Glutathione S-transferases (GST, EC2.5.1.18) are a family of multifunctional, dimeric enzymes that catalyse the nucleophilic attack of the tripeptide glutathione (&amp / #947 / -L-glutamyl-L-cysteinyl-L-glycine) on lipophilic compounds with electrophilic centres. The primary function of GSTs is generally considered to be the detoxification of both endogenous and xenobiotic compounds. Cytosolic GSTs have been grouped into eleven distinct classes as: (A) / Alpha, (M) / Mu, (P) / Pi, (S) / Sigma, (T) / Theta, (Z) / Zeta, (F) / Phi, (U) / Tau, (B) / Beta, (O) / Omega and (L) / Lambda. iv In this study, the total RNAs from Pinus brutia needles were isolated, GST Zeta cDNA was prepared by RT-PCR, the length of the insert was elongated by applying 5&#039 / RACE (Rapid Amplification for cDNA ends) method and the identity of the insert was checked by sequencing. The amino acid sequence of GST-Zeta was deduced as composed of 226 amino acids. The genomic DNA was also isolated from Pinus brutia needles, amplified by PCR and sequenced, and compared to the sequence of cDNA. The expression level of GST-Zeta in individual trees of Pinus brutia were examined by Northern blot analysis, and compared to their thiol contents. The mRNA levels varied up to three-fold, whereas GSH amounts varied approximately 1.8 fold, and there were no correlation between the GST-Zeta expression and GSH concentration. GST enzyme with activity towards CDNB was isolated and purified from Pinus brutia needles in 1.95 % yield with a purification factor of 15.45-fold. The purification protocol included a sequential chromatography on Sephadex G-25 column, DEAE cellulose anion exchanger liquid chromatography column, and S-hexylglutathione agarose affinity columns. The purified GST showed specific activity towards CDNB as 2022 nmole/min/mg. The GST purified from needles had a molecular weight (Mr) value of about 24.000 which was confirmed by SDS-PAGE.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Resumo: A beta talassemia heterozigota pode ser decorrente de mutações β 0 ou β + , de acordo com ausência ou redução na produção das cadeias da beta globina, respectivamente. Poucos estudos na literatura correlacionam possíveis efeitos deletérios dessas mutações, quando em heterozigose. A coerança com mutações em genes, não localizados no cluster do gene da β-globina, podem modificar o fenótipo talassêmico. O estresse oxidativo gerado pelo aumento de ERO é um dos mecanismos responsáveis pala aceleração da apoptose celular. Há evidências que o excesso de cadeias alfa, mesmo em pequenas quantidades, seja suficiente para iniciar a geração de ERO e, portanto, estresse oxidativo em beta talassêmicos heterozigotos. Nas formas homozigotas ou com dupla heterozigose os pacientes apresentam anemia grave, transfusão dependente, e a sobrecarga de ferro é uma das mais frequentes e graves complicações nesses indivíduos. Na forma heterozigota a ocorrência de sobrecarga de ferro é rara, porém alterações no perfil do ferro são observadas. O objetivo desse trabalho foi analisar a influência dos níveis de ferritina sérica, das mutações no gene da β-globina, das mutações no gene HFE, da atividade das enzimas de detoxificação GST e CAT e dos polimorfismos das GSTs na expressão fenotípica de indivíduos beta talassêmicos heterozigotos. Foram avaliados 158 portadores de beta talassemia heterozigota diagnosticados previamente por metodologias clássicas. Todos adultos, de origem caucasóide, residentes na região Central do Estado de São Paulo e acompanhados em serviços públicos ou privados da região. O grupo controle foi composto de 24 adultos, caucasóides e residentes na mesma região. A avaliação hematológica foi realizada pelo hemograma, em contadores automatizados de células e quantificação das sub frações de Hb A2 e Hb F por HPLC. O perfil... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Beta thalassemia may occur due to mutations β 0 or β +, according to absence or reduction in the production of beta globin chains, respectively. Few studies have been done correlating possible deleterious effects of these mutations, when in heterozygous. Co-inheritance with mutations in the genes, not located in the cluster of β-globin gene may modify the phenotype of the thalassemic. Stress oxidative generated by the increase of ROS is one of the mechanisms responsible for the acceleration of cell apoptosis. There are evidences that alpha chains in excess, even in small quantities, are sufficient to start ROS generation and thus oxidative stress in heterozygous beta thalassemic. In homozygous form or genetic heterozygous compound present severe anemia, transfusion dependent and iron overload is one of the complications more frequent