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.

Made available in DSpace on 2015-04-11T13:54:30Z (GMT). No. of bitstreams: 1 Brena de Lourdes Aguiar Lima.pdf: 1248497 bytes, checksum: d14922a8c86943653012f0ea3e3818cf (MD5) Previous issue date: 2013-04-08 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Plasmodium vivax threatens ~40% of the world s population, with a wide spectrum of asymptomatic to severe clinical manifestations. Glutathione S-transferase (GST) gene polymorphisms have been shown to influence their ability to reduce oxidative stress. Using molecular tools we identified and compared GSTM1, GSTT1 and GSTP1 gene polymorphism in 175 uncomplicated (n=118) and severe (n=57) vivax patients. There were no differences in the frequency of GST alleles between uncomplicated and severe malaria. Nevertheless, a comparison of biochemical and hematological parameters in severe malaria, we observed patients with GSTM1 double-deletion had more platelets (p<0.04) suggesting a decreased risk of thrombocytopenia. In addition, patients with single and/or double deletion of GSTT1 and GSTM1 also had a reduced risk for thrombocytopenia (p<0.045, p<0.026, respectively) compared to wild type allele. In contrast, individuals with GSTP1 heterozygous or homozygous A313G mutation had an increased risk of jaundice (p<0.034, p<0.022) and anemia as observed with decreased RBCs (p<0.008), hemoglobin (p<0.019) and hematocrit (p<0.008) levels in serum. Our results, not only indicate a direct influence of GST polymorphism on biochemical parameters but also its diagnostic potential in assessing disease progression during clinical malaria. / Plasmodium vivax acomete aproximadamente 40% da população mundial, com um amplo espectro de manifestações clínicas, desde formas assintomáticas a infecções graves. Polimorfismos nos genes da Glutationa S-transferase (GST) influenciam a capacidade das isoenzimas de reduzir o estresse oxidativo. Usando métodos moleculares nós identificamos e comparamos polimorfismos nos genes da GST em 175 pacientes com malária vivax não-grave (n=118) e grave (n=57). Não houve diferenças estatísticas nas freqüências alélicas das GSTs entre os pacientes com malária não grave e grave. No entanto, guando comparou-se os parâmetros bioquímicos e hematológicos no grupo com malária grave, observamos que pacientes com deleção no gene GSTM1 apresentaram maior contagem plaquetária (p˂0.04), sugerindo menor risco de trombocitopenia. Além disso, pacientes com deleção única e/ou dupla nos genes GSTT1 e GSTM1 demonstraram menor risco de desenvolverem trombocitopenia (p˂0.045, p˂0.026, respectivamente) em comparação com os pacientes portadores do genótipo selvagem. Em contraste, indivíduos com malária grave e portadores dos genótipos heterozigotos ou homozigotos para a mutação A313G no gene GSTP1 apresentaram maior risco de desenvolverem icterícia (p˂0.034, p˂0.022, respectivamente) e anemia, como demonstrado pelos menores níveis de hemácias (p˂0.008, p˂0.019, respectivamente) e hematócrito (p˂0.008). Nossos resultados não indicam apenas uma influência direta dos polimorfismos das GSTs nos parâmetros bioquímicos e hematológicos, mas também o seu potencial de avaliar a progressão da doença.

Em insetos, as enzimas glutationa S-transferases (GSTs) são conhecidas pela capacidade de degradar inseticidas, pesticidas e outros compostos químicos, naturais ou não naturais, estranhos ao organismo, podendo também promover o transporte intracelular de hormônios, metabólitos, e atuar na proteção celular contra o estresse oxidativo. Além disto, a função de uma GST tem sido associada ao processo de sequestro, pelo corpo gorduroso, de um tipo de proteína (hexamerinas) estocada na hemolinfa larval para ser utilizada como fonte de aminoácidos durante a metamorfose. Os objetivos deste trabalho consistiram em caracterizar a estrutura, a expressão e aspectos da função dos genes codificadores de GSTs em abelhas operárias Apis mellifera, além de investigar a possível função de um destes genes, hp191(GSTS1), na dinâmica de sequestro de hexamerinas durante a metamorfose. A metodologia utilizada abrangeu técnicas de biologia molecular, como RT-PCR semiquantitativa e em tempo real, sequenciamento de nucleotídeos, western blot, silenciamento gênico. Resumidamente os resultados mostraram (1) diferenças estruturais (número e organização de íntrons e éxons) entre os dez genes GSTs de A. mellifera, (2) aumento da atividade destes genes relacionado ao envelhecimento e intensa atividade de forrageamento, (3) níveis de expressão dependente do tipo de dieta alimentar, (4) perfil de expressão de hp191(GSTS1), assim como sua resposta aos hormônios morfogenéticos (hormônio juvenil e 20-hidroxiecdisona), consistentes com função na metamorfose, (4) diminuição dos níveis de hexamerina HEX 70a na hemolinfa em consequência do silenciamento de hp191(GSTS1) mediado por RNAi. Em conjunto, estes dados