a research area that has not been intensively pursued. It is generally believed that cancer cells are persistently oxidative stressed. The consequence of this phenomenon will result in changes of the characteristic of a cancer cell. Whether or not the oxidative stress has a role in regulating the metastatic potential of a cancer cell is a research area that has been totally neglected. Using a group of human hepatocellular carcinoma (HCC) cell lines with distinct disparity in their differentiation status, established by their morphological observation and the abilities of synthesizing at least 15 plasma proteins, as the experimental model, we proposed to investigate the possibility that the constitutive oxidative stress status of these HCC cells may be modulated by their differentiation status. As a result, varying degrees of oxidative stress status of these cells will affect the propensity of expression of a few redox-sensitive transcription factors, such as NF-kB and AP-1, which may eventually modulate the metastasis- related gene expression. If these proposed hypotheses turn out to be true, we will then investigate the underlying mechanism(s) associated with the phenomena observed. Firstly, we demonstrated previously (Liu et al., 2000) that the total antioxidative capacity (as expressed by the composite propensities of expressing 4 antioxidant enzymes and the intracellular glutathione contents) as well as GSH/GSSG ratios of the HCC cells we studied were excellently correlated with their differentiation status, with an order of HepG2 > Hep3B > J5 > SK-Hep-I. To further confirm this observed phenomenon, we quantified the steady state mRNA expressions of the four antioxidant enzymes by duplex RT-PCR method. In this study, we further confirmed that well-differentiated HCC cells, such as HepG2 and Hep3B expressed higher levels of extracellular GPx (eGPx) and catalase mRNAs. Conversely, the expression of mRNA for both enzymes in a poorly-differentiated HCC cells, such as SK-Hep-I and Mahlavu, was trace or even negligible. Since GSH biosynthesis is controlled by g-glutamylcysteine synthetase (g-GCS), a rate-limiting enzyme composing of a catalytic heavy subunit ( gGCS h ) and a regulatory light subunit ( gGCSl) ,we wanted to further substantiate that differentiation status-mediated upregulation of GSH is regulated by this enzyme. We demonstrated that the g-GCSh expression was again differentiation status regulated, established by using either a HPLC or a duplex RT-PCR method. The order of ranking for the expression of g-GCS was HepG2 > Hep3B > J5 > SK-Hep-I. In contrast, we found that g-GCSl mRNA seemed not to be influenced by the differentiation status. It has been documented that the transcription factors NF-kB and AP-1 are redox-sensitive. Thus, we wanted to see if both transcription factors could constitutively be activated. Also, is the expression propensity of these transcription factors modulated by the oxidative stress status of these HCC cells? Using EMSA and supershift techniques, we demonstrated that varying degrees of expressions of both transcription factors can be seen, with an order of expression propensity of SK-Hep-I > Mahlavu > J5 > Hep3B > HepG2. Having known that both NF-kB and AP-1 could modulate a group of metastasis-related gene expression, we then investigate if the constitutive metastatic potential of these HCC cells can be varied depending upon how extensive the expression propensity of both NF-kB and AP-1, we used a panel of markers for evaluating the metastatic potential, namely: Matrix metalloproteinase (MMP), interleukin 8 (IL-8) and an adhesion molecular E-cadherin. Using both activity and duplex RT-PCR methods, we demonstrated that only a poorly differentiated HCC cells were capable of expressing MMPs (SK-Hep-I predominately expressed a large amount of MMP-9 and mRNA; Mahlavu predominately expressed MMP-2 along with a trace amount of MMP-9). HepG2, Hep3B and J5 were completely devoid of MMP expression. Using ELISA assay, we measured the secretion of IL-8 in the culture media by these HCC cells and demonstrated that the propensity of secretion having an order of SK-Hep-I > HepG2 > Hep3B (J5 and Mahlavu expressed only a trace amount). Next, we used western blotting and duplex RT-PCR techniques to demonstrate that the expressions of E-cadherin were predominately existed in only well-differentiated cell lines, HepG2 and Hep3B. J5, Mahlavu and SK-Hep-I were all devoid of expression. Finally, for the purpose of demonstrating that the intracellular oxidative stress status does have a role in regulating the above-mentioned metastasis-related gene expression, we transfected g-GCSh cDNA to SK-Hep-I cell and obtained a cell type, termed GCS 30, in which its g-GCSh activity, mRNA and GSH content has been proved to be higher than its untransfected counterpart, SK-Hep-I. We then measured the oxidative stress status of GCS 30 using DCFDA-flowcytometric method. From our data, we did demonstrate that the oxidative-stress status of GCS 30 was shown to be decreased as compared to its counterpart. However, we were surprised to find out that GSH/GSSG ratio remained unchange in GCS-30 as compared to SK-Hep-I cells. Using EMSA technique, we showed that GCS 30 cells only exhibited relative strong binding activity to NF-kB, but not for AP-1 binding. Surprisingly, we found that the MMPs activities in GCS 30 cells were relatively comparable to SK-Hep-I, indicating that MMP expression might be regulated by a pathway other than AP-1. The mechanism(s) underlying this observed phenomenon await further clarification.