and severe in these patients. In the heterozygous form the presence of iron overload is rare, but alterations in the profile of iron are observed. The aim of this study was to examine influence of the levels of serum ferritin, the mutations in the β-globin gene, the mutations in the HFE gene, activity of enzymes detoxification, GST and CAT, and polymorphisms of GSTs in the expression phenotypic of individuals heterozygous beta thalassemic. We evaluated 158 patients with beta thalassemia heterozygous previously diagnosed by classic methodologies. They were all adults, Caucasian origin, residents in the Central Region of São Paulo state and accompanied in public or private services of the region. The control group consisted of 24 adults, Caucasians and residents in the same region. Hematologic evaluation was carried out by hemogram, in automated counters of cells and quantification of the subfractions of Hb A2 and Hb F by HPLC; the iron profile was based on the transferrin saturation , calculated on the serum iron and total iron binding capacity... (Complete abstract click electronic access below) / Orientador: Claudia Regina Bonini-Domingos / Coorientador: Eduardo Alves de Almeida / Banca: Elvira Maria Guerra Shinohara / Banca: Ivan Lucena Angulo / Banca: Maria Stella Figueiredo / Banca: Maria Tercília Vilela de Azeredo Oliveira / Doutor

Os carrapatos Rhipicephalus (Boophilus) microplus e Haemaphysalis longicornis são ectoparasitas hematófagos que infestam bovinos. O uso de vacinas anticarrapato tem mostrado ser uma estratégia alternativa promissora para o controle desses parasitos. As glutationa-S-transferases (GSTs) pertencem a uma família de enzimas multifuncionais presentes em organismos animais e vegetais, e, entre suas funções, pode-se destacar o transporte intracelular, a participação em processos digestivos, a síntese de prostaglandinas, a detoxificação de substâncias tóxicas e a proteção contra o estresse oxidativo. Em trabalhos anteriores, verificou-se em camundongos que a GST de H. longicornis (rGST-Hl) é mais imunogênica que a GST de R. microplus (rGST-Bm). O objetivo do presente estudo foi investigar a proteção cruzada conferida pela imunização de bovinos com a rGST-Hl frente a uma infestação de R. microplus. Por Western blot, verificou-se que os bovinos vacinados com a rGST-Hl apresentaram um aumento nos níveis de anticorpos anti-rGST-Hl, os quais foram capazes de reconhecer a GST-Bm nativa de extratos de larva, ovário e glândula salivar. Posteriormente, os bovinos foram infestados com 20.000 larvas de R. microplus. O percentual de proteção conferido foi calculado em função da variação entre os grupos tratados e controle, em relação ao número e peso de carrapatos que terminaram a alimentação, a capacidade de postura e a fertilidade dos ovos. O grupo dos bovinos vacinados com rGST-Hl mostrou uma redução de 53% e 52% no número e no peso de teleóginas, respectivamente. A postura de ovos e a eclodibilidade apresentaram uma redução no grupo vacinado de 0,6% e 8%, respectivamente. A eficácia total contra a infestação de R. microplus foi de 57%, quando comparado ao grupo controle. / The ticks Rhipicephalus (Boophilus) microplus and Haemaphysalis longicornis are hematophagous ectoparasites that infest bovines. Vaccine has been considered one of the most promising methods for the control of these parasites. Glutathione Stransferases (GSTs) enzymes are present in animal and vegetal organisms and are important in intracellular transport, digestive process, synthesis of prostaglandins, detoxification and protection against oxidative stress. In a previous study with mice we showed that H. longicornis GST (rGST-Hl) is more immunogenic than R. microplus GST (rGST-Bm). The objective of the present study was to investigate the crossprotection against R. microplus infestation in bovine vaccinated with rGST-Hl. By Western blot, the immunized bovines showed an increase in antibodies anti-rGST-Hl. The native GST-Bm in larvae, ovary and salivary gland were recognized by sera of bovines immunized with rGST-Hl, indicating that the antibodies induced by rGST-Hl immunization recognize native R. microplus GSTs. The bovines were challenged with 20,000 larvae of R. microplus. Protection was calculated by variation in number and weight of fully engorged ticks, egg laying capacity and egg fertility. The bovines vaccinated with rGST-Hl showed a reduction of 53% and 52% in number and weight of engorged ticks, respectively. Egg laying capacity and egg fertility of ticks from vaccinated bovines were respectively 0.6% and 8% lower, as compared to ticks from control animals. The overall efficacy ratio against R. microplus was 57.03%, as compared with the control group.

