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.

Occupational exposure-related hearing loss is a significant health concern for affected workers. Organic solvent exposure has emerged as an important contributor to hearing loss. It is thought that hearing loss related to solvent and noise exposure is mediated by reactive oxygen species (ROS). The glutathione associated enzymes and the manganese superoxide dismutase enzymes (SOD2) are important components of the cochlear hair cellâs defense against oxidative stress. This study is aimed to determine whether polymorphisms within the glutathione S-transferases (GST) P1 and GSTM1, glutathione peroxidase 1 (GPX1), and SOD2 are associated with hearing status in solvent exposed factory workers. Genotypes for the GSTM1 + vs. null, GSTP1 Ile105Val, GPX1 Pro198Leu, SOD2 Val16Ala polymorphisms along with hearing status were determined in factory workers exposed to organic solvents. Hearing tests consisted of pure tone audiometric (PTA) thresholds from 3-6 kHz and distortion product otoacoustic emissions (DPOAEs) for 3-6 kHz. Bivariate and multivariate regression analysis was undertaken to assess for association between polymorphisms and hearing outcomes. The GSTP1 Val/Ile genotype at position 105 was associated with higher PTA thresholds (β=12.41, P value= 0.01) from 3-6 kHz in workers below age 22-43. The analysis showed a protective association of the SOD2 Ala/Val genotype (β= -26.42, P value= 0.025) and The GPX1 Leu/Leu genotypes (β=47.81, P value= 0.034) with audiometric thresholds from 3-6 kHz in individuals above age 43. This small cross-sectional study suggests that polymorphisms within the antioxidant system may alter susceptibility to hearing loss in workers exposed to organic solvents. These results also suggest the mechanisms by which this affect are mediated are complex and should be further investigated.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

A virus-like particle (VLP) is a biological nanoparticle. It consists of the protective protein shell of a virus that is devoid of the nucleic acid required for viral replication. VLPs have two key uses: they can act as vaccines by inducing an immune response similar to their native virions, or they can facilitate gene therapy and drug delivery by encapsulating non-viral molecules and efficiently transporting them into cells. Manufacture of VLPs involves cell-based expression of virus-shell protein, with particle assembly and purification following one of two paradigms: (i) in vivo VLP assembly, followed by purification of full particles from cell lysate; (ii) partially assembled protein is recovered from cell lysate and assembled into VLPs in vitro. The flexibility and efficiency of both of these VLP manufacturing paradigms can be improved by first gaining a fundamental understanding of what is happening at key process steps. These improvements will lower the cost of VLP manufacture and enhance the viability of VLP products in the biopharmaceutical marketplace. The research reported here yielded positive outcomes for two key steps of the VLP manufacturing process, using murine polyomavirus VLPs for all experimentation. Firstly, enhanced understanding concerning the capture of virus shell protein in pentamer form (capsomeres) from cell lysate using glutathione-S-transferase (GST) affinity chromatography was obtained. It was discovered that prokaryotic expression of GST-tagged capsomeres yielded soluble aggregates having variable size distribution. Methods were developed to physically and chemically characterise these soluble aggregates, and the mechanism by which they adsorb to the chromatography resin was described using an established mathematical model. Secondly, particle characterisation of whole VLPs isolated from cell lysate was undertaken. Methods utilizing three orthogonal and quantitative techniques were developed to suggest that encapsulation of non-viral molecules (nucleic acids or proteins) during in vivo assembly causes distinct changes to the size distribution of isolated VLPs: transmission electron microscopy (TEM), asymmetrical flow field-flow fractionation with multiple-angle light scattering (AFFFF-MALS) and electrospray differential mobility analysis (ES-DMA). The understanding gained from the research presented in this work enables the enhanced capture of partially assembled virus shell protein from cell lysate, as well as a method to efficiently and cost-effectively analyse VLP solutions for the presence of desirable or undesirable encapsulated material.