<p> Biological thiols are a class of labile and redox-active metabolite with significant interest to biomedical research due to their involvement in redox mechanisms of cell signaling and physiological control. As a result of oxidative stress, levels of various reduced thiols and oxidized disulfides are altered, which disrupts major cellular regulation pathways modulating protein function and gene expression. Thus, analysis of thiols in biological fluids is essential for understanding the role of oxidative stress and thiol dysregulation in aging and human diseases. However, reliable ex-vivo thiol determination is challenging due to their low abundance and susceptibility to auto-oxidation and thiol-disulfide exchange reactions. In this thesis, capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) in conjunction with maleimide labeling is developed as an integrative strategy for comprehensive plasma thiol redox status analysis for metabolomics. Maleimide labeling helps to address both major constraints in thiol analysis by stabilizing free sulfhydryl groups as their thioether adducts while improving ionization efficiency and analytical sensitivity. This enhancement in ionization efficiency can be quantitatively predicted based on relative changes in fundamental physicochemical properties of thiols that occur upon covalent derivatization when using multivariate calibration. On-line sample preconcentration together with thiol-selective labeling using a cationic quaternary ammonium maleimide analog allowed for simultaneous analysis of reduced thiols and intact oxidized disulfides by CE-ESI-MS with low nanomolar detection limits of 8-30 nM. Improved identification of unknown low abundance thiols and other classes of polar metabolites is also demonstrated by prediction of relative migration times in CE that is complementary to ESI-MS. Comprehensive plasma thiol speciation together with untargeted profiling of polar metabolites provides a novel platform for holistic understanding of complex changes in metabolic networks associated with thiol dysregulation and/or nutritional intervention for the prevention or treatment of human disorders. </p> / Thesis / Master of Science (MSc)

Contrôle de l'oxydation et de la dégradation du pool de vitamine C chez la tomate et impact sur la qualité du fruit et la tolérance au stress. Le métabolisme de l’ascorbate et plus principalement le statut redox du pool d’ascorbate sont impliqués dans la tolérance au stress et dans les processus primaires de croissance et de développement de la plante. La teneur et le statut redox de l’ascorbate chez les plantes sont régulés par (i) ses voies de biosynthèse, (ii) par le cycle ascorbate-glutathion permettant le recyclage des formes semi-oxydées et oxydées de l’ascorbate et (iii) par sa dégradation, l’ensemble de ces processus étant sous le contrôle de l’environnement. Au cours de ce travail de thèse, des méthodes de transgénèse nous ont permis d’identifier, chez différents génotypes de tomate à petit et gros fruits, les bouleversements physiologiques et métaboliques permettant de compenser des modifications de l’activité des enzymes monodéhydroascorbate réductase (impliqué dans le cycle ascorbate-glutathion) et ascorbate oxydase. Nous avons observé d’importantes modifications phénotypiques altérant le rendement en fruits de la plante sous conditions de culture pouvant générer un stress et également en condition normale de culture. Des liens entre l’activité des enzymes précités avec le métabolisme des sucres, la photosynthèse et la conductance stomatique sont révélés. Le déséquilibre entre les activités oxydantes et réductrices de ces enzymes constitue la première étape vers une dégradation de l’ascorbate. Le taux de dégradation se révèle très faible à la lumière, tandis qu’à l’obscurité une forte accumulation des produits de dégradation l’oxalate, le thréonate ainsi que l’oxalyl-thréonate est observé dans les feuilles de tomate. Enfin, l’activité de l’enzyme MDHAR est corrélée au taux de dégradation à l’obscurité. Les travaux de cette thèse mettent en avant l’importance du statut redox du couple ascorbate / monodéhydroascorbate dans les processus de croissance cellulaire et entre dans la régulation du rendement chez la tomate, et influe la dégradation de l’ascorbate. / Ascorbate metabolism and particularly ascorbate redox status are involved in stress tolerance and growth processes of plant cells. The concentration of ascorbate and its redox status are under control of (i) its biosynthetic pathways, (ii) the ascorbate-glutathione cycle allowing recycling of semi-oxidized and oxidized forms of ascorbate and (iii) its degradation rate. These processes are under environmental control. Transgenic lines modified for the activity of monodehydroascorbate reductase (involved in ascorbate-glutathione cycle) and ascorbate oxidase were generated in cherry and large-fruited genotypes of tomato. Physiological and metabolic modifications related to the modification of these enzyme activities were studied. We observed large phenotypic alterations that affected fruit yield under both stress conditions and normal growth conditions. Links between ascorbate recycling and sugar metabolism, photosynthesis and stomatal conductance were also revealed. An imbalance between the oxidizing and reducing activities of these enzymes is the first step leading to ascorbate degradation. We have shown that the degradation rate was very low under light, whereas under darkness the degradation compounds oxalate, threonate and oxalyl-threonate accumulated in tomato leaves. Also, the degradation rate is correlated with MDHAR activity. These results highlight the crucial role of the redox status of the ascorbate / monodehydroascorbate couple in growth processes and yield stability in tomato, and the impact on ascorbate degradation.