of study named: Effects of selected natural substances on the antioxidant system of an organism Developed: Mgr. Anna Hodková Department of Pharmacology and Toxikology, Faculty of Medicine in Pilsen, Charles University in Prague Pilsen 2016 The aim of this study was to compare the effects of selected natural substances on the antioxidant defense system under comparable conditions, focusing on influencing the activity of selenoenzymes thioredoxin reductase (TrxR-1) and glutathione peroxidase (GPx-1). Experiments were performed in rats (Wistar, male). Livers, and in some cases kidneys were collected in all experiments. Homogenates were created from the collected organs and subsequently the activity of TrxR-1 and GPx-1, glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD), and reduced glutathione (GSH) and lipid peroxidation (LP) levels were determined. We demonstrated significant effects of selected natural substances on the redox system, including influences of selenoenzymes thioredoxin reductase and glutathione peroxidase. The biggest influence on the activity of selenoenzymes thioredoxin reductase and glutathione peroxidase had hydroxytyrosol (HT) and oleuropein (OLEU). In rat liver tissue there was a significant decrease of the activity of both above mentioned enzymes after...

O Lúpus Eritematoso Sistêmico (LES) é uma doença inflamatória crônica autoimune que apresenta uma ampla variedade de manifestações clínicas e anormalidades imunológicas, afetando principalmente mulheres. É caracterizado pela perturbação da homeostase imunológica, que envolve a indução e produção de autoanticorpos, bem como pela formação e deposição de complexos imunes, que conduzem a uma intensa resposta inflamatória e dano tecidual. Há evidências de que fatores imunológicos, ambientais, hormonais e genéticos estão implicados na patogênese da doença. Genes e proteínas envolvidas na metabolização/detoxificação de xenobióticos são frequentemente utilizados como marcadores de susceptibilidade para o desenvolvimento de doenças, cuja etiologia está relacionada à exposição a fatores de risco ambientais. Enzimas do Citocromo P450 (CYP) são as principais responsáveis pela fase I de detoxificação, na qual ativam o xenobiótico, tornando-o mais eletrofílico e, desta forma, mais reativo. As Glutationa S-transferases (GST) são enzimas detoxificantes de fase II e normalmente conjugam a glutationa reduzida com uma variedade de compostos eletrofílicos, como espécies reativas de oxigênio, facilitando a excreção de produtos tóxicos. Polimorfismos nos genes CYP e GST são capazes de alterar a expressão e a atividade catalítica das enzimas, sendo responsáveis por diferenças interindividuais quanto à capacidade de biotransformação de xenobióticos. O objetivo do nosso trabalho foi avaliar a influência de três polimorfismos GST (GSTM1 nulo, GSTT1 nulo, e GSTP1*Val) e dois polimorfismos CYP (CYP1A1*2C e CYP2E1*5B) na predisposição ao LES em uma amostra de 370 pacientes com LES e 329 doadores de sangue saudáveis provenientes da região sul do Brasil. Os polimorfismos CYP foram genotipados por PCR-RFLP, enquanto que os polimorfismos GST foram genotipados por PCR multiplex (GSTM1 nulo, GSTT1 nulo) e PCR-RFLP para GSTP1. As freqüências alélicas e genotípicas foram comparadas entre pacientes e controles usando o teste de Qui-Quadrado ou o teste Exato de Fisher. As análises foram realizadas subdividindo os indivíduos de acordo com sua origem étnica. Entre os indivíduos Euro-descendentes, observou-se uma menor freqüência de genótipos heterozigotos GSTP1*Val