A virus-like particle (VLP) is a biological nanoparticle. It consists of the protective protein shell of a virus that is devoid of the nucleic acid required for viral replication. VLPs have two key uses: they can act as vaccines by inducing an immune response similar to their native virions, or they can facilitate gene therapy and drug delivery by encapsulating non-viral molecules and efficiently transporting them into cells. Manufacture of VLPs involves cell-based expression of virus-shell protein, with particle assembly and purification following one of two paradigms: (i) in vivo VLP assembly, followed by purification of full particles from cell lysate; (ii) partially assembled protein is recovered from cell lysate and assembled into VLPs in vitro. The flexibility and efficiency of both of these VLP manufacturing paradigms can be improved by first gaining a fundamental understanding of what is happening at key process steps. These improvements will lower the cost of VLP manufacture and enhance the viability of VLP products in the biopharmaceutical marketplace. The research reported here yielded positive outcomes for two key steps of the VLP manufacturing process, using murine polyomavirus VLPs for all experimentation. Firstly, enhanced understanding concerning the capture of virus shell protein in pentamer form (capsomeres) from cell lysate using glutathione-S-transferase (GST) affinity chromatography was obtained. It was discovered that prokaryotic expression of GST-tagged capsomeres yielded soluble aggregates having variable size distribution. Methods were developed to physically and chemically characterise these soluble aggregates, and the mechanism by which they adsorb to the chromatography resin was described using an established mathematical model. Secondly, particle characterisation of whole VLPs isolated from cell lysate was undertaken. Methods utilizing three orthogonal and quantitative techniques were developed to suggest that encapsulation of non-viral molecules (nucleic acids or proteins) during in vivo assembly causes distinct changes to the size distribution of isolated VLPs: transmission electron microscopy (TEM), asymmetrical flow field-flow fractionation with multiple-angle light scattering (AFFFF-MALS) and electrospray differential mobility analysis (ES-DMA). The understanding gained from the research presented in this work enables the enhanced capture of partially assembled virus shell protein from cell lysate, as well as a method to efficiently and cost-effectively analyse VLP solutions for the presence of desirable or undesirable encapsulated material.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-20T20:46:00Z No. of bitstreams: 1 Joelma Figueiredo Menezes Estudo de polimorfismos....pdf: 11425878 bytes, checksum: 175372593b635c3bd50b3a10af52fc30 (MD5) / Made available in DSpace on 2012-07-20T20:46:00Z (GMT). No. of bitstreams: 1 Joelma Figueiredo Menezes Estudo de polimorfismos....pdf: 11425878 bytes, checksum: 175372593b635c3bd50b3a10af52fc30 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Indivíduos com doença falciforme (DF) apresentam estresse oxidativo (EO) constante, que é decorrente da deficiência nos sistemas antioxidantes, caracterizados pela produção de espécies reativas do oxigênio (EROS) de origem multifatorial, principalmente geradas pelo processo hemolítico. Objetivo. Investigar polimorfismos relacionados ao estresse oxidativo nos genes HFE da hemocromatose hereditária (c.282C>Y, c.63H>D e c.65S>C); haptoglobina (HP), glutationa S-transferase (GST) (GSTT1 e GSTM1) e paraoxonase (PON1) (c.55L>M e c.192Q>R), associando-os aos dados hematológicos e bioquímicos, aos níveis séricos de vitamina C, paraoxonase 1 e receptor de transferrina e histórico clínico de pacientes com doença falciforme. Casuística e Métodos. A casuística foi composta por 153 indivíduos com DF (HbSS e HbSC) e 196 crianças saudáveis. O