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-05-04T18:36:57Z No. of bitstreams: 1 2016 - CRISTINA SANTOS DA SILVA.pdf: 3106410 bytes, checksum: 18949ec17e0df834beb420705591824d (MD5) / Made available in DSpace on 2017-05-04T18:36:57Z (GMT). No. of bitstreams: 1 2016 - CRISTINA SANTOS DA SILVA.pdf: 3106410 bytes, checksum: 18949ec17e0df834beb420705591824d (MD5) Previous issue date: 2016-03-31 / CAPES / Chagas? Disease or American trypanosomiasis is an important parasitic disease in the Americas and is still considered one of the major neglected tropical diseases affecting millions of people worldwide due to lack of effective control. Thus, it has a significant impact on human health. This disease has its epidemiology conditioned by triatomines and mammals and its etiological agent is the protozoan Trypanosoma cruzi.Investigations on the adaptation mechanisms, gene regulation and parasite interaction vs. vector evolved, which proves the need for the use of molecular tools for the study and elucidation on adaptive changes in these parasites. Recently, it was found evidence where studies indicate that the expression of T. cruzi?s antioxidant enzymes such as cytosolic peroxiredoxin (TcCPx), mitochondrial peroxiredoxin (TcMPx), (TcAPX) and trypanothione synthetase (TXNI, TXNII), superoxide dismutase (SOD A and SOD B) can be part of parasite?s protective system and indicate factors related to different levels of virulence of the etiological agent.Thus, proteomics analysis and evaluation of the expression of antioxidant enzymes from different subcellular compartments can corroborate to the studies related to virulence indices and expression of proteins involved in this process. In this sense, the objectives of this study were to evaluate the morphology of the isolated samples SMM98, SMM36 and SMM1, analyze beyond the proteomic profile, the expression of antioxidant enzymes (TcCPx; TcMPx; TcAPx; TcTXNI; TcTXNII, SOD A and SOD B) and observe the redox status expression using isolates SMM36 and SMM98 in comparison with strains TCC, Silvio and DM28c. The results showed different morphology from the isolated, high levels of virulence, varied protein profile comparison between SMM98 isolated and SMM36, when treated with NAC and Heme changes were observed in the development of the parasites, indicating the participation of the Redox Status of the Rio de Janeiro used in this study. / A doen?a de Chagas ou tripanossom?ase americana ? uma importante doen?a parasit?ria nas Am?ricas e ainda hoje ? considerada uma das principais doen?as tropicais negligenciadas acometendo milh?es de pessoas no mundo devido ? falta de controle efetivo. Desta forma, apresenta um impacto significativo sobre a sa?de humana. Esta enfermidade tem sua epidemiologia condicionada pelos triatom?neos e os mam?feros e tem como agente etiol?gico o protozo?rio Trypanosoma cruzi.Investiga??es sobre os mecanismos de adapta??o, regula??o g?nica e intera??o parasito x vetor evolu?ram, o que comprova a necessidade da utiliza??o de ferramentas moleculares para o estudo e elucida??es sobre as mudan?as adaptativas ocorridas nestes parasitos. Recentemente evid?ncias surgiram neste sentido, onde estudos indicam que a express?o de enzimas antioxidantes do T. cruzi tais como: peroxirredoxinas citos?lica (TcCPx), mitocondriais (TcMPx), (TcAPX) e tripanotiona sintetase (TXNI, TXNII), super?xido dismutase (SOD A e SOD B) podem fazer parte do sistema protetivo do parasito e indicar fatores relacionados aos diferentes n?veis de virul?ncia deste agente etiol?gico. Desta forma, an?lises prote?mica e avalia??o da express?o de enzimas antioxidantes de diferentes compartimentos subcelulares podem corroborar para com os estudos relacionados aos ?ndices de virul?ncia e express?o de prote?nas envolvidas neste processo. Neste sentido, os objetivos deste trabalho foram avaliar a morfologia dos isolados das amostras SMM98, 36 e 1, analisar al?m do perfil prote?mico, a express?o de enzimas antioxidantes (TcCPx; TcMPx; TcAPx; TcTXNI; TcTXNII, SOD A e SOD B) e observar a express?o do status redox utilizando os isolados SMM36 e SMM98 em compara??o com as cepas TCC, Silvio e DM28c. Os resultados obtidos demonstraram aspectos morfol?gicos diferenciados dentre os isolados, n?veis de virul?ncia elevados, perfil de prote?nas variados quando comparados entre os isolados SMM98 e SMM36. E, quando tratados com Heme e NAC foram observadas altera??es no desenvolvimento dos parasitos, denotando a participa??o do Status Redox frente aos isolados do Rio de Janeiro utilizados neste estudo.