informam sobre estrutura, expressão e função dos genes GSTs de A. mellifera com particular foco na potencial participação de hp191(GSTS1) na metamorfose. / In insects, the enzymes glutathione S-transferases (GSTs) are known for their ability to degrade insecticides, pesticides and other chemical compounds, natural or not, which are not normally produced or expected to be present in the organism. GSTs can also promote the intracellular transportation of hormones and metabolites as well as act in the cellular protection against oxidative stress. In addition, the GST function has been associated with the process of sequestration, by the fat body, of one type of protein (hexamerin) which is stored in the larval hemolymph to be used as a source of amino acids during metamorphosis. The aims of this study were (1) to characterize structure and expression, and explore the roles of the GST encoding genes in Apis mellifera worker bees and, (2) to investigate the potential role of one of these genes, hp191(GSTS1), in the dynamics of hexamerin sequestration during metamorphosis. The methodology included molecular biology techniques, such as semiquantitative and real time RT-PCR, gene sequencing and silencing, and western blot. Briefly, the results revealed structural differences (number and organization of introns and exons) among the ten GSTs genes of A. mellifera, increased activity of these genes associated to bee aging and the intense foraging activity, and modulation of the expression levels of GST genes by the type of diet. The results also revealed that the expression profile of hp191(GSTS1), as well as its response to the morphogenetic hormones (juvenile hormone and 20-hydroxyecdysone), are consistent with a function in metamorphosis. Furthermore, hp191(GSTS1) silencing mediated by RNAi resulted in decreased hemolymph levels of a hexamerin (HEX 70a) with an essential function in metamorphosis. Altogether, these data provide novel findings concerning the structure, expression and function of the GSTs genes of A. mellifera with a special focus on the potential participation of hp191(GSTS1) in metamorphosis.

Diabetes mellitus is a chronic autoimmune disease in which the immune system attacks the insulin-secreting ß-cells in the pancreas. It leads to an absolute deficiency of insulin. Chronic hyperglycemia induces increased production of reactive oxygen species, which leads to a decrease of natural antioxidant level in blood, and it contributes to genesis of diabetes complications (e.g. vascular or pulmonic). Moreover, the oxidative stress results in onset of pancreas inflammations and the damage of its ß-cells. Aims: Our aim was to assess whether or not certain genotypes or their combinations occur with higher frequency among groups of patients of type 1 diabetes (T1D) and type 2 diabetes and in a control group of healthy individuals. Methods: The study included groups of 40 T1D patients, 40 T2D patients and 45 healthy individuals. The polymorphisms of genes involved in the oxidative stress response were analyzedby using RFLP, PCR with TaqMan probes and allele specific PCR. The target genes involved superoxide dismutase SOD1 and SOD3 genes; glutathione-S-transferase GSTM1, GSTT1, GSTP1 genes; glutathioneperoxidase gene GPX1 and catalase gene CAT. The levels of plasma malondialdehyde were measured by using liquid chromatography. Results: Statistically significant differences were found in the...

Le mélanome malin (MM) est un cancer très réfractaire aux thérapies anticancéreuses, dont les vincalcaloïdes (VAs). Afin d'étudier le rôle de la GSTM1 (glutathion S-transférase 1) et la MRP1 (multidrug resistance protein 1) dans la résistance acquise du MM aux VAs, nous avons établi 4 modèles cellulaires de résistance à la vincristine (CAL1R-VCR), à la vindésine (CAL1R-VDS), à la vinorelbine (CAL1R-VRB) et à la vinflunine (CAL1R-VFL), par exposition continue de cellules du MM (CAL1-wt), pendant un an, à ces anticancéreux. L'expression d'ne GSTM1 fonctionnelle est spécifiquement observée (RT-PCR, western blot, activité GST totale) dans les cellules résistantes. Le curcumin (inhibiteur de GSTM1), la BSO (inhibiteur de synthèse de glutathion) et le MK571 (inhibiteur de MRP1), réduisent considérablement le résistance acquise à la VCR et à la VDS mais pas à la VRB ou à la VFL. Toutefois, tous ces VAs réduisent spécifiquement l'activité GSTM1. Ces données montrent l'implication différentielle de GSTM1 et MRP1 dans la résistance aux VAs. Pour déterminer les mécanismes moléculaires de cette chimiorésistance, nous avons réalisé une étude pangénomique (biopuces Affymetrix HG-U133 Plus 2.00) sur les lignées CAL1 (wt et R). Le regroupement hiérarchique (par Cluster et TreeView) des données des puces a révélé une similarité entre les profils d'expression génique de CAL1R-VRB et CAL1-wt mais aussi entre ceux de CAL1R-VCR et CAL1R-VDS. L'analyse bioinformatique (par IPA) des transcrits