perfil de hemoglobinas foi determinado por HPLC. Os polimorfismos nos genes HFE, HP, GST, PON1, a presença da talassemia alfa 3,7Kb e 4,2Kb e dos haplótipos ligados ao grupo de genes da globina beta foram investigados por PCR e PCR-RFLP. A avaliação do perfil lipídico, hepático, função renal, inflamatório e metabolismo do ferro foram realizadas por técnicas espectrofotométricas. Os níveis séricos de vitamina C, receptor de transferrina solúvel e hemeoxigenase 1 total foram detectados por ELISA. Resultados. O estudo de polimorfismos no gene da GST demostrou que o genótipo Mu nulo foi o mais frequente em ambos os grupos estudados; a análise hematológica , nos indivíduos HbSS revelou diferença significante para os parâmetros de leucócitos (p=0,0452), de segmentados neutrófilos (p=0,0224) e o evento de STA (p=0,0423); nos indivíduos com DF foram encontradas diferenças para a uréia (p=0,0288) e ferritina (p=0,0316). No estudo do gene da Haptoglobina nos indivíduos com DF encontrou-se diferença significativa para contagem de reticulócitos (p=0,0167), ferritina sérica (p=0,0493); contagem de linfócitos típicos (p=0.0422) e LDH (p=0,0181); nos HbSS encontrou-se diferença para creatinina (p=0,0178) e ALT (p=0,0127). Para o gene HFE foi encontrada diferença significativa entre os alelos selvagem e mutante para o polimorfismo c.63H>D e contagem de plaquetas (p=0,0311) em indivíduos com DF; para o polimorfismo c.65S>C observou-se diferenças estatisticamente significativas para hemoglobina (p=0,0044) e hematócrito (p=0,0078). Nos HbSC para o polimorfismo c.63H>D observou-se entre os alelos selvagem e mutante diferenças significativas com o VCM (p=0,0032), HCM (p=0,0010) e linfócitos típicos (p=0,0242). Entre os indivíduos HbSS observou-se diferença significativa entre os alelos estudados e plaquetas (p=0,0078). Para a mutação c.65S>C observou-se diferenças significativas para os parâmetros de hemoglobina (p=0,0265) e hematócrito (p=0,0407) nos indivíduos HbSS e hemoglobina (p=0,0051) e hematócrito (p=0,0060) nos indivíduos HbSC. A análise do histórico clínico dos indivíduos com DF e o polimorfismo c.63H>D demonstrou diferença significativa para esplenomegalia, OR=3,38 (0,93 -12,22) e p=0,0402. Nos indivíduos HbSS foi encontrada diferença significativa entre os alelos c.63H>D e infecção, com OR=0,29 (0.07 -1,18) e p=0,0455. Para o gene PON1, a avaliação da atividade da PON1 nos indivíduos DF entre os alelos selvagem e mutante revelou diferença estatisticamente significativa, tendo o alelo selvagem maior atividade da PON1 que alelo mutante; para o polimorfismo PON1c.192Q>R observou-se entre os alelos selvagem e mutante diferenças significativas para as variáveis bioquímicas VLDL-C (p=0,0267) e triglicérides (p=0,0127). Nos HbSC verificou-se diferença estatística significativa para o PON1c.192Q>R e concentração de hemoglobina (p=0,0459), hematócrito (p=0,0225) e contagem de linfócitos típicos (p=0,0364). Para o PON1c.55L>M observou-se diferença significativa com a idade (p=0,0139), plaquetas (p=0,0109), creatinina (p=0,0329) e PCR (p=0,0141). Observou-se associação entre atividade da PON1 e esplenectomia (p=0,001); para hemeoxigenase e ferro sérico (p=0,023); e para vitamina C observamos correlação com colesterol HDL (p=0,037). Há correlação entre glutationa e vitamina C (p=0,035).Conclusões: Os polimorfismos estudados podem estar agindo sinergicamente com a hemoglobina variante S e assim influenciar na gravidade da doença, necessitando de outros estudos para validar estes resultados, uma vez que algumas destas investigações foram realizadas pela primeira vez neste grupo de indivíduos. / Patients with sickle cell disease (SCD) has continued oxidative stress (OS), which is resulting from ineffective antioxidant systems, characterized by production of reactive oxygen species (ROS) of multifactorial origin, mainly generated by the hemolytic process. Objective: To investigate polymorphism related to oxidative stress in hereditary hemochromatosis HFE gene (c.282C>Y, c.63H>D c.65S and D>C), haptoglobin (HP), glutathione S-transferase (GST) (GSTT1 and GSTM1) and paraoxonase (PON1) (c.55L>M and c.192Q>R), in association with the hematological and biochemical data, serum levels of vitamin C, paraoxonase 1, transferrin receptor and clinical manifestations of SCD patients. Methods. The sample included 153 individuals with SCD (HbSS and HbSC) and 196 healthy children. The profile of hemoglobin was determined by HPLC. The HFE gene polymorphisms, HP, GST, PON1, the presence of alpha thalassemia 3.7 Kb, 4.2 Kb and haplotypes of the beta gene cluster were investigated by PCR and PCR-RFLP. The lipid profile, liver, renal function, inflammation and iron metabolism was performed by spectrophotometric techniques. Serum levels of vitamin C, soluble transferrin receptor and total hemeoxigenase were investigated by ELISA. Results: The GST Mu null genotype was more frequent in both groups and both polymorphisms were in Hardy-Weinberg equilibrium. In the HbSS observed significant differences in the parameters of leukocytes (p = 0.0452) and segmented neutrophils (p = 0.0224) and event STA (p = 0.0423). Individuals with SCD had differences for urea (p = 0.0288) and ferritin (p = 0.0316). Evaluating the Haptoglobin gene in individuals with SCD found a significant difference for reticulocyte count (p = 0.0167), serum ferritin (p = 0.0493); typical lymphocyte count (p = 0.0422) and LDH (p = 0.0181); in the HbSS were found differences for creatinine (p = 0.0178) and ALT (p = 0.0127). For the HFE gene was found significant differences were observed between wild and mutant alleles for the polymorphism c.63H> D and platelet count (p = 0.0311) in individuals with SCD; for polymorphism c.65S> C were differences were statistically significant for hemoglobin (p = 0.0044) and hematocrit (p = 0.0078). In the HBSC observed significant differences in MCV (p = 0.0032), MCH (p= 0.0010) and typical lymphocytes (p = 0.0242). Among subjects HbSS were observed significant differences between alleles and platelets (p = 0.0078). For mutation c.65S> C showed significant differences in the parameters of hemoglobin (p=0.0265) and hematocrit (p=0.0407) in HbSS individuals, and hemoglobin (p=0.0051) and hematocrit (p=0.0060) in subjects HbSC. The analysis of the clinical history of individuals with SCD and polymorphism c.63H> D there was a significant difference for splenomegaly, OR = 3.38 (0.93 -12.22) and p = 0.0402. In HbSS individuals significant difference was found between alleles c.63H> D and infection, with OR = 0.29 (0.07 -1.18) p = 0.0455. For the PON1 gene, The activity assessment of PON1 in the individuals DF with wild and mutant genotypes showed statistically significant differences, the wild-type allele showed higher PON1 activity than the mutant allele; for the biochemical parameters to polymorphism PON1c.192Q> R and the values of VLDL-C (p = 0.0267) and triglycerides (p = 0.0127). In HbSC there was a statistically significant difference for PON1c.192Q> R and hemoglobin (p = 0.0459), hematocrit (p = 0.0225) and typical lymphocyte count (p = 0.0364). For PON1c.55L> M there was significant difference with age (p = 0.0139), platelets (p = 0.0109), creatinine (p = 0.0329) and CRP (p= 0.0141). There was an association between PON1 activity and splenectomy (p = 0.001) for hemeoxigenase and serum iron (p = 0.023), and vitamin C showed a correlation with HDL cholesterol (p = 0.037). There is a correlation between glutathione and vitamin C (p=0.035) Conclusions: The polymorphisms could be acting synergistically with this hemoglobin variant S, and influence the severity of disease, have been necessary further studies to validate these results, since some of these investigations were performed for the first time in this group of individuals.

Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-18Bitstream added on 2014-06-13T19:20:17Z : No. of bitstreams: 1 gerardi_dg_dr_jabo.pdf: 906206 bytes, checksum: 29e6a477f11cbcccee68cc2a21495f1a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Tendo em vista a expressão da proteína associada à resistência a múltiplas drogas (MRP1), da enzima glutationa-S-transferase pi (GSTpi) e da proteína p53 com o desenvolvimento da resistência a múltiplas drogas (RMD) nas células neoplásicas, objetivou-se neste estudo avaliar a expressão desses três marcadores, pela imunoistoquímica, em 68 espécimes de neoplasias caninas, incluindo tumores venéreos transmissíveis (TVTC), mastocitomas, carcinomas mamários e de glândula hepatóides e linfomas. Os espécimes foram subdivididos em: TVT, Tumor venéreo transmissível (n=9); TVTR, TVTs resistentes à quimioterapia (n=5); MASTI, mastocitomas cutâneos grau I (n=8); MASTIII, mastocitomas cutâneos grau III (n=8); CARM, carcinomas mamários (n=14); CARH, carcinomas de glândulas hepatóides (n=8); LINFB, linfomas de células B (n=9); LINFT, linfomas de células T (n=7). Resultados mostraram que as expressões da MRP1, GSTpi e p53 foram observadas em 38 (55%), 43 (62,3%) e 50 (72,4%) espécimes, respectivamente. Em 27 (39,1%) espécimes houve coexpressão dos três marcadores. A expressão da MRP1, GSTpi e p53 isoladas ou associadas pôde ser observada em todos os grupos experimentais, exceto o grupo TVTC que não expressou a MRP1. A localização da marcação nas células tumorais foi citoplasmática para MRP1 e nuclear e/ou citoplasmática para a GSTpi e p53. Não foi observada diferença na expressão dos marcadores de resistência a quimioterapia em relação à resistência a quimioterapia (TVT e TVTR), gradação histológica (MASTI e III) e imunofenótipo (LINFB e T). Há relação direta entre o aumento da expressão da MRP1 e da GSTpi nos linfomas T. / Multidrug resistance in tumors involves the expression of multidrug resistance protein-1 MRP1, enzyme glutathione-S-transferase pi (GSTpi), and p53 protein. Therefore, this study aimed at evaluating the expression of these three markers, by immunohistochemistry, in neoplasic cells. Sixty-eight canine tumor samples, including transmissible venereal tumor (TVTC), cutaneous mast cell tumor, mammary carcinoma, hepatoid gland carcinoma, and lymphoma were studied. Samples were assigned into one of the following subgroups: TVT, transmissible venereal tumor (n=9); TVTR, chemoresistant TVT (n=5), MASTI, grade-I cutaneous mast cell tumor (n=8); MASTIII, grade-III cutaneous mast cell tumor (n=8); CARM, mammary carcinoma (n=14); CARH, hepatoid gland carcinoma (n=8); LINFB, B-cell lymphoma (n=9); LINFT, T-cell lymphoma (n=7). We observed that 38 (55%), 43 (62.3%), and 50 (72.4%) samples expressed MRP1, GSTpi, and p53, respectively. Co-expression of the three markers was present in 27 (39.1%) samples. Expression of MRP1, GSTpi, and p53 alone or associated could be observed in all experimental groups, except for TVT group which did not express MRP1. Staining in the tumor cells was cytoplasmatic to MRP1, and both nuclear and cytoplasmatic to GSTpi and p53. No significant difference in the expression of the markers could be observed with relation to chemoresistance (TVT and TVTR), histological grade (MASTI and MASTIII) and immunophenotype (LINFB and LINFT). A direct relation was present between the raise of the expression of MRP1 and GSTpi in T cells lymphoma.

