Cytochromes P450 are heme enzymes with very broad substrate specificity, they can oxidize tens to hundreds of different substrates including both eobiotics and xenobiotics, but with varying efficiencies and kinetics. They are responsible for the metabolic activation of many carcinogens resulting in formation of reactive intermediates, these reactive species participate in the formation of DNA adducts and also increase of oxidative stress. Eukaryotic cytochromes P450 are membrane bound enzymes found mostly in the endoplasmic reticulum. In vertebrates, they are expressed in a variety of tissues mostly in the liver, but also in kidney, lung, skin and others. The cytochrome P450 1B1 is an inducible enzyme which occurs mainly in the lung and skin. Its expression is induced predominantly by the presence of polycyclic aromatic hydrocarbons, dioxins and heterocyclic amines. The human cytochromes P450 are typically obtained using heterologous expression and isolated as a C-terminally modified hexa-histaq constructs using immobilized metal affinity chromatography. This thesis focuses on effect of C-terminal modifications on activity of human cytochrome P450 1B1. First, the two expression vectors encoding the human form of cytochrome P450 1B1 were prepared, one contained a classical C-terminal hexa-histaq...

No / The cytochrome P450 family of enzymes is involved in the Phase I metabolism of a wide variety of compounds. Although generally involved with detoxification, overexpression of one family member, cytochrome P450 1B1 (CYP1B1), has been associated with human epithelial tumors. As such, CYP1B1 was hypothesized to be a novel target for the development of anticancer therapies. We investigated expression of CYP1B1 protein in 61 human colorectal adenocarcinomas and compared this to that observed in 14 histologically normal human large bowel samples removed from patients undergoing surgery for large bowel tumors. Although we confirmed that CYP1B1 was expressed at high levels in human colorectal tumor epithelia, we also found that CYP1B1 was not absent from normal colonic epithelia but was expressed at low levels. The expression of CYP1B1 in colon tumors does not correlate with tumor stage or degree of lymph node invasion in this study. Furthermore, in addition to expression in colon epithelia, CYP1B1 is also observed in blood vessels within the colon. As with the epithelia, levels of CYP1B1 were higher in tumor vasculature than that of the normal colon. Although these observations greatly support the development of CYP1B1 targeted anticancer therapies, they also indicate the caution that should be observed when developing such drugs.

O objetivo deste trabalho foi a geração e avaliação de ortoimagens ALOS/PRISM, utilizandose para isso MDEs (Modelos Digitais de Elevação) obtidos através das diferentes combinações de visadas desse sensor de uma imagem com nível de processamento 1B1. Na primeira etapa foram gerados os MDEs a partir das combinações das três visadas, utilizando-se para isso pontos de controle obtidos através de levantamento de campo, onde foram testados a quantidade mínima necessária de pontos de controle para geração de MDE e outros parâmetros tais como nível de processamento, tamanho de janela, entre outros. Na etapa seguinte, comparou-se o resultado da extração com os Modelos Digitais de Elevação do ASTER (GDEM) e o SRTM (Shuttle Radar Topographic Mission), este gerado a partir do Projeto TOPODATA do INPE (Instituto Nacional de Pesquisas Espaciais). Na etapa final avaliaram-se os MDEs com os pontos de checagem. Através de análises estatísticas selecionaram-se os melhores MDEs para ortorretificação. Os resultados demonstraram que, para este caso específico, todos os MDES da combinação de visada Backward – Foward (gerados a partir de 6 pontos de controle) seriam aprovados por apresentarem os menores valores de RMS (entre 2,94 e 3m) e que, ao se utilizar outros MDEs oriundos de outras combinações, as ortoimagens obtidas também atendem ao Padrão de Exatidão Cartográfica (PEC) Classe A para escala 1:25.000, bem como as ortoimagens geradas com os Modelos de Elevação Globais SRTM e GDEM. A área de estudo escolhida foi uma região rural do município de São Gabriel – RS, que possui relevo com média altimétrica de 160m, podendo ser considerado plano, o que justifica o bom desempenho do SRTM em conjunto com os MDEs gerados a partir do PRISM. / The aim of this work was the generation and evaluation of orthoimages ALOS / PRISM, using the MDEs (Digital Elevation Models) obtained using different combinations of sensor views of an image with 1B1 processing level. In the first step the MDEs were created from combinations of three views, using for it the ground control points obtained by field survey, where the minimum amount necessary of control points for DEM generation and other parameters as processing level, size of the windows, among others were tested. In the further step, the result of extraction was compared to Digital Elevation Models from ASTER (GDEM) and SRTM (Shuttle Radar Topographic Mission), this one generated from the Project TOPODATA from INPE (National Institute for Space Research). In the final step, the MDEs were evaluated with the checkpoints. Through statistical analysis the best MDEs were selected for orthorectification. The results showed that, for this specific case, all MDEs combination of Backward - Foward (generated from 6 control points) would be approved by submitting the lowest values of RMS (between 2.94 and 3 m) and, when using other MDEs from another combinations, obtained orthoimages also satisfies the Cartographic Accuracy Standard (PEC) for Class A 1:25,000 scale, as the orthoimages generated from the SRTM and GDEM. The chosen study area was a rural region of São Gabriel - RS, which has average altitude of 160 m, can be considered plane, which explains the good performance of the SRTM in conjunction with the MDEs generated from PRISM.

O objetivo deste trabalho foi a geração e avaliação de ortoimagens ALOS/PRISM, utilizandose para isso MDEs (Modelos Digitais de Elevação) obtidos através das diferentes combinações de visadas desse sensor de uma imagem com nível de processamento 1B1. Na primeira etapa foram gerados os MDEs a partir das combinações das três visadas, utilizando-se para isso pontos de controle obtidos através de levantamento de campo, onde foram testados a quantidade mínima necessária de pontos de controle para geração de MDE e outros parâmetros tais como nível de processamento, tamanho de janela, entre outros. Na etapa seguinte, comparou-se o resultado da extração com os Modelos Digitais de Elevação do ASTER (GDEM) e o SRTM (Shuttle Radar Topographic Mission), este gerado a partir do Projeto TOPODATA do INPE (Instituto Nacional de Pesquisas Espaciais). Na etapa final avaliaram-se os MDEs com os pontos de checagem. Através de análises estatísticas selecionaram-se os melhores MDEs para ortorretificação. Os resultados demonstraram que, para este caso específico, todos os MDES da combinação de visada Backward – Foward (gerados a partir de 6 pontos de controle) seriam aprovados por apresentarem os menores valores de RMS (entre 2,94 e 3m) e que, ao se utilizar outros MDEs oriundos de outras combinações, as ortoimagens obtidas também atendem ao Padrão de Exatidão Cartográfica (PEC) Classe A para escala 1:25.000, bem como as ortoimagens geradas com os Modelos de Elevação Globais SRTM e GDEM. A área de estudo escolhida foi uma região rural do município de São Gabriel – RS, que possui relevo com média altimétrica de 160m, podendo ser considerado plano, o que justifica o bom desempenho do SRTM em conjunto com os MDEs gerados a partir do PRISM. / The aim of this work was the generation and evaluation of orthoimages ALOS / PRISM, using the MDEs (Digital Elevation Models) obtained using different combinations of sensor views of an image with 1B1 processing level. In the first step the MDEs were created from combinations of three