em pacientes com LES em comparação aos controles (p=0,0047; OR 0,63 CI 95% 0,43 – 0,93 em relação a GSTP1*Ile/Ile e OR 0,49 CI 95% 0,26 – 0,92 em relação a GSTP1*Val/Val). No grupo Afro-descendente, houve tendência a uma maior freqüência do alelo GSTP1*Val em pacientes quando comparados aos controles (p=0,061). O alelo CYP2E1*5B foi significativamente mais freqüente nos pacientes do que em controles (p=0,038; OR 2,69 CI 95% 1,00 – 8,42). Não foi observada qualquer implicação clínica dos polimorfismos CYP e GST nos pacientes com LES. Nossos dados sugerem um papel protetor do genótipo heterozigoto GSTP1*105Ile/Val em Euro-descendentes e uma possível influência do alelo CYP2E1*5B na susceptibilidade ao LES entre Afro-descendentes. / Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease that presents a variety of clinical manifestations and immunological abnormalities, particularly affecting women. It is characterized by disruption of immunologic homeostasis, which results in the induction and production of autoantibodies, as well as the formation and deposition of immune complexes, leading to an intense inflammatory response and tissue damage. There is evidence that immunological, environmental, hormonal and genetic factors are involved in the pathogenesis of the disease. Genes and proteins involved in metabolism/detoxification of xenobiotics are often used as markers of susceptibility to the development of diseases whose etiology is related to exposure to environmental risk factors. Cytochrome P450 (CYP) enzymes are primarily responsible for phase I detoxification, in which activate the xenobiotic, making it more electrophilic and thus more reactive. The Glutathione S-transferases (GST) are phase II detoxifying enzymes and usually conjugate reduced glutathione with a variety of electrophilic compounds, such as reactive oxygen species, facilitating the excretion of toxic products. Polymorphisms in the CYP and GST genes can alter the expression and catalytic activity of enzymes, being responsible for interindividual differences regarding the capacity of xenobiotics biotransformation. The aim of our study was to evaluate the influence of three GST polymorphisms (GSTM1 null, GSTT1 null and GSTP1*Val) and two CYP polymorphisms (CYP1A1*2C and CYP2E1*5B) in SLE predisposition in a sample of 370 SLE patients and 329 healthy blood donors, both from southern Brazil. The CYP polymorphisms were genotyped by PCR-RFLP, while the GST polymorphisms were genotyped by multiplex PCR and PCR-RFLP for GSTP1. Allelic and genotypic frequencies were compared between patients and controls using the Chi-square test or Fisher´s exact test. Analyses were performed subdividing the individuals according to their ethnic origin. Among European-derived individuals, it was observed a lower frequency of GSTP1*Val heterozygous genotypes in SLE patients compared to controls (p = 0.0047; OR 0.63 CI 95% 0.43 - 0.93 in relation to GSTP1*Ile/Ile) and (OR 0.49 95% CI 0.26 - 0.92 in relation to GSTP1*Val/Val). In African-derived group, there was a trend to a higher frequency of GSTP1*Val allele in patients when compared to controls (p=0.061). The CYP2E1*5B allele was significantly more frequent in patients than controls (p=0.038, OR 2.69 95% CI 1.00 - 8.42). We did not observe any clinical implication of the CYP and GST polymorphisms in patients with SLE. Our data suggest a protective role of the GSTP1*105Ile/Val heterozygous genotype in European-derived and a possible influence of the CYP2E1*5B allele in SLE susceptibility among African-derived.