Orientadora: Mirela Tinucci Costa / Banca: Renée Laufer Amorim / Banca: Carlos Roberto Daleck / Banca: Felipe Augusto Ruiz Sueiro / Banca: Noeme Sousa Rocha / Resumo: Tendo em vista a expressão da proteína associada à resistência a múltiplas drogas (MRP1), da enzima glutationa-S-transferase pi (GSTpi) e da proteína p53 com o desenvolvimento da resistência a múltiplas drogas (RMD) nas células neoplásicas, objetivou-se neste estudo avaliar a expressão desses três marcadores, pela imunoistoquímica, em 68 espécimes de neoplasias caninas, incluindo tumores venéreos transmissíveis (TVTC), mastocitomas, carcinomas mamários e de glândula hepatóides e linfomas. Os espécimes foram subdivididos em: TVT, Tumor venéreo transmissível (n=9); TVTR, TVTs resistentes à quimioterapia (n=5); MASTI, mastocitomas cutâneos grau I (n=8); MASTIII, mastocitomas cutâneos grau III (n=8); CARM, carcinomas mamários (n=14); CARH, carcinomas de glândulas hepatóides (n=8); LINFB, linfomas de células B (n=9); LINFT, linfomas de células T (n=7). Resultados mostraram que as expressões da MRP1, GSTpi e p53 foram observadas em 38 (55%), 43 (62,3%) e 50 (72,4%) espécimes, respectivamente. Em 27 (39,1%) espécimes houve coexpressão dos três marcadores. A expressão da MRP1, GSTpi e p53 isoladas ou associadas pôde ser observada em todos os grupos experimentais, exceto o grupo TVTC que não expressou a MRP1. A localização da marcação nas células tumorais foi citoplasmática para MRP1 e nuclear e/ou citoplasmática para a GSTpi e p53. Não foi observada diferença na expressão dos marcadores de resistência a quimioterapia em relação à resistência a quimioterapia (TVT e TVTR), gradação histológica (MASTI e III) e imunofenótipo (LINFB e T). Há relação direta entre o aumento da expressão da MRP1 e da GSTpi nos linfomas T. / Abstract: Multidrug resistance in tumors involves the expression of multidrug resistance protein-1 MRP1, enzyme glutathione-S-transferase pi (GSTpi), and p53 protein. Therefore, this study aimed at evaluating the expression of these three markers, by immunohistochemistry, in neoplasic cells. Sixty-eight canine tumor samples, including transmissible venereal tumor (TVTC), cutaneous mast cell tumor, mammary carcinoma, hepatoid gland carcinoma, and lymphoma were studied. Samples were assigned into one of the following subgroups: TVT, transmissible venereal tumor (n=9); TVTR, chemoresistant TVT (n=5), MASTI, grade-I cutaneous mast cell tumor (n=8); MASTIII, grade-III cutaneous mast cell tumor (n=8); CARM, mammary carcinoma (n=14); CARH, hepatoid gland carcinoma (n=8); LINFB, B-cell lymphoma (n=9); LINFT, T-cell lymphoma (n=7). We observed that 38 (55%), 43 (62.3%), and 50 (72.4%) samples expressed MRP1, GSTpi, and p53, respectively. Co-expression of the three markers was present in 27 (39.1%) samples. Expression of MRP1, GSTpi, and p53 alone or associated could be observed in all experimental groups, except for TVT group which did not express MRP1. Staining in the tumor cells was cytoplasmatic to MRP1, and both nuclear and cytoplasmatic to GSTpi and p53. No significant difference in the expression of the markers could be observed with relation to chemoresistance (TVT and TVTR), histological grade (MASTI and MASTIII) and immunophenotype (LINFB and LINFT). A direct relation was present between the raise of the expression of MRP1 and GSTpi in T cells lymphoma. / Doutor

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O presente trabalho ilustra o desenvolvimento de uma rota sintÃtica para a obtenÃÃo de uma meso-metaloporfirina, a partir de uma meso-porfirina base livre, macromolÃcula derivada do LÃquido da Casca da Castanha de Caju (LCC), subproduto do agronegÃcio do caju. A obtenÃÃo dessas espÃcies seguida de estudos preliminares da modificaÃÃo da superfÃcie do eletrodo de ouro com a meso-metaloporfirina de cobre com aplicaÃÃo para sensor de NO (Ãxido NÃtrico) tambÃm compÃe os objetivos desse trabalho. A partir do processo de metalaÃÃo,foi sintetizada uma meso-metaloporfirina utilizando Cu (II) como centro metÃlico, a partir da macromolÃcula base livre, e testes voltamÃtricos foram realizados utilizando o eletrodo de ouro, em meio de diclorometano e PTBA 0,1 mol L-1, obtido a 100 mV s-1.Estudos foram realizados a cerca do comportamento eletroquÃmico dos