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-09-27T19:30:21Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) amanda_escobar_teixeira.pdf: 1867154 bytes, checksum: a9782b1752452b0bdb34a3ba10ae56bb (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-10-01T17:48:46Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) amanda_escobar_teixeira.pdf: 1867154 bytes, checksum: a9782b1752452b0bdb34a3ba10ae56bb (MD5) / Made available in DSpace on 2018-10-01T17:48:46Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) amanda_escobar_teixeira.pdf: 1867154 bytes, checksum: a9782b1752452b0bdb34a3ba10ae56bb (MD5) Previous issue date: 2018 / Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / O objetivo deste trabalho foi avaliar os efeitos da dieta de cafeteria desde a lacta??o nos par?metros nutricionais e na a??o da risperidona nos comportamentos de ansiedade, locomo??o, mem?ria e intera??o social. Foram utilizadas 14 ninhadas de ratos da linhagem Wistar (Rattus novergicus) alojados em gaiolas individuais, sob condi??es padr?o. Os ratos machos de cada ninhada formaram, da lacta??o at? a fase adulta, os grupos: Controle (CTRL) ? receberam ra??o padr?o e ?gua ad libitum (n = 42); Cafeteria (CAF) ? receberam dieta de cafeteria e ?gua ad libitum (n = 42). Entre o 114? e o 119? dia de vida, os animais foram subdividos (n = 21) para receberem o tratamento com salina (CTRL-S e CAF-S) ou risperidona (CTRL-R e CAF-R). Nesse per?odo foram realizados os testes comportamentais do labirinto em cruz elevado, campo aberto, teste de reconhecimento de objetos e intera??o social. No 119? os animais foram colocados em jejum, para que, no 120? dia fossem anestesiados e eutanasiados por exsanguina??o. Foram avaliados: consumo de ra??o, ingest?o cal?rica, peso corporal, ganho de peso, coeficiente de efici?ncia alimentar (CEA), comprimento naso-anal e ?ndice de massa corporal (IMC); peso dos ?rg?os e do tecido adiposo abdominal; teores de colesterol total e fra??es, triacilglicerol e glicemia do soro; teor de lip?dios, colesterol total e triacilglicerol do f?gado e das fezes; estado redox do f?gado e de diferentes por??es do enc?falo (c?rtex pr?-frontal e hipocampo). Foi utilizada an?lide de vari?ncia (ANOVA) seguida de Newman Keuls quando necess?rio (P<0,05). O grupo CAF demonstrou menor ingest?o alimentar e maior ingest?o cal?rica, devido ? densidade energ?tica da dieta. O maior consumo cal?rico se configurou em maior ganho de peso, CEA, IMC e ac?mulo de tecido adiposo abdominal. O grupo CAF tamb?m obteve eleva??o dos n?veis de triacilglicerol plasm?ticoa, al?m de lip?dios e triacilglicerol hep?tico, que foram classificados em esteatose hep?tica. Ao mesmo tempo, houve uma rela??o ruim entre as fra??es do colesterol plasm?tico (HDL-c, LDL-c e VLDL-c), com aumento do risco de doen?as aterog?nicas. Portanto, a dieta de cafeteria foi capaz de reproduzir um modelo de obesidade humana e de s?ndrome metab?lica. Os animais do grupo de cafeteria apresentaram caracter?sticas de estresse oxidativo no hipocampo, o que pode comprometer a fun??o dessa estrutura e promover altera??es comportamentais. O grupo CAF - S obteve efeito ansiol?tico devido ao maior n?mero de entradas e tempo gasto no centro do campo aberto, al?m de maior locomo??o atrav?s do maior n?mero de quadrantes percorridos. CAF - S ainda demonstrou preju?zos na mem?ria avaliados atrav?s do teste de reconhecimento de objetos e diminui??o da intera??o social. Tais efeitos podem estar associados a altera??es no sistema serotonin?rgico desses animais. CAF - R demonstrou efeito ansiog?nico e diminui??o na locomo??o no campo aberto. No teste de reconhecimento de objetos, houve diminui??o na intera??o com os objetos, sem, no entanto, melhorar o ?ndice de discrimina??o em rela??o a CAF - S. No teste de intera??o social, CAF - R obteve maior sociabilidade. Os resultados da administra??o da risperidona indicam que esta droga possui um efeito diferente quando aplicada em animais que foram tratados com dieta de cafeteria desde a lacta??o. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2018. / The objective of this work was to evaluate the effects of the cafeteria diet from lactation on the nutritional parameters and the action of risperidone on the behaviors of anxiety, locomotion, memory and social interaction. Fourteen litters of Wistar rats (Rattus novergicus) housed in individual cages were used under standard conditions. The male rats from each litter formed from the lactation to the adult phase, the groups: Control (CTRL) - received standard chow and water ad libitum (n = 42); Cafeteria (CAF) - received cafeteria diet and water ad libitum (n = 42). Between days 114 and 119, animals were subdivided (n = 21) to receive treatment with saline (CTRL-S, CAF-S) or risperidone (CTRL-R and CAF-R). During this period, the behavioral tests of the plus maze size, open field, test of object recognition and social interaction were performed. At 119? the animals were fasted, so that on the 120? day they were anesthetized and euthanized by exsanguination. The following were evaluated: feed intake, caloric intake, body weight, weight gain, food efficiency coefficient (FEC), naso-anal length and body mass index (BMI); weight of organs and abdominal adipose tissue; levels of total cholesterol and fractions, triacylglycerol and serum glycemia; lipid content, total cholesterol, and triacylglycerol of liver and faeces; redox status of the liver and different portions of the encephalon (prefrontal cortex and hippocampus). Variance analysis (ANOVA) was used followed by Newman Keuls when necessary (p<0,05). The CAF group demonstrated lower dietary intake and higher caloric intake, due to the energy density of the diet. The highest caloric intake was in greater weight gain, FEC, BMI and abdominal adipose tissue accumulation. The CAF group also achieved elevations in plasma triglyceride levels, besides