Les champignons décomposeurs du bois jouent un rôle important dans le cycle du carbone en participant notamment au recyclage de la matière organique. Outre leur aptitude à minéraliser la biomasse lignocellulosique, ces organismes ont la capacité de dégrader des molécules potentiellement toxiques libérées lors de ce processus. Leur système de détoxication comprend différentes familles multigéniques dont les glutathion transférases. Ces enzymes ubiquitaires, sont regroupées en différentes classes dans le règne fongique, certaines d’entre elles étant étendues chez ces champignons. Dans ce contexte, l’objectif principal de cette thèse consistait à appréhender les fonctions des glutathion transférases de la classe Omega (GSTOs) étendue chez Trametes versicolor, un champignon de pourriture blanche. Une approche biochimique et structurale a été menée sur neuf protéines produites de façon recombinante. Dans un premier temps, une caractérisation enzymatique de ces isoformes a été réalisée à l’aide de substrats synthétiques montrant une similarité des propriétés catalytiques. Puis, à partir d’une banque de molécules pures et de mélanges complexes issus de différentes essences forestières, une méthode de screening à haut débit a permis d’identifier des ligands potentiels de ces enzymes. La résolution de la structure tridimensionnelle de trois isoformes a démontré l’état homodimérique de ces protéines et l’implication de deux sites de fixation dans la reconnaissance de ces ligands : le site H (présent dans chaque monomère) et le site L (à l’interface du dimère). Par exemple, l’isoforme TvGSTO3S est capable de fixer dans son site H plusieurs hydroxybenzophénones, mais également un flavonoïde, la dihydrowogonine. Dans ce dernier cas, cette interaction avec un ligand naturel issu d’extraits de bois de merisier a été démontré par une approche de cristallographie d’affinité. D’autre part, des expériences de co-cristallisation ont permis de détecter deux molécules d’un autre flavonoïde, la naringénine, dans le site L de l’isoforme TvGSTO6S. Enfin, une interaction spécifique impliquant les sites H et L de l’isoforme TvGSTO2S a été démontrée avec l’oxyresvératrol. L’analyse structurale a révélé que les deux configurations du stilbène étaient liées à la protéine : la configuration trans dans le site H et la configuration cis dans le site L. Ainsi, malgré une redondance fonctionnelle partielle, ces recherches ont démontré l’existence d’un spectre d’interactions spécifiques pour chaque isoforme testée. Le caractère étendu de la classe Omega indiquerait que ces enzymes seraient impliquées dans l’adaptation du champignon à son environnement. En effet, les ligands identifiés au cours de ces travaux suggèrent que les propriétés « ligandines » des TvGSTOs joueraient un rôle dans la détoxication des produits issus de dégradation du bois / Wood decaying fungi play an important role in the carbon cycle by participating in the recycling of organic matter. In addition to their ability to mineralize lignocellulosic biomass, these organisms have the ability to degrade potentially toxic molecules released during this process. Their detoxification system involves several multigenic families including glutathione transferases. These ubiquitous enzymes are grouped into several classes in the fungal kingdom, some of them are widespread in these fungi. In this context, the main objective of this thesis was to understand the functions of glutathione transferases of the Omega class (GSTOs) extended in Trametes versicolor, a white rot fungus. A biochemical and structural approach was led using nine recombinant proteins. Firstly, enzymatic characterization of these isoforms was performed using synthetic substrates, the obtained results demonstrating a similarity of catalytic properties. Then, using a library of pure molecules and another one of complex mixtures from different forest species, a high throughput screening method was applied to identify potential ligands for these enzymes. The resolution of the three-dimensional structure of three isoforms demonstrated the homodimeric state of these proteins and the involvement of two binding sites in the recognition of these ligands: the H site (present in each monomer) and the L site (at the dimer interface). For example, the isoform TvGSTO3S is able to bind several hydroxybenzophenones in its H site, but also a flavonoid, dihydrowogonin. In this case, this interaction with a natural ligand derived from wild-cherry tree extract was demonstrated by an affinity crystallography approach. On the other hand, co-crystallization experiments detected two molecules of another flavonoid, naringenin, in the L site of the isoform TvGSTO6S. Finally, a specific interaction involving the H and L sites of the isoform TvGSTO2S was demonstrated with oxyresveratrol. Structural analysis revealed that the presence of both configurations of the stilbene in the protein: the trans configuration in the H site and the cis configuration in the L site. Thus, despite partial functional redundancy, this research demonstrated the existence of a specific pattern of interactions for each tested isoform. The expansion of the Omega class could indicate that these enzymes are involved in the adaptation of the fungus in its environment. Indeed, the ligands identified during this work suggest that the "ligandin" properties of TvGSTOs play a role in detoxifying wood degradation products