les plus différemment exprimés entre les lignées cellulaires, a mis en évidence 6 réseaux géniques connus pour leur rôle dans la chimiorésistance tumorale. Le programme FatiGO a révélé 3 termes biologiques sur-représentés (> 60%) dans CAL1R (ribosome, filaments intermédiaires du cytosquelette, récepteurs olfactifs) tandis que l'étude fonctionnelle (invalidation génique par siRNA, test de viabilité) de GPR143, KIT et SLC45A2 (gènes interagissant avec NF-κB et CCND1 (facteurs de la chimiorésistance tumorale), très exprimés dans CAL1-wt et muets dans CAL1R) a montré la faible tendance des deux premiers à être impliqués dans la résistance aux VAs. / Malignant melanoma (MM) is a very refractory tumor to anticancer therapies, including vinca alkaloïds (VAs). To investigate the role of GSTM1 (glutathione S-transferase μ1) and MRP1 (multidrug resistance protein 1) in MM acquired resistance to VAs, we established 4 cellular models of resistance to vincristine (CAL1R-VCR), to vindesine (CAL1R-VDS), to vinorelbine (CAL1R-VRB) and to vinflunine (CAL1R-VFL), by continuous exposure of MM cells (CAL1-wt), for one year, to these anticancer agents. The expression of a functional GSTM1 is specifically observed (RT-PCR, western blot, total GST activity) in resistant cells. Curcumin (GSTM1 inhibitor), BSO (glutathione synthesis inhibitor) and MK571 (MRP1 inhibitor), considerably reduce the acquired resistance to VCR and VDS but not that to VRB or VFL. However, all these VAs specifically reduce GSTM1 activity. These data show the differential involvement of GSTM1 and MRP1 in resistance to VAs. To determine the molecular mechanisms of this chemoresistance, we performed a pangenomic study (Affymetrix HG-U133 Plus 2.00 microarrays) on the CAL1 lines (wt and R). The hierarchical clustering (by Cluster and TreeView) of array data revealed a similarity between the gene expression profiles of CAL1R-VRB and CAL1-wt, but also between those of CAL1R-VCR and CAL1R-VDS. The bioinformatic analysis (by IPA) of the most differentially expressed transcripts between cell lines, highlighted 6 gene networks known for their role in tumor chemoresistance. FatiGO program revealed 3 biological terms overrepresented (>60%) in CAL1R (ribosome, intermediate filaments of cytoskeleton, olfactory receptors), while functional study (gene invalidation by siRNA, viability test) of GPR143, KIT and SLC45A2 (genes interacting with NF-kB and CCND1 (tumor chemoresistance factors), highly expressed in CAL1-wt and mute in CAL1R) showed the weak trend of the two formers to be involved in resistance to VAs.

O carcinoma epidermóide de cabeça e pescoço (CECP) é considerado um dos tumores mais freqüentes em países em desenvolvimento, detectado quase sempre em estádios mais avançados, com poucas opções eficazes de tratamento, o que diminui a taxa de sobrevida para cinco anos em 50%. Os principais fatores de risco associados são consumo de tabaco e de álcool, seguidos da dieta e infecções virais. Não parecem existir até o momento métodos de vigilância, biomarcadores moleculares ou quimioprevenção que tenham se mostrado eficientes. A identificação de polimorfismos em genes de metabolização do tabaco e do álcool tem sido sugerida na busca de marcadores de susceptibilidade individual ao CECP. Dentre as enzimas envolvidos na fase I de metabolização incluem-se as da família do citocromo P450 (CYPs) e da fase II de metabolização as enzimas glutationa S-transferases (GSTs). Incluem-se também os polimorfismos em genes de reparo que podem alterar o risco para desenvolvimento CECP, sobretudo nos indivíduos consumidores de álcool e tabaco. Polimorfismos em CYPs e GSTs podem alterar a dinâmica de metabolização e excreção de carcinógenos aumentando o risco de mutações como adutos de DNA e conseqüentemente o câncer. No presente estudo foram avaliados polimorfismos em genes da fase I (CYP1A1 MspI, CYP2E1 PstI) da fase II (GSTM1, GSTT1, GSTP1 BsmA) e no gene de reparo XRCC1, em 207 pacientes portadores de CECP, dos quais 92 (44%) com carcinoma epidermóide da cavidade bucal (CECB), e em 244 controles, selecionados no mesmo hospital. A identificação dos polimorfismos a partir do DNA de linfócitos periféricos dos pacientes e controles (PCR-RFLP) revelou maior freqüência do genótipo GSTM1 nulo em pacientes com CECP (53,2%) quando comparados aos controles (37,7%), aumentando em aproximadamente cinco vezes o risco da doença (OR = 4,75; IC 95%, 3,06-7,40). Resultados semelhantes para o genótipo GSTM1 nulo foram encontrados nos casos de CECB (OR = 2,15; IC 95%, 1,28-3,6). Verificou-se também aumento no risco de CECB com o genótipo XRCC1-194Trp (OR = 2,33; IC 95%, 1,08-4,98) e proteção associada ao genótipo XRCC1-399Gln (OR, 0.35; IC 95%, 0.12-0.96) que foi mais prevalente nos controles do que nos pacientes com CECB. A presença simultânea de dois genes desfavoráveis (associação gene-gene) aumentou em torno de duas vezes o risco de CECB quando