lipids and hepatic triacylglycerol, which were classified as hepatic steatosis. At the same time, there was a poor relationship between fractions of plasma cholesterol (HDL-c, LDL-c and VLDL-c), with increased risk of atherogenic diseases. Therefore, the cafeteria diet was able to reproduce a model of human obesity and metabolic syndrome. The animals in the cafeteria group presented oxidative stress characteristics in the hippocampus, which may compromise the function of this structure and promote behavioral changes. The CAF - S group had an anxiolytic effect due to the greater number of entrances and time spent in the center of the open field, besides greater locomotion through the greater number of quadrants traveled. CAF - S also demonstrated memory impairments assessed through the object recognition test and decreased social interaction. Such effects may be associated with changes in the serotonergic system of these animals. CAF - R demonstrated an anxiogenic effect and decreased locomotion in the open field. In the object recognition test, there was a decrease in the interaction with the objects, without, however, improving the discrimination index in relation to CAF - S. In the social interaction test, CAF - R obtained greater sociability. The results of the administration of risperidone indicate that this drug has a different effect when applied in animals that have been treated with cafeteria diet since lactation.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / As formas epimastigotas de Trypanosoma cruzi proliferam e se diferenciam no interior de diferentes compartimentos do trato digestivo dos triatomíneos. Esses ambientes antagônicos, no que diz respeito à concentração de nutrientes, pH e status redox, constituem um desafio para o protozoário por conterem moléculas e fatores capazes de deflagrar diferentes sinalizações e respostas no parasito. Por isso, testamos a influência de produtos abundantes do metabolismo do vetor e de status redox distintos, frente aos processos de proliferação e diferenciação in vivo e in vitro. Como exemplo temos o heme e a hemozoína, subprodutos da digestão da hemoglobina, e o urato, rico na urina dos insetos. O heme é uma importante molécula em todos os seres vivos. Nosso grupo mostrou seu papel na proliferação in vitro de T. cruzi e que esse sinal é governado pela enzima redox-sensível CaMKII (Lara et al., 2007; Souza et al., 2009). Esse efeito parece depender de uma sinalização redox, onde o heme e não seus análigos induz a formação de EROs, modulando a atividade da CaMKII (Nogueira et al, 2011). Apesar de gerar espécies reativas de oxigênio (EROs) em formas epimastigotas, o heme não alterou a ultraestrutura desses parasitos mostrando uma adaptação a ambientes oxidantes. Além disso, a adição de FCCP inibiu a formação de EROs mitocondrial, diminuindo a proliferação dos parasitos. Em contrapartida, a AA aumentou drasticamente a produção de EROs mitocondrial levando à morte dos epimastigotas. Estes resultados confirmam a hipótese de regulação redox do crescimento de epimastigotas. A formação de &#946;- hematina (hemozoína) constitui uma elegante estratégia para minimizar o efeito tóxico do heme nos insetos hematófagos. Contudo, a &#946;-hematina não influenciou a proliferação ou a metaciclogênese in vitro. Já o urato, e outros antioxidantes clássicos como o GSH e o NAC prejudicaram a proliferação in vitro de epimastigotas. Estes efeitos foram parcialmente revertidos quando os antioxidantes foram incubados juntamente com o heme. Durante a metaciclogênese in vitro, o NAC e o urato induziram um aumento significativo das formas tripomastigotas e levaram a diminuição da porcentagem de formas epimastigotas. Em contrapartida, o heme e a &#946;-hematina apresentaram o efeito oposto, diminuindo a porcentagem de formas tripomastigotas e aumentando a de epimastigotas. No intuito de confirmar a influencia do status redox na biologia do parasito in vivo, nós quantificamos a carga parasitária nas porções anterior e posterior e no reto do triatomíneo alimentado na presença ou na ausência de NAC e urato por qPCR. O tratamento com os antioxidantes aumentou a carga parasitária em todas as partes do intestino analisadas. Posteriormente, para diferenciar as formas evolutivas responsáveis pelo incremento da carga parasitária, foram realizadas contagens diferenciais nas mesmas porções do intestino do inseto vetor. Cinco dias após a infecção foi observado aumento significativo de formas tripomastigotas e diminuição de formas epimastigotas in vivo. Em conjunto, estes dados sugerem que, assim como a concentração de nutrientes e o pH, o status redox também pode influenciar a biologia do T. cruzi no interior do inseto vetor. Neste cenário, moléculas oxidantes agiriam a favor da proliferação, e em contraste, antioxidantes parecem favorecer a metaciclogênese. / Trypanosoma cruzi epimastigotes proliferate and differentiate inside different compartments of the triatomines gut. These environments are antagonic in terms of nutrient content, pH and redox status. All these factors represent a challenge to the protozoan due to the presence of molecules and factors which are able to induce different signals to the parasite. Thus, we tested the influence of abundant metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis in vitro and in vivo. These molecules are heme and hemozoin, both byproducts of hemoglobin digestion, and urate, present in the urine of insects. Heme is a ubiquitous