A exposição ao chumbo ainda permanece como um sério problema de saúde pública, especialmente nos países em desenvolvimento como o Brasil. Apesar do número crescente de estudos mostrando déficits neurocomportamentais em indivíduos expostos ocupacionalmente ao chumbo, os mecanismos envolvidos com a manifestação destes transtornos permanecem pouco conhecidos. Desta forma, o uso de modelos animais abre grandes possibilidades, não somente de investigação dos mecanismos biológicos envolvidos com a toxicidade do chumbo, mas também na elaboração de estratégias para proteção e/ou reversão dos seus efeitos. Portanto, o objetivo central deste trabalho foi avaliar o potencial do comportamento rotacional, medido em testes de nado livre, como um modelo em roedores para estudo dos efeitos neurocomportamentais da exposição ao chumbo durante a vida adulta. Para tanto, foram realizados três estudos. No primeiro, avaliamos os efeitos da exposição subcrônica (60-90 dias) ao chumbo sobre a atividade locomotora no teste de nado livre e em um teste tradicionalmente utilizado para avaliar a ação de substâncias neurotóxicas sobre a locomoção, o campo aberto. Enquanto no campo aberto não foram encontradas diferenças significativas entre o grupo exposto e o grupo controle, no teste de nado livre, os animais expostos ao chumbo apresentaram maior atividade natatória e menor quantidade de alternâncias espontâneas no sentido de natação que o grupo controle. No segundo estudo, observamos que a magnitude dos efeitos do chumbo no teste de nado livre variou em função da consistência da lateralidade, sendo os resultados mais pronunciados no grupo de animais não consistentes (com menor lateralidade). Nos dois estudos, os efeitos do chumbo foram independentes do nível de chumbo em sangue (PbS) dos animais expostos. No terceiro estudo, mostramos um aumento dos níveis de glutationa oxidada no cérebro somente no grupo de animais com PbS acima de 60 μg/dL. Com base nestes resultados, podemos concluir que: 1) O comportamento rotacional medido em teste de nado livre é um modelo interessante para estudar os efeitos neurocomportamentais do chumbo; 2) A consistência da lateralidade pode ser um fator determinante da susceptibilidade aos efeitos tóxicos do chumbo no sistema nervoso; e 3) Os efeitos do chumbo no teste de nado livre ocorrem mesmo em níveis de estresse oxidativo que não afetam os níveis de glutationa oxidada no cérebro. / Lead exposure continues to be a serious public health problem, particularly in developing countries such as Brazil. Despite the increasing number of studies showing neurobehavioral deficits in people occupationally exposed to lead, the mechanisms involved in the manifestation of these problems remain largely unknown. In this sense, the use of animal models opens up the possibility of not only investigating the biological mechanisms associated with lead toxicity but also of elaborating strategies aiming at protecting individuals against its ill effects or even reversing those that already present. Therefore, the main objective of the present work was to assess the potential of the rotatory behavior in the free-swimming test as a rodent model for studying of the neurobehavioral effects adult life lead exposure. In the first study, we analyzed the effect of subchronic lead exposure on motor activity in the freeswimming test and in open-field test, which has been traditionally used to assess the effects of toxic substances on locomotion. While no significant differences were observed between the exposed and control groups in the open-field, in the free-swimming test, lead exposed animals displayed increased natatory activity and fewer spontaneous alterations in the direction of locomotion in comparison with the control group. In the second study, we observed that the magnitude of the effects of lead exposure varied as a function of the consistency of the lateralization, being the results more pronounced in inconsistent animals (lesser degree of lateralization). In both studies, the effects of lead were not dependent on blood levels of lead exposed animals. In the third study, we demonstrated a significant increase in the levels of oxidized glutathione in the brain (indicative of oxidative stress) only in the group of animals that had lead levels above 60 μg/dL. Based in these results, we can conclude that: 1) Rotatory behavior in the free-swimming test constitutes an interesting model for the study of the neurobehavioral effects of lead exposure, 2) Consistency of laterality may be a relevant factor regarding the susceptibility to the toxic effects of lead on the nervous system and 3) The effects of lead are observable in the free-swimming test even at oxidative stress levels that do not affect brain levels of oxidized glutathione.