associados GSTM1-CYP1A1 (OR=1,93; IC 95%, 1,1-3,3), GSTM1-CYP2E1 (OR=2,2; IC 95%, 1,36-3,87), GSTM1-XRCC1-194 (OR=2,44; IC 95%, 1,44-4,14) e CYP2E1-XRCC1-194 (OR=2,0; IC 95%, 1,1 3,62). Quando considerada na análise a interação gene-ambiente verificamos que pacientes com CECB que consumiam acima de 39 maços/ano tinham risco aumentado de câncer associado aos genótipos GSTP1 BmsA (OR = 5,0; IC 95%, 1,9 -12,4) e acima de 20 maços/ano com o genótipo CYP1A1MstI (OR = 53,7; IC 95%, 1,0 -14,2). Por outro lado, o consumo acima de 30 g/l/d, associado ao genótipo XRCC1-194 aumentou oito vezes o risco de CECB (OR = 8,8,; IC 95%, 1,3-45,7) e o genótipo XRCC1-399 parece ter sido um fator de proteção ao CECB mesmo com consumo de álcool acima de 5 a 30 g/l/d (OR = 0,1; IC 95%, 0,03 0,7). Os resultados obtidos parecem sugerir uma contribuição dos polimorfismos genéticos de metabolização do álcool e do tabaco no risco de CECP e da cavidade bucal. A identificação de marcadores genéticos de suscetibilidade individual ao câncer pode auxiliar o médico na indicação de medidas de prevenção e de controle no acompanhamento de pacientes ou da população de risco ao CECP, principalmente fumantes e alcoolistas crônicos. / The squamous cell carcinoma of the head and neck (HNSCC) is considered one of the most prevalent cancers in developing countries, detected nearly always in well-advanced phases with few effective treatment options, which reduce the survival rate to five years in 50% of the patients. The main risk factors associated with it are tobacco and alcohol intake, followed by diet and viral infections. For the moment, there seems not to be vigilance methods, molecular biomarkers or chemioprevention that have been shown to be efficient. The identification of polymorphisms in tobacco and alcohol metabolizing genes has been suggested in the search for markers of individual susceptibility to the HNSCC. Among the enzymes involved in the metabolizing phase I are those belonging to the P450 cytochrome family (CYPs) while in the metabolizing phase II are included the glutathione S-transferases (GSTs). The repair gene polymorphisms which may alter the risk of developing HNSCC, especially in alcohol and tobacco consumers are also included. Polymorphisms in CYPs and GSTs may alter the metabolizing dynamics and carcinogen excretion increasing the risk of mutations like DNA adducts and, as a consequence, the risk of cancer. In the present study, polymorphisms in phase I genes (CYP1A1 MspI, CYP2E1 PstI) and in Phase II genes (GSTM1, GSTT1, GSTP1 BsmA) were evaluated, and the repair gene XRCC1 as well, in 207 HNSCC carriers, 92 of them (44%) with oral squamous cell carcinoma (OSCC), and also in 244 controls selected in the same hospital. The identification of polymorphisms from the DNA of peripheral lymphocytes in patients and control subjects (PCR-RFLP) showed greater prevalence of the GSTM1 null genotype which was higher in patients with HNSCC (53.2%) when compared to the controls (37.7%), increasing about five times as much the risk for the disease (OR = 4.75; 95% CI, 3.06-7.40). Similar results for the GSTM1 null genotype were found in the OSCC (OR = 2.15; 95% CI, 1.28-3.6). It was also observed that there was a rise in the risk for OSCC with the XRCC1-194Trp (OR = 2.33; 95% CI, 1.08-4.98) genotype and some protection associated with the XRCC1-399Gln (OR, 0.35; 95% CI, 0.12-0.96) genotype, which was more prevalent in the control subjects than in the OSCC patients. The simultaneous presence of two unfavourable genes (gene-gene association) approximately doubled the risk for OSCC when GSTM1-CYP1A1 (0R=1.93; 95% CI, 1.1-3.3), GSTM1-CYP2E1 (0R=2.2; CI 95%, 1.36-3.87), GSTM1-XRCC1-194 (OR=2.44; CI 95%, 1.44-4.14) and CYP2E1-XRCC1-194 (OR=2.0; CI 95%, 1.1 3.62) were associated. When the interaction gene-environment was considered in the analysis, it was found that OSCC patients who were used to consuming above 39 cigarette packs a year had an increase in the risk of getting cancer associated with the GSTP1 BmsA (OR=5.0; CI 95%, 1.9-12.4) genotypes and above 20 packs a year associated with the CYP1A1MstI (OR = 53.7; CI 95%, 1.0 - 14.2) genotype. On the other hand, the above-30-g/l/d consumption associated with the XRCC1-194 genotype raised eight times as much the risk for ECOC (OR=8,8; CI 95%, 1.3-45.7) and the XRCC1-399 genotype seems to have been a protection factor against ECOC even with alcohol consumption above 5 to 30 g/l/d (OR=0.1; CI 95%, 0.03 0.7). The results obtained seem to suggest a contribution of the genetic polymorphisms of the alcohol-and-tobacco-related metabolizing enzymes to HNSCC and the OSCC. The identification of genetic markers of individual susceptibility to cancer may help the clinician with the indication of prevention and control measures for the medical follow-up of patients or the population at risk of contracting SCCHN, mainly chronic smokers and alcoholics.

