The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.

The MdfA is a 410 amino acid-long integral membrane protein, which belongs to the Major Facilitator superfamily of multidrug transporters. It is predicted to consist of 12 transmembrane helices. MdfA uses the energy of the transmembrane proton gradient to pump a variety of toxic compounds out of E. coli cells. No high resolution structure of MdfA is available. The goals of this research project were to develop a practical method for purification of MdfA, to evaluate the feasibility of structure determination by Nuclear Magnetic Resonance (NMR) and X-ray crystallography, and to develop an activity assay for purified MdfA. To this end, MdfA, with a hexa-histidine tag attached to facilitate protein purification, was successfully expressed and incorporated into the cell membrane using an E. coli expression system. MdfA was extracted from the cell membrane with the detergents 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), n-dodecyl-B-D-maltoside (DDM), and 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LMPG) and purified by affinity chromatography on nickel-nitrilotriacetic acid agarose. Pure protein was found to be monodisperse in DHPC, DDM and LMPG micelles. To achieve simple amino acid selective isotope labeling for high-resolution NMR studies, MdfA was expressed in a cell-free translation system. To determine if the purified protein was properly folded, 19F NMR experiments were carried out on 5-fluoro-tryptophan-labeled MdfA while titrating the MdfA substrates ethidium bromide and chloramphenicol into the fluoro-tryptophan-labeled MdfA sample. An activity assay was developed for MdfA incorporated into liposomes using the fluorescent dye 9-amino-6-chloro-2-methoxyacridine (ACMA) to detect proton translocation coupled to substrate transport. Results from both the 19F NMR and the transport activity assay indicated that the purified MdfA was properly folded and functional. NMR experiments with pure MdfA yielded spectra of insufficient quality for high-resolution structure determination but did indicate that structural studies of MdfA by NMR are feasible. Crystallization trials yielded crystals that are likely to contain protein and will serve as a starting point for further optimization of crystallization conditions for X-ray structure determination.

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

La pollution croissante des milieux aquatiques nécessite la mise au point de nouvelles technologies permettant d’optimiser la surveillance de la qualité de l’eau. Dans ce contexte, nous avons développé un biomarqueur de susceptibilité du degré de la pollution globale des milieux aquatiques intitulé « Sentinelle ». Le principe du biomarqueur Sentinelle est basé sur le niveau de coexpression de deux protéines « Multidrug Resistance » (MDR), la protéine ABCG2-like et la P-gp, dans les érythrocytes de la truite Salmo trutta fario. Le biomarqueur sentinelle a été validé en conditions in vitro grâce au développement des cultures primaires d’érythrocytes de truite. Après l’exposition des globules rouges de truites à des concentrations croissantes d’un polluant modèle, le Benzo-a-pyrène, l’expression de la protéine ABCG2-like et de la P-gp augmente d’une manière dose dépendante. Le biomarqueur Sentinelle a ensuite été validé en milieu naturel sur des truites fario en provenance de différents cours d’eau d’Auvergne. En milieu naturel, les deux protéines MDR sont exprimées différemment dans les érythrocytes de truites fario selon le degré de contamination du cours d’eau. En effet, dans une rivière où la pollution est faible voire nulle, seule la protéine ABCG2-like est exprimée, alors que dans une rivière présentant une contamination plus importante, la P-gp et l’ABCG2-like sont toutes les deux coexprimées par une réponse de type relais. Les expériences menées en conditions in vitro et en milieu naturel, laissent supposer que la protéine ABCG2-like assure une fonction de garde alors que la P-gp assurerait une fonction de protection défensive. En conséquence, selon le niveau d’expression de la protéine de garde et de la protéine de défense, le degré de contamination de la rivière pourrait être évalué. L’intérêt de l’utilisation du biomarqueur Sentinelle a aussi été validé sur des Salmonidés en provenance de pisciculture. Ce nouvel outil biologique apporte des informations plus intégratives et plus précoces sur la qualité des milieux aquatiques, informations essentielles pour une meilleure gestion des ressources en eau. / Increasing aquatic pollution requires the development of new technologies for to optimize the monitoring of water quality. In this context, we have developed a “biomarker of susceptibility” designating the degree of global pollution in aquatic medium, entitled "Sentinel". The Sentinel biomarker is based on the co-expression level of two major "Multidrug resistance" (MDR) proteins, such as ABCG2-like protein and P-gp, in erythrocytes of brown trout’s in response to aquatic pollution. After developing a primary erythrocyte culture, the Sentinel biomarker was validated in a controlled medium. Trout erythrocytes exposure to increasing concentrations of Benzo-a-pyrene, a model pollutant, induced an increase expression of ABCG2-like protein and P-gp by a dose-dependent response. The Sentinel biomarker was then developed in a natural environment, using the erythrocytes of brown trout collected from the various rivers located in the Auvergne region of France. In the natural environment, both MDR proteins are differentially expressed in the erythrocytes of brown trout depending on the degree of contamination of rivers. Indeed, wild brown trout erythrocytes in an uncontaminated river, expressed only the ABCG2-like protein, whereas, in a river presenting a higher contamination, P-gp and ABCG2-like were both coexpressed with a relay response. Experiments in vitro conditions and natural environment, suggest that ABCG2-like protein acts as a vanguard protective protein, in complement to P-gp which acts as a “defensive” protective protein. Consequently, according to the expression level of the vanguard and defense proteins, the degree of contamination of the river could be evaluated. The use advantage of the Sentinel biomarker has also been validated on Salmonidae erythrocytes form farmed fish. This new tool provides biological information more early and integrative on the quality of aquatic environments. These informations are essential for better management of water resources.