filmes formados em presenÃa de NO em meio aquoso de Na2SO4 0,5 mol L-1 obtendo o voltamograma cÃclico para o eletrodo de ouro modificado com a porfirina de cobre(II) na detecÃÃo de NO e foi observado no voltamograma para NO sobre uma intensidade de corrente 7,6 vezes maior comparada à do pico observado para o mesmo processo do NO na superfÃcie de ouro nÃo modificado demonstrando elevado potencial para aplicaÃÃo como sensor eletroquÃmico. Outra metodologia eletroanalÃtica foi desenvolvida para a obtenÃÃo de um biossensor, baseado em uma enzima, glutationa-S-transferase (GST), para determinaÃÃo do pesticida molinato, um herbicida prÃ-emergente, em amostras reais de Ãgua de campos de arrozais da cidade do Porto, em Portugal. A construÃÃo deste biossensor baseou-se a imobilizaÃÃo de GST em um eletrodo de carbono vÃtreo (GCE), atravÃs da ligaÃÃo covalente glutaraldeÃdo-amino-silano (APTES/GA). O princÃpio deste biossensor consistiu no processo de inibiÃÃo da GST promovida pelo molinato. A curva de calibraÃÃo foi obtida por meio da tÃcnica de voltametria de pulso diferencial (VPD) variando a concentraÃÃo do pesticida entre 1,01x10-6 â 4,20x10-5 mol L-1 apresentando um limite de detecÃÃo (LD) de 0,064 mg L-1. O biossensor baseado na GST foi aplicado para quantificar o molinato nas amostras de Ãgua das lavouras de arrozais. Os resultados obtidos com este biossensor foram comparados com aqueles obtidos por HPLC e nÃo houve diferenÃas estatisticamente significativas comprovando, entÃo, que a metodologia desenvolvida foi precisa no nÃvel de concentraÃÃo estudada. / This study illustrates the development of synthetic route will be obtaining meso-metalloporphyrin, from porphyrin meso-free base macromolecule derived from Shell Liquid Cashew Nut (CNSL), byproduct of cashew agribusiness. Obtaining these species followed by preliminary studies of surface modification of gold electrode with copper meso-metalloporphyrin with application to sensor of NO (Nitric Oxide) also composed the objectives of this work. From metalation process, was synthesized by meso-metalloporphyrin using Cu (II) the a metal to center, from the macromolecule free base, and voltametric tests were carried out using the gold electrode in the middle of dichloromethane and TBAP 0,1 mol L-1, obtained at 100 mV s-1. Studies have been conducted about the electrochemical behavior of the films formed in the presence of NO in aqueous Na2SO4 0,5 mol L-1 the cyclic voltammogram obtained will be the gold electrode modified with porphyrin to copper (II) the detection of NO and voltammogram was observed in about one NO to current intensity 7,6 times larger compared to the peak observed for the same process of NO in unmodified gold surface showing high potential for application as an electrochemical sensor. Electroanalytical another methodology was developed will be obtaining the biosensor, based on an enzyme, glutathione-S-transferase (GST), will be determining the pesticide molinate, the real daily pre-emergent herbicide in samples of to water of paddy fields City Porto, in Portugal. The construction of this biosensor was based on the immobilization of GST in glassy carbon electrode (GCE) by covalent glutaraldehyde-amino-silane (APTES/GA). The principle of this biosensor consisted of the process promoted by inhibition of GST molinate. The calibration curve was obtained by the technique of differential pulse voltammetry (DPV) varying the concentration of the pesticide between 1,01 x10-6 to 4,20 x10-5 mol L-1 having a limit of detection (DL) of 0,064 mg L-1. The biosensor based on GST was applied to quantify the molinate in water samples of paddy crop. The results obtained with this biosensor were compared with those obtained by HPLC and no statistically significant differences proving therefore that the developed methodology has been studied in terms of concentration.