Made available in DSpace on 2017-07-21T20:00:00Z (GMT). No. of bitstreams: 1 Luiz Ricardo Olchanheski.pdf: 1069422 bytes, checksum: 4a8faf74d88ea768a689fc8a9a055a56 (MD5) Previous issue date: 2014-02-25 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / The intensive use of agrochemicals has assumed an important role in increasing agricultural production; however, one of the impacts has been changes in population structure of microbiota in the soil, which need to tolerate the xenobiotics that are applied in order to survive. A pertinent question is whether these mechanisms are selected by the presence of different agrochemicals in soils, or if there are constitutive mechanisms of adaptation. The aim of this study was to evaluate the defense system of an un-environmental strain, E. coli DH5-α, in relation to the herbicide mesotrione, which had no prior contact with the soil. The results showed that this strain was able to tolerate higher doses of the herbicide, and that the determination of the herbicide by a method developed and validated by high performance liquid chromatography (HPLC), made it possible to determine the complete disappearance of mesotrione in the sample after 3 h of exposure. Growth rates in the treatment with the herbicide were lower than the control, prior to the period of degradation, showing the toxic effect on the bacterial cells. As regards defense systems, it was noted that changes in the saturation of the membrane lipids reduced the damage caused by reactive oxygen species and possibly hindered the entry of xenobiotics in the cell, as well as activating GST enzyme activity in the antioxidant system and in the metabolizing process of the herbicide. Considering that E. coli DH5-α showed no previous contact with mesotrione, the defense system found in this strain can be considered as general and non-specific. This strategy may be interesting in the adaptation of bacterial strains in agricultural soils, which are subject to changing herbicides in an ever more intense manner. / O uso intensivo de agroquímicos tem assumido um papel relevante no aumento da produção agrícola, mas um dos impactos gerados é a mudança na estrutura populacional de microbiotas de solo, que precisam tolerar os xenobióticos aplicados para sobreviver. Uma pergunta pertinente é se os mecanismos de tolerância são selecionados pela presença de diferentes agroquímicos em solo, ou se há mecanismos constitutivos de adaptação. O objetivo deste trabalho foi avaliar o sistema de defesa de uma linhagem não ambiental, a E. coli DH5-α, em relação ao herbicida mesotrione, com o qual não houve contato prévio em solo. Resultados obtidos mostraram que esta linhagem foi capaz de tolerar elevadas doses do herbicida, e a determinação do herbicida por um método desenvolvido e validado por cromatografia líquida de alta eficiência (CLAE) permitiu determinar que após 3 horas não foi possível detectar o herbicida nas amostras avaliadas. Taxas de crescimento no tratamento com o herbicida foram menores em relação ao controle nos tempos anteriores ao período de degradação, mostrando o efeito tóxico sobre as células bacterianas. Como sistemas de defesa, constataram-se a alteração na saturação dos lipídeos de membrana, diminuindo os danos causados pelas espécies ativas de oxigênio e possivelmente dificultando a entrada do xenobiótico na célula, além da atuação da enzima GST no sistema antioxidante e no processo de metabolização do herbicida. Considerando que a E. coli DH5-α não apresentou um contato prévio com o mesotrione, o sistema de defesa encontrado nesta linhagem pode ser considerado como geral e não específico. Esta estratégia pode ser interessante na adaptação de linhagens bacterianas em solos agrícolas, os quais são submetidos a regimes de trocas de herbicidas de maneira cada vez mais intensa.