molecule present in all living organisms. Our group studied its role in T. cruzi growth in vitro, showing that this signal is governed by the redox-sensitive enzyme CaMKII (Lara et al., 2007; Souza et al., 2009). Indeed, it seems to rely on a redox signaling pathway in which heme, but not its analogs, induces ROS formation, thus modulating CaMKII activity (Nogueira et al., 2011). Although it induces ROS in epimastigotes, the heme molecule had no deleterious effect upon the parasites ultrastructure, suggesting an adaptation to oxidative environments. In addition, FCCP inhibited mitochondrial ROS formation, then decreasing the parasite proliferation. On the other hand, AA drastically increased mitochondrial ROS production leading to cell death. These results corroborate the hypothesis of redox regulation of epimastigotes proliferation. Hemozoin (&#946;- hematin) formation is an elegant strategy to minimize the toxic effect of heme in hematophagous insects. However, &#946;-hematin had no influence upon the proliferation or metacyclogenesis in vitro. Also, urate, GSH and NAC impaired epimastigote proliferation. These effects were partially reversed when the antioxidants were incubated along with heme. During metacyclogenesis in vitro, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. Heme and &#946;-hematin presented the opposite effect diminishing the percentage of trypomastigotes and increasing the percentage of epimastigotes. To confirm the influence of the redox status in the parasite biology in vivo, we quantified the parasite loads in the anterior and posterior midguts and in the rectum of the triatomine vector fed with or without NAC and urate by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. Afterwards, in order to distinguish the evolutive forms responsible for the increment of parasite loads, we performed differential counting of the midgut sections. Five days post infection we observed an increment of trypomastigotes and a decrease of epimastigote forms in vivo. Taken together, these data suggests that, as well as nutrient content and pH, the redox status may also influence T. cruzi biology in the vector. In this scenario, oxidants act to turn on proliferation and in contrast, antioxidants seem to switch the cycle towards metacyclogenesis.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / As formas epimastigotas de Trypanosoma cruzi proliferam e se diferenciam no interior de diferentes compartimentos do trato digestivo dos triatomíneos. Esses ambientes antagônicos, no que diz respeito à concentração de nutrientes, pH e status redox, constituem um desafio para o protozoário por conterem moléculas e fatores capazes de deflagrar diferentes sinalizações e respostas no parasito. Por isso, testamos a influência de produtos abundantes do metabolismo do vetor e de status redox distintos, frente aos processos de proliferação e diferenciação in vivo e in vitro. Como exemplo temos o heme e a hemozoína, subprodutos da digestão da hemoglobina, e o urato, rico na urina dos insetos. O heme é uma importante molécula em todos os seres vivos. Nosso grupo mostrou seu papel na proliferação in vitro de T. cruzi e que esse sinal é governado pela enzima redox-sensível CaMKII (Lara et al., 2007; Souza et al., 2009). Esse efeito parece depender de uma sinalização redox, onde o heme e não seus análigos induz a formação de EROs, modulando a atividade da CaMKII (Nogueira et al, 2011). Apesar de gerar espécies reativas de oxigênio (EROs) em formas epimastigotas, o heme não alterou a ultraestrutura desses parasitos mostrando uma adaptação a ambientes oxidantes. Além disso, a adição de FCCP inibiu a formação de EROs mitocondrial, diminuindo a proliferação dos parasitos. Em contrapartida, a AA aumentou drasticamente a produção de EROs mitocondrial levando à morte dos epimastigotas. Estes resultados confirmam a hipótese de regulação redox do crescimento de epimastigotas. A formação de &#946;- hematina (hemozoína) constitui uma elegante estratégia para minimizar o efeito tóxico do heme nos insetos hematófagos. Contudo, a &#946;-hematina não influenciou a proliferação ou a metaciclogênese in vitro. Já o urato, e outros antioxidantes clássicos como o GSH e o NAC prejudicaram a proliferação in vitro de epimastigotas. Estes efeitos foram parcialmente revertidos quando os antioxidantes foram incubados juntamente com o heme. Durante a metaciclogênese in vitro, o NAC e o urato induziram um aumento significativo das formas tripomastigotas e levaram a diminuição da porcentagem de formas epimastigotas. Em contrapartida, o heme e a &#946;-hematina apresentaram o efeito oposto, diminuindo a porcentagem de formas tripomastigotas e aumentando a de epimastigotas. No intuito de confirmar a influencia do status redox na biologia do parasito in vivo, nós quantificamos a carga parasitária nas porções anterior e posterior e no reto do triatomíneo alimentado na presença ou na ausência de NAC e urato por qPCR. O tratamento com os antioxidantes aumentou a carga parasitária em todas as partes do intestino analisadas. Posteriormente, para diferenciar as formas evolutivas responsáveis pelo incremento da carga parasitária, foram realizadas contagens diferenciais nas mesmas porções do intestino do inseto vetor. Cinco dias após a infecção foi observado aumento significativo de formas tripomastigotas e diminuição de formas epimastigotas in vivo. Em conjunto, estes dados sugerem que, assim como a concentração de nutrientes e o pH, o status