Indiana University-Purdue University Indianapolis (IUPUI) / Many cancers, including breast cancer, often develop resistance to chemotherapeutic drugs over a course of treatment. Many factors, including ABC transporter-mediated drug efflux, have been shown to play a role in acquired drug resistance. Fatty acid synthase (FASN), the key enzyme of lipid synthesis pathway, was found to be over-produced in an Adiamycin resistant breast cancer cell line MCF7/AdrVp3000, compared to its parental drug sensitive MCF7 cell line. Inhibition of FASN expression increased the drug sensitivity in breast cancer cells (MCF7/AdrVp3000 and MDA-MB-468), but not in the normal breast epithelia cell line MCF10A1. Enforced overexpression of FASN in MCF7 breast cancer cells decreased its drug sensitivity. These results indicated that FASN overexpression can induce drug resistance in breast cancers. Ectopic overexpression of FASN in MCF7 cells did not affect cell membrane permeability, transporter activity, nor did it affect cell proliferation rate. However, FASN overexpression protects cancer cells from drug-induced apoptosis by decreasing caspase-8 activation. In FASN over-expressing MCF7 cells, I discovered the positive feedback relationship between FASN and activation of Akt as previously reported. However, activation of Akt did not mediate FASN-induced drug resistance. Together with the findings that FASN expression associates with poor prognosis in several types of cancers, my investigations suggest that FASN overexpression is a novel mechanism of drug resistance in breast cancer chemotherapy. Inhibitors of FASN can be used as sensitizing agents in breast cancer chemotherapy.

The purpose of the study was to investigate the problem of defaulting of tuberculosis (TB) treatment in the Khomas region of Namibia. A quantitative, descriptive research approach was used to investigate the reasons for defaulting of TB treatment under the DOTS strategy in Khomas Region of Namibia. Data was collected by using a structured interview schedule with 54 participants who were on DOTS strategy and defaulted. Data were analysed by using the Epi info computer program. The major findings from the data obtained, revealed that the respondents did not have an in-depth knowledge of TB and the health education was not successful. This contributed to the defaulting of their treatment. Based on the study findings nurses should improve health education to TB patients on DOTS and also educate members of the community to address the stigmatisation of TB. Recommendations for further improvement in the compliance of TB treatment were made. / Public Health / M.A. (Public Health)

Decentralisation of the multidrug resistant tuberculosis (MDR TB) programme to primary health care (PHC) facilities in the Nelson Mandela Metropolitan was implemented in order to improve the effectiveness of MDR TB services. This study explored the knowledge gaps of nurses at PHC facilities as regards MDR TB. A quantitative, cross-sectional descriptive study was conducted; data was collected using a structured questionnaire. Non-probability sampling was applied in this study. A convenient sampling technique was used and 25 of the 42 facilities were selected. Thirty-two respondents completed the questionnaire with a response rate of 64%. Descriptive statistics were used to describe the data. Only 38% of the nurses had been trained on MDR TB. Overall scores were high with a mean knowledge score of 61%. However there were knowledge gaps regarding side effects of MDR TB medication. This study revealed gaps in knowledge of certain areas of MDR TB management / Health Studies / M.A. (Public Health)

Vaults, recently discovered in 1986, are multi-subunit organelles with a molecular mass of ,--,13 MDa. The specific function of vaults is unknown, although they are believed to be involved in internal transport. These ribonucleoproteins are composed of the major vault protein, which comprises ' 70% of the vault's mass, two minor proteins, TEP1 and vPARP, and untranslated RNA(s). It is believed that the protein components of the vault are structural while the RNAs are the functional components. Implications of the vault's involvement in multi-drug resistance in cancer have been made. In some resistant cancer cells, the major vault protein and vRNA(s) are up-regulated up to 15 times when cells are exposed to a cytotoxic drug. Cytotoxic drugs such as doxorubicin are administered as a cancer treatment, but may be ineffective because the drug is actively pumped out of the cell. Multi-drug resistance is the most common failure of chemotherapeutic cancer treatment. In order to prevent the development of multi-drug resistance this research employed the use of small interfering RNA technology to down-regulate the expression of one of the vault RNAs, vRNA3, in cultured GLC4 cells, a small-cell lung cancer cell line. If the vRNA(s) are the functional portion of the vault and a cloned siRNA prevents their up-regulation after drug exposure, the cells should lose their multi-drug resistance, stimulating apoptosis. If successful, this approach may provide an alternative approach to cancer treatment in cells which respond to chemotherapy by increasing the number of vault particles.Initially, the transfection of a plasmid into GLC4 cells was optimized. The best transfection efficiency (N20%) was obtained by using GeneTherapySystems' GenePORTER2 transfection reagent in serum free conditions. To determine if the vault RNAs are the functional portion of the vault complex that confers multi-drug resistance to a cell, a small interfering RNA fragment was designed to specifically knock-down the expression of human vault RNA 3. The siRNA sequence homologous to a portion of vault RNA3 was cloned into an expression vector, and using optimized transfection protocols was transfected into GLC4/REV cells. A Western analysis using caspase-8 antibodies showed no difference in caspase-8 expression in doxorubicin treated and untreated cells. Preliminary results yielded by reverse transcriptase polymerase chain reaction amplification of isolated RNA indicated that the vRNAs were not down-regulated by the siRNAs. / Department of Biology

Background: Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model. Methods: Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O-2, 60 min), hypoxia (Hx, 6% O-2, 60 min) or H/R (6% O-2, 60 min followed by 21% O-2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean +/- SD and analyzed via ANOVA followed by the post hoc Bonferroni t test. Results: We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation. Conclusions: Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.