O carcinoma epidermóide de cabeça e pescoço (CECP) é considerado um dos tumores mais freqüentes em países em desenvolvimento, detectado quase sempre em estádios mais avançados, com poucas opções eficazes de tratamento, o que diminui a taxa de sobrevida para cinco anos em 50%. Os principais fatores de risco associados são consumo de tabaco e de álcool, seguidos da dieta e infecções virais. Não parecem existir até o momento métodos de vigilância, biomarcadores moleculares ou quimioprevenção que tenham se mostrado eficientes. A identificação de polimorfismos em genes de metabolização do tabaco e do álcool tem sido sugerida na busca de marcadores de susceptibilidade individual ao CECP. Dentre as enzimas envolvidos na fase I de metabolização incluem-se as da família do citocromo P450 (CYPs) e da fase II de metabolização as enzimas glutationa S-transferases (GSTs). Incluem-se também os polimorfismos em genes de reparo que podem alterar o risco para desenvolvimento CECP, sobretudo nos indivíduos consumidores de álcool e tabaco. Polimorfismos em CYPs e GSTs podem alterar a dinâmica de metabolização e excreção de carcinógenos aumentando o risco de mutações como adutos de DNA e conseqüentemente o câncer. No presente estudo foram avaliados polimorfismos em genes da fase I (CYP1A1 MspI, CYP2E1 PstI) da fase II (GSTM1, GSTT1, GSTP1 BsmA) e no gene de reparo XRCC1, em 207 pacientes portadores de CECP, dos quais 92 (44%) com carcinoma epidermóide da cavidade bucal (CECB), e em 244 controles, selecionados no mesmo hospital. A identificação dos polimorfismos a partir do DNA de linfócitos periféricos dos pacientes e controles (PCR-RFLP) revelou maior freqüência do genótipo GSTM1 nulo em pacientes com CECP (53,2%) quando comparados aos controles (37,7%), aumentando em aproximadamente cinco vezes o risco da doença (OR = 4,75; IC 95%, 3,06-7,40). Resultados semelhantes para o genótipo GSTM1 nulo foram encontrados nos casos de CECB (OR = 2,15; IC 95%, 1,28-3,6). Verificou-se também aumento no risco de CECB com o genótipo XRCC1-194Trp (OR = 2,33; IC 95%, 1,08-4,98) e proteção associada ao genótipo XRCC1-399Gln (OR, 0.35; IC 95%, 0.12-0.96) que foi mais prevalente nos controles do que nos pacientes com CECB. A presença simultânea de dois genes desfavoráveis (associação gene-gene) aumentou em torno de duas vezes o risco de CECB quando associados GSTM1-CYP1A1 (OR=1,93; IC 95%, 1,1-3,3), GSTM1-CYP2E1 (OR=2,2; IC 95%, 1,36-3,87), GSTM1-XRCC1-194 (OR=2,44; IC 95%, 1,44-4,14) e CYP2E1-XRCC1-194 (OR=2,0; IC 95%, 1,1 3,62). Quando considerada na análise a interação gene-ambiente verificamos que pacientes com CECB que consumiam acima de 39 maços/ano tinham risco aumentado de câncer associado aos genótipos GSTP1 BmsA (OR = 5,0; IC 95%, 1,9 -12,4) e acima de 20 maços/ano com o genótipo CYP1A1MstI (OR = 53,7; IC 95%, 1,0 -14,2). Por outro lado, o consumo acima de 30 g/l/d, associado ao genótipo XRCC1-194 aumentou oito vezes o risco de CECB (OR = 8,8,; IC 95%, 1,3-45,7) e o genótipo XRCC1-399 parece ter sido um fator de proteção ao CECB mesmo com consumo de álcool acima de 5 a 30 g/l/d (OR = 0,1; IC 95%, 0,03 0,7). Os resultados obtidos parecem sugerir uma contribuição dos polimorfismos genéticos de metabolização do álcool e do tabaco no risco de CECP e da cavidade bucal. A identificação de marcadores genéticos de suscetibilidade individual ao câncer pode auxiliar o médico na indicação de medidas de prevenção e de controle no acompanhamento de pacientes ou da população de risco ao CECP, principalmente fumantes e alcoolistas crônicos. / The squamous cell carcinoma of the head and neck (HNSCC) is considered one of the most prevalent cancers in developing countries, detected nearly always in well-advanced phases with few effective treatment options, which reduce the survival rate to five years in 50% of the patients. The main risk factors associated with it are tobacco and alcohol intake, followed by diet and viral infections. For the moment, there seems not to be vigilance methods, molecular biomarkers or chemioprevention that have been shown to be efficient. The identification of polymorphisms in tobacco and alcohol metabolizing genes has been suggested in the search for markers of individual susceptibility to the HNSCC. Among the enzymes involved in the metabolizing phase I are those belonging to the P450 cytochrome family (CYPs) while in the metabolizing phase II are included the glutathione S-transferases (GSTs). The repair gene polymorphisms which may alter the risk of developing HNSCC, especially in alcohol and tobacco consumers are also included. Polymorphisms in CYPs and GSTs may alter the metabolizing dynamics and carcinogen excretion increasing the risk of mutations like DNA adducts and, as a consequence, the risk of cancer. In the present study, polymorphisms in phase I genes (CYP1A1 MspI, CYP2E1 PstI) and in Phase II genes (GSTM1, GSTT1, GSTP1 BsmA) were evaluated, and the repair gene XRCC1 as well, in 207 HNSCC carriers, 92 of them (44%) with oral squamous cell carcinoma (OSCC), and also in 244 controls selected in the same hospital. The identification of polymorphisms from the DNA of peripheral lymphocytes in patients and control subjects (PCR-RFLP) showed greater prevalence of the