redox também pode influenciar a biologia do T. cruzi no interior do inseto vetor. Neste cenário, moléculas oxidantes agiriam a favor da proliferação, e em contraste, antioxidantes parecem favorecer a metaciclogênese. / Trypanosoma cruzi epimastigotes proliferate and differentiate inside different compartments of the triatomines gut. These environments are antagonic in terms of nutrient content, pH and redox status. All these factors represent a challenge to the protozoan due to the presence of molecules and factors which are able to induce different signals to the parasite. Thus, we tested the influence of abundant metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis in vitro and in vivo. These molecules are heme and hemozoin, both byproducts of hemoglobin digestion, and urate, present in the urine of insects. Heme is a ubiquitous molecule present in all living organisms. Our group studied its role in T. cruzi growth in vitro, showing that this signal is governed by the redox-sensitive enzyme CaMKII (Lara et al., 2007; Souza et al., 2009). Indeed, it seems to rely on a redox signaling pathway in which heme, but not its analogs, induces ROS formation, thus modulating CaMKII activity (Nogueira et al., 2011). Although it induces ROS in epimastigotes, the heme molecule had no deleterious effect upon the parasites ultrastructure, suggesting an adaptation to oxidative environments. In addition, FCCP inhibited mitochondrial ROS formation, then decreasing the parasite proliferation. On the other hand, AA drastically increased mitochondrial ROS production leading to cell death. These results corroborate the hypothesis of redox regulation of epimastigotes proliferation. Hemozoin (&#946;- hematin) formation is an elegant strategy to minimize the toxic effect of heme in hematophagous insects. However, &#946;-hematin had no influence upon the proliferation or metacyclogenesis in vitro. Also, urate, GSH and NAC impaired epimastigote proliferation. These effects were partially reversed when the antioxidants were incubated along with heme. During metacyclogenesis in vitro, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. Heme and &#946;-hematin presented the opposite effect diminishing the percentage of trypomastigotes and increasing the percentage of epimastigotes. To confirm the influence of the redox status in the parasite biology in vivo, we quantified the parasite loads in the anterior and posterior midguts and in the rectum of the triatomine vector fed with or without NAC and urate by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. Afterwards, in order to distinguish the evolutive forms responsible for the increment of parasite loads, we performed differential counting of the midgut sections. Five days post infection we observed an increment of trypomastigotes and a decrease of epimastigote forms in vivo. Taken together, these data suggests that, as well as nutrient content and pH, the redox status may also influence T. cruzi biology in the vector. In this scenario, oxidants act to turn on proliferation and in contrast, antioxidants seem to switch the cycle towards metacyclogenesis.

INTRODUCTION: Inflammatory bowel disease (IBD) is a complex, chronic, autoimmune disease of the gastrointestinal tract. Reactive oxygen species (ROS), a product of active leukocytes, have been implicated in the pathogenesis of IBD. The ability to reliably measure ROS in blood, urine, and stool samples could represent a new approach to assessing disease activity and response to therapy in pediatric patients with IBD. OBJECTIVES: To assess the relationship between redox measurements and clinical disease activity in pediatric patients with IBD. METHODS: Biological specimens, including stool, urine, blood plasma, and intestinal aspirates, were collected from patients at Boston Children’s Hospital. Each sample’s oxidation-reduction potential was measured by two oxidation-reduction potential probes (an Arrowdox probe and a Mettler Toledo probe). Probes were directly immersed into the sample, returning a millivolt measurement of oxidation-reduction potential. Linear regression was performed to explore the relationship between patient-reported outcome measures (PROMs) and redox measurements of biological specimens. Patients were also stratified by disease severity, and ANOVA testing was performed to test for differences in oxidation-reduction potential observed in patients with remittent, mild, moderate, and severe disease activity. RESULTS: Redox values in stool, urine, plasma, and intestinal aspirate did not significantly correlate with PROMs or differ significantly among groups categorized by disease severity. CONCLUSIONS: Measurements of oxidation-reduction potential from stool, urine, plasma, and intestinal aspirate do not appear to be useful for assessing disease severity in pediatric patients with inflammatory bowel disease.