Os carrapatos Rhipicephalus (Boophilus) microplus e Haemaphysalis longicornis são ectoparasitas hematófagos que infestam bovinos. O uso de vacinas anticarrapato tem mostrado ser uma estratégia alternativa promissora para o controle desses parasitos. As glutationa-S-transferases (GSTs) pertencem a uma família de enzimas multifuncionais presentes em organismos animais e vegetais, e, entre suas funções, pode-se destacar o transporte intracelular, a participação em processos digestivos, a síntese de prostaglandinas, a detoxificação de substâncias tóxicas e a proteção contra o estresse oxidativo. Em trabalhos anteriores, verificou-se em camundongos que a GST de H. longicornis (rGST-Hl) é mais imunogênica que a GST de R. microplus (rGST-Bm). O objetivo do presente estudo foi investigar a proteção cruzada conferida pela imunização de bovinos com a rGST-Hl frente a uma infestação de R. microplus. Por Western blot, verificou-se que os bovinos vacinados com a rGST-Hl apresentaram um aumento nos níveis de anticorpos anti-rGST-Hl, os quais foram capazes de reconhecer a GST-Bm nativa de extratos de larva, ovário e glândula salivar. Posteriormente, os bovinos foram infestados com 20.000 larvas de R. microplus. O percentual de proteção conferido foi calculado em função da variação entre os grupos tratados e controle, em relação ao número e peso de carrapatos que terminaram a alimentação, a capacidade de postura e a fertilidade dos ovos. O grupo dos bovinos vacinados com rGST-Hl mostrou uma redução de 53% e 52% no número e no peso de teleóginas, respectivamente. A postura de ovos e a eclodibilidade apresentaram uma redução no grupo vacinado de 0,6% e 8%, respectivamente. A eficácia total contra a infestação de R. microplus foi de 57%, quando comparado ao grupo controle. / The ticks Rhipicephalus (Boophilus) microplus and Haemaphysalis longicornis are hematophagous ectoparasites that infest bovines. Vaccine has been considered one of the most promising methods for the control of these parasites. Glutathione Stransferases (GSTs) enzymes are present in animal and vegetal organisms and are important in intracellular transport, digestive process, synthesis of prostaglandins, detoxification and protection against oxidative stress. In a previous study with mice we showed that H. longicornis GST (rGST-Hl) is more immunogenic than R. microplus GST (rGST-Bm). The objective of the present study was to investigate the crossprotection against R. microplus infestation in bovine vaccinated with rGST-Hl. By Western blot, the immunized bovines showed an increase in antibodies anti-rGST-Hl. The native GST-Bm in larvae, ovary and salivary gland were recognized by sera of bovines immunized with rGST-Hl, indicating that the antibodies induced by rGST-Hl immunization recognize native R. microplus GSTs. The bovines were challenged with 20,000 larvae of R. microplus. Protection was calculated by variation in number and weight of fully engorged ticks, egg laying capacity and egg fertility. The bovines vaccinated with rGST-Hl showed a reduction of 53% and 52% in number and weight of engorged ticks, respectively. Egg laying capacity and egg fertility of ticks from vaccinated bovines were respectively 0.6% and 8% lower, as compared to ticks from control animals. The overall efficacy ratio against R. microplus was 57.03%, as compared with the control group.