GSTM1 null genotype which was higher in patients with HNSCC (53.2%) when compared to the controls (37.7%), increasing about five times as much the risk for the disease (OR = 4.75; 95% CI, 3.06-7.40). Similar results for the GSTM1 null genotype were found in the OSCC (OR = 2.15; 95% CI, 1.28-3.6). It was also observed that there was a rise in the risk for OSCC with the XRCC1-194Trp (OR = 2.33; 95% CI, 1.08-4.98) genotype and some protection associated with the XRCC1-399Gln (OR, 0.35; 95% CI, 0.12-0.96) genotype, which was more prevalent in the control subjects than in the OSCC patients. The simultaneous presence of two unfavourable genes (gene-gene association) approximately doubled the risk for OSCC when GSTM1-CYP1A1 (0R=1.93; 95% CI, 1.1-3.3), GSTM1-CYP2E1 (0R=2.2; CI 95%, 1.36-3.87), GSTM1-XRCC1-194 (OR=2.44; CI 95%, 1.44-4.14) and CYP2E1-XRCC1-194 (OR=2.0; CI 95%, 1.1 3.62) were associated. When the interaction gene-environment was considered in the analysis, it was found that OSCC patients who were used to consuming above 39 cigarette packs a year had an increase in the risk of getting cancer associated with the GSTP1 BmsA (OR=5.0; CI 95%, 1.9-12.4) genotypes and above 20 packs a year associated with the CYP1A1MstI (OR = 53.7; CI 95%, 1.0 - 14.2) genotype. On the other hand, the above-30-g/l/d consumption associated with the XRCC1-194 genotype raised eight times as much the risk for ECOC (OR=8,8; CI 95%, 1.3-45.7) and the XRCC1-399 genotype seems to have been a protection factor against ECOC even with alcohol consumption above 5 to 30 g/l/d (OR=0.1; CI 95%, 0.03 0.7). The results obtained seem to suggest a contribution of the genetic polymorphisms of the alcohol-and-tobacco-related metabolizing enzymes to HNSCC and the OSCC. The identification of genetic markers of individual susceptibility to cancer may help the clinician with the indication of prevention and control measures for the medical follow-up of patients or the population at risk of contracting SCCHN, mainly chronic smokers and alcoholics.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-08-14T18:09:29Z No. of bitstreams: 1 LucianaFarias.pdf: 24712291 bytes, checksum: d8760f57e945d0cde298c31a44b38539 (MD5) / Made available in DSpace on 2017-08-14T18:09:29Z (GMT). No. of bitstreams: 1 LucianaFarias.pdf: 24712291 bytes, checksum: d8760f57e945d0cde298c31a44b38539 (MD5) Previous issue date: 2009-11-04 / This work is discussed the conceptual model of a multi-agent system for environmental monitoring with the use of biomarkers of aquatic organisms present in the port complex of São Luís-Maranhão-Brasil, second most important in the country in cargo handling. Located in the São Marcos Bay, this complex have an estuarine ecosystems which have suffered attacks in the current process of economic development, caused by intense port handling, dumping of ballast water and washing the vessels, overfishing, introduction of exotic species in the middle pollution in urban and industrial effluents, subject to severe environmental impacts that should be monitored. Methodologically, the modeling of the monitoring solution, we used the existing environmental conditions and aquatic life caught in two different sites of the port complex, the first in a potentially contaminated area and the second in a contamination-free (control), proposing the creation of a network of sensors in these locations. Invest conceded data by Carvalho-Neta (2007) whose research includes to catch fish in these perimeters, then submitting them for laboratory analysis to measure the enzyme activity of glutathione S-transferase (GST) and Catalase (CAT), the result was processed and recorded in bio-ontology . The core of the solution of Multi-agent system is based on the results derived from the biochemical analysis of GST, inspiring the modeling software agent that simulates the behavior of this enzyme. The solution also includes an application running on mobile devices that makes the collection of environmental variables in the selected points, validates them and makes the inference of those who could not be collected. Multi-agent System for Environmental Monitoring of the Port Complex of the Island of São Luís-Maranhão- Brasil, therefore, is made up of the bio-ontology, sensor networking, mobile application collection and inference of data from environmental conditions, software agents to simulate biochemical analysis, calculation of GST activity and other staff related to the maintenance and security of the SMA. / Nesta dissertação é discutido o modelo conceitual de um sistema multiagente para monitoramento ambiental com uso de marcadores biológicos de organismos aquáticos presentes no complexo portuário de São Luís-MA, segundo mais importante do país em movimentação de carga. Situado na Baía de São Marcos, esse complexo possui um dos ecossistemas estuarinos que mais têm sofrido agressões no atual processo de desenvolvimento econômico, provocadas pela intensa movimentação portuária, despejo de água de lastro e lavagem dos navios, pesca predatória, introdução de espécies exóticas no meio, poluição por efluentes domésticos e industriais, sujeitando o ambiente a fortes impactos ambientais que devem ser monitorados. Metodologicamente, na