Dissertação(mestrado)- Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2011. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-08-08T15:13:55Z No. of bitstreams: 1 dissertao - tila clivea martins em pdf.pdf: 3894921 bytes, checksum: 4ecfe10594f0b4dda190edc8958a9750 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-09-18T03:53:39Z (GMT) No. of bitstreams: 1 dissertao - tila clivea martins em pdf.pdf: 3894921 bytes, checksum: 4ecfe10594f0b4dda190edc8958a9750 (MD5) / Made available in DSpace on 2012-09-18T03:53:39Z (GMT). No. of bitstreams: 1 dissertao - tila clivea martins em pdf.pdf: 3894921 bytes, checksum: 4ecfe10594f0b4dda190edc8958a9750 (MD5) Previous issue date: 2011 / Os fatores abióticos são um dos principais problemas na aquicultura, pois a alteração destes pode resultar em baixo nível de crescimento ou induzir efeitos deletérios que podem eventualmente levar a morte dos organismos. Neste trabalho, fez-se uma simulação da variação de oxigênio dissolvido com desligamento da aeração até que os níveis nos tanques atingissem 3 mg/L (condição de hipóxia) quando então a aeração era religada. Com base nestas condições foi avaliado o aumento na competência antioxidante do camarão Litopenaeus vannamei (Boone, 1931) como resultado da ação do ácido lipóico (AL) aplicado a ração, nas doses de 35, 70 e 140 mg de AL para cada 1 kg de ração, quando exposto a uma situação de hipóxia/re-oxigenação. Foram utilizados camarões machos e fêmeas com peso inicial de 2,07 g (+ 0,24). A atividade da enzima glutationa-S-transferase (GST) aumentou na dose de 70 mg de AL/kg nas brânquias, no entanto, no hepatopâncreas a resposta foi bifásica, as vezes aumentando e as vezes diminuindo. As análises dos níveis de peroxidação lipídica (TBARS) em brânquias constatou que o AL induziu um efeito fortemente antioxidante na dose de 70 mg/kg após 4 h de re-oxigenação. No hepatopâncreas o AL reduziu os níveis de TBARS após de 0,5 h de re-oxigenação na dose de 35 mg AL/kg, e logo após 4 h apresentou em efeito pró-oxidante. Na avaliação da capacidade antioxidante total contra peroxi-radicais foi constatado que em normóxia as doses de 70 e 140 mg AL/kg induziram um efeito antioxidante nas brânquias do camarão. No hepatopâncreas, em normóxia, a dose de 70 mg AL/kg promoveu um efeito antioxidante, enquanto que no organismos submetidos a hipóxia/re-oxigenação foi constatado que a dose de 140 mg AL/kg promoveu um aumento da capacidade antioxidante. Ainda, nas duas doses mais altas de AL (70 e 140 mg/kg) foi verificado um aumento de peso dos camarões. Sugere-se que a dose mais indicada seja a de 70 mg de AL para 1 kg de ração, por apresentar melhores resultados nas análises bioquímicas e por induzir aumento de peso ao camarão os quais atingiram peso final de 7,94 g (+ 0,15). / The abiotic factors are a major problem in aquaculture, because changing them can result in low growth or induce deleterious effects that may eventually lead to death of organisms. In this work, it was a simulation of the variation of dissolved oxygen by turning off the aeration tanks in which the levels reached 3 mg/L (hypoxic condition) when the aeration was then restarted. Under these conditions we evaluated the increase in antioxidant power of the shrimp Litopenaeus vannamei (Boone 1931) as a result of action of lipoic acid (LA) applied to food at doses of 35, 70 and 140 mg of LA for each 1 kg ration when exposed to a hypoxia/re-oxygenation. We used juvenile male and female shrimps with initial weight of 2.07 g (+ 0.24). The activity of glutathione-S-transferase (GST) increased in a dose of 70 mg AL/kg in the gills; however, in the hepatopancreas response was biphasic, sometimes increasing and sometimes decreasing. The analysis of the levels of lipid peroxidation (TBARS) in gills showed that LA induced a strong antioxidant effect at a dose of 70 mg/kg after 4 h of re-oxygenation. In the AL hepatopancreas decreased levels of TBARS after 0.5 h re-oxygenation in a dose of 35 mg AL/kg, and after 4 hours resulted in pro-oxidant effect. In assessing the total antioxidant capacity against peroxy-radicals was found that in normoxia the doses of 70 and 140 mg AL/kg induced an antioxidant effect in the gills of the shrimp. In the hepatopancreas, in normoxia, the dose of 70 mg AL/kg provided an antioxidant effect, whereas in organisms subjected to hypoxia/re-oxygenation was found that the dose of 140 mg AL/kg caused an increase in antioxidant capacity. Still, the two higher doses of LA (70 and 140 mg/kg) there was an increase in weight of shrimp. It is suggested that the optimal dose is 70 mg of the AL to 1 kg, by offering better results in biochemical analysis and to induce weight gain to shrimp which reached the final weight of 7.94 g (+ 0, 15).