Made available in DSpace on 2014-06-11T19:35:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-23Bitstream added on 2014-06-13T20:46:13Z : No. of bitstreams: 1 barretto_lss_dr_jabo.pdf: 796760 bytes, checksum: 1b897563a911eb0bb996aa629ab300d1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo deste trabalho foi avaliar os efeitos da suplementação do meio de maturação com roscovitina, butirolactona I, cisteamina e b-mercaptoetanol na maturação oocitária e suas conseqüências na fecundação e desenvolvimento embrionário bovino in vitro, além da variabilidade da freqüência de danos ao DNA embrionário. Oócitos foram maturados a 38,8oC em atmosfera de 5% CO2 em ar em meio TCM-199 suplementado com 0,6% BSA, 0,5 mg FSH, 100 UI hCG e 1 mg estradiol/mL (controle - C), adicionado de 50 æM de cisteamina (CC) ou 50 æM de bmercaptoetanol (CM). O grupo padrão do laboratório (S) foi suplementado com 10% de SFB como fonte protéica. Os oócitos em meio suplementado com butirolactona I ou roscovitina foram pré-maturados por 24 horas em meio TCM-199 suplementado com 0,3% BSA, antibiótico e piruvato, de acordo com os grupos: 100 æM de butirolactona I (CB), 100 æM de butirolactona I + 50 æM de cisteamina (CBC), 100 æM de butirolactona I + 50 æM de b-mercaptoetanol (CBM), 25 æM de roscovitina (CR), 25 æM de roscovitina + 50 æM de cisteamina (CRC) e 25 æM de roscovitina + 50 æM de b-mercaptoetanol (CRM). Após o cultivo de pré-maturação, foram transferidos para gotas com meio controle para maturação por 8, 16 e 24 horas, para avaliação da maturação nuclear e citoplasmática, e dosagem da [GSHi]. A fecundação foi realizada após 24 horas do início do cultivo de maturação (grupos sem inibidores), e 20 horas (grupos com inibidores). O cultivo de desenvolvimento foi realizado em meio SOF-Modificado suplementado com 0,5% de BSA e 2,5% de SFB à temperatura de 38,8ºC por 192 horas. Observou-se que a butirolactona I é capaz de bloquear a maturação meiótica de maneira reversível e mais eficiente que a roscovitina, e que os antioxidantes não interferiram na retomada da maturação nuclear... / The aim of this work was to evaluate the effects of maturation media supplementation with roscovitine, butyrolactone I, cysteamine and - mercaptoethanol on oocyte maturation and the consequences on fertilization and bovine embryo development in vitro, and also DNA damage frequency in embryos. Oocytes were matured at 38.8°C under a 5% CO2 in air environment, in TCM-199 medium supplemented with 0.6% BSA, 0.5 mg FSH, 100 IU hCG and 1 mg estradiol/mL (control group - C) and with either 50 æM cysteamine (CC group) or 50 æM b-mercaptoethanol (CM group). The standard group of the laboratory (S group) was supplemented with 10% FCS as protein source. The oocytes in which groups were pre-incubated for 24 hours with butyrolactone I and roscovitine in TCM-199 medium supplemented with 0.3% BSA, antibiotic and pyruvate were culturedaccording to the groups: 100 æM butyrolactone I (CB), 100 æM butyrolactone I + 50 æM cysteamine (CBC), 100 æM butyrolactone I + 50 æM b-mercaptoethanol (CBM), 25 æM roscovitine (CR), 25 æM roscovitine + 50 æM cysteamine (CRC) and 25 æM roscovitine + 50 æM bmercaptoethanol (CRM). After the pre-incubation, the oocytes were transferred to the control maturation medium for 8, 16 and 24 hours for evaluation of nuclear and cytoplasmic maturation and [GSHi] dosage. For IVF, the oocytes were fertilized at 24 hours after the beginning of maturation (groups without inhibition), or 20 hours (groups with inhibition). Embryo culture was in SOF-Modified medium supplemented with 0.5% BSA and 2.5% FCS at 38.8ºC for 192 hours. We observed that butyrolactone I is capable of reversibly blocking meiotic maturation more effectively than roscovitine, and that addition of antioxidants did not interferewith resumption of nuclear maturation...(Complete abstract, acess undermentioned eletronic address)