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Exercise can represent a physical stress that disrupts the homeostasis. Elevated muscle oxygen consumption increases reactive oxygen species (ROS) and inflammatory proteins production, leading to oxidative stress and systemic inflammation. Nowadays, both caffeine, a commonly compound present in many commercial beverages and medicines, and the non-steroidalanti-inflammatory drugs (NSAIDs), including diclofenac, have been used in sports competitions events. Considering that athletes intent to improve their sports performance, mainly on high intensity competitions and short duration that can lead to inflammation and pain, a great number of athletes consume caffeine and NSAIDs due they ergogenic effects or avoid inflammation and loss of performance. However, little is known about the physical exercise and concomitant use of caffeine/diclofenac. Considering the sport specificity, most authors have focused exercise in skeletal muscle studies. In view of the important role of liver during physical activities, one of the goals of this work was to analyze the effect of caffeine and diclofenac on cell damage, hepatic inflammation and oxidative stress markers in exercised rats. In two papers, we highlight the effect of caffeine on the oxidative damage, inflammation and tissue adaptation in liver, muscle and plasma of training rats. The experimental protocol included four groups: sedentary-saline, sedentary-diclofenac, exercise-saline, and exercise-diclofenac. The exercised groups performed a 4-week aerobic swimming training protocol and were treated with saline or caffeine (6 mg/kg). We found significant changes on citrate synthase (CS), superoxide desmutase (SOD), glutathione peroxidase (GPx) and acetylcholinesterase (AChE) enzyme activities and aspartate aminotransferase (AST) and thiobarbituric acid reactive substances (TBARS) levels after training. These modifications were reverted by caffeine treatment. In the manuscript, we highlight the effects of diclofenac (10 mg/kg) on the inflammation induced by an acute exercise. Rats were divided in 4 groups: control-saline (CS), control-diclofenac (CD), exercise-saline (ES) and exercise-diclofenac (ED). The animals from the C and E groups received saline, while the groups CD and ED received diclofenac treatment during seven days previous to the exercise bout, which consisted in an acute bout of eccentric exercise lasting 90 min. We identified an increase in both gene expression and levels of proteins TLR4, MyD88, TRIF, NFκB, p65, IL-6, TNF-α and iNOS in the exercised groups. Diclofenac treatment blunted these responses exercised-induced. Taken together, the data indicate that diclofenac and caffeine interfere on oxidative and inflammation responses induced by physical exercise, altering the adaptive mechanisms of tissues under exercise. / O exercício físico pode representar um tipo de estresse por alterar a homeostase corporal. O elevado consumo de oxigênio pelo músculo esquelético durante o exercício físico aumenta a produção de espécies reativas de oxigênio (EROs) e proteínas inflamatórias, desencadeando estresse oxidativo e inflamação sistêmica. Atualmente, a cafeína, um composto presente em diversas bebidas e medicamentos, e os anti-inflamatórios não-esteroidais (AINEs), incluindo o diclofenaco, têm sido amplamente usados em competições esportivas. Considerando que os atletas treinam com o objetivo de melhorar o seu desempenho esportivo, principalmente em provas de alta intensidade e curta duração e que essas podem levar à sensação de dor e processo inflamatório, um grande número desses atletas usa tanto cafeína e AINEs como recursos ergogênicos ou até mesmo para evitar perdas de performance em suas provas. No entanto, pouco se sabe sobre os efeitos da associação entre treinamento físico e o uso concomitante de cafeína/diclofenaco nos tecidos. Levando em consideração a especificidade esporte, a maioria dos estudos são referentes a associação exercício físico e músculo esquelético. Entretanto, as respostas adaptativas ao exercício físico não são restritas ao tecido muscular. Considerando o importante papel do fígado durante a atividade física, um objetivo desta tese foi analisar os efeitos da cafeína e, em um segundo momento, do diclofenaco, sobre marcadores hepáticos de estresse oxidativo, dano tecidual e inflamação em ratos treinados. Nos artigos destacamos o papel da cafeína em modular as respostas de dano, estresse oxidativo, inflamação e adaptação causadas pelo treinamento físico em fígado, músculo e plasma. O protocolo experimental foi realizado com 4 grupos distintos: sedentário-salina, sedentário-cafeína, exercício-salina e exercício-cafeína. Os grupos exercício foram submetidos a 4 semanas de treinamento aeróbio de natação e os grupos tratados foram suplementados com cafeína (6 mg/kg), durante o treinamento. Identificamos mudanças significativas na atividade das enzimas citrato sintase (CS), superóxido desmutase (SOD), glutationa peroxidase (GPx) e acetilcolinesterase (AChE), e nos níveis da aspartato aminotransferase (AST) e substâncias reativas ao ácido tiobarbitúrico (TBARS) após treinamento. Todas essas alterações foram revertidas pelo tratamento com cafeína. No manuscrito destacamos o papel modulador do diclofenaco sobre a inflamação gerada por exercício agudo. O protocolo consistiu em uma sessão de 90 mim de exercício excêntrico agudo em esteira em ratos tratados previamente com salina ou diclofenaco (10mg/kg) (grupos: controle-salina, exercício-salina, controle-diclofenaco, exercício-diclofenaco). Após o exercício, foi identificado um aumento na expressão gênica e níveis da proteínas TLR4, MyD88, TRIF, NFκB p65, IL-6, TNF-α e iNOS. Essas respostas geradas pelo exercício excêntrico foram bloqueadas pelo tratamento com diclofenaco. Os dados obtidos nos permitem concluir que o diclofenaco e a cafeína interferem nas respostas adaptativas de cunho oxidativo/inflamatório geradas pelo exercício físico, podendo assim alterar os mecanismos de hormesis dos tecidos ao exercício físico.