modelagem da solução de monitoramento, utilizou-se o registro das condições ambientais e de organismos aquáticos capturados em dois pontos distintos do complexo portuário: o primeiro, em uma área potencialmente contaminada e o segundo em uma livre de contaminação (controle), propondo-se a criação de uma rede de sensores nesses locais. Empregou-se dados cedidos por Carvalho-Neta (2007) cuja pesquisa contou com a captura de peixes nesses perímetros, submetendo-os posteriormente a análise laboratorial para medição da atividade enzimática da Glutationa s- Transferase (GST) e Catalase (CAT), tendo todos os resultados processados e registrados em bio-ontologia. O núcleo da solução do Sistema Multiagente baseia-se nos resultados oriundos da análise bioquímica da GST, inspirando a modelagem de agente de software que simula o comportamento desta enzima. Todos esses dados foram registrados em bio-ontologia. A solução contempla também uma aplicação executada em dispositivos móveis que realiza a coleta das variáveis abióticas nos pontos selecionados, valida-as e realiza a inferência daquelas que não puderam ser coletadas. O Sistema Multiagente para Monitoramento Ambiental do Complexo Portuário da Ilha de São Luís, portanto, é constituído pelo conjunto da bio-ontologia, rede de sensores, aplicação móvel de coleta e inferência de dados das condições do meio ambiente, agentes de software para simulação de análise bioquímica, cálculo da atividade da GST e outros agentes relacionados à manutenção e segurança do SMA.

ABSTRACT MICROARRAY APPLICATIONS FOR DETERMINATION OF THE EFFECTS OF EMODIN ON BREAST CANCER CELL LINES Ekenel Qomi, Emilia M.S., Department of Biotechnology Supervisor: Prof. Dr. Mesude Iscan Co-Supervisor: Assoc. Prof. Dr. Nursen &Ccedil / oruh February 2012, 191 pages Cancer is a genetic disease that is characterized by uncontrolled cells growth. Breast cancer is a type of cancer originating from breast tissue. Some breast cancers are sensitive to hormones such as estrogen which makes it possible to treat them by blocking the effects of these hormones in the target tissues. These require less aggressive treatment than hormone negative cancers. Breast cancers without hormone receptors, are higher-risk, and are treated more aggressively. The aim of our study is to investigate the effect of emodin on MCF-7 which is ER (estrogen receptor) positive, and MDA-MB-231 (ER negative) cancerous cell lines. Emodin which is a phytoestrogen component, extracted from rheum (genus) plant, has been reported to suppress the growth of tumor in some clinical situation, and it&rsquo / s found that emodin induced apoptosis through the decrease of Bcl-2/Bax ratio and the increase of cytoplasm cytochrome c concentration in human breast cancer Bcap-37 cells. Comparing the effect of emodin between ER positive and ER negative cells at the molecular level was investigated by Microarray analysis of gene expressions using Affymetrix Human Genome U133 plus 2.0 Array. The microarray data analysis was performed by using BRB-Array Tools, v.4.2.0. GST and its classes / Alpha, Mu, Pi, Theta, Sigma, Omega, Zeta and Kappa is our interested genes because of its role in regulating susceptibility to cancer, by their ability to metabolize reactive electrophilic intermediates to usually less reactive and more water soluble glutathione conjugates. And also its have a role in detoxifying the damage caused by oxidative stress which is a result of the radiotherapy. v The differentially expressed genes from emodin treated and untreated control breast cancer cell lines were compared after normalization and filtering and annotated, it was shown that the top 10 highly (significantly) varied genes belong to the biological processes such as (namely) cell cycle, cell division, cell proliferation, mitosis and meiosis, this insure the relation of emodin to the cell growth processes in the cancerous cells. The analysis of the change on the cell growth confirmed the anti-tumor effect of emodin. About the effect of emodin treatment on MCF-7 and MDA-MB-231 cancerous cell lines separately / Both cells its significant genes was belong to cell growth biological processes, in MCF-7 cells in-addition other biological processes was shown, for example / stimulus to estradoil response, and the metabolism of xenobiotic by cytochrome p450, so CYP1A1 gene code for a protein which is used in emodin metabolism. The varied gene number was nearly 4400 gene from the scatter plot result in MCF-7 cells while in MDA-MB-231 cells it was nearly 3400 gene, these result insured the effect of emodin as a phytoestrogenic component as MCF-7 cells are ER positive cells, so emodin bind to the ER in MCF-7 cells and affected more gene number than MDA-MB-231. More number of GST enzyme classes changed in MCF-7 cells than MDA-MB-231, and the effect of emodin as anti-cancer showed different change of GST genes between MCF-7 and MDA-MB-231. The results confirmed by network analysis done, to find the most related genes to our top 10 regulated gene list, and these genes were analyzed / most of them where in our gene list, and their regulation after emodin treatment analyzed and the result was supported to emodin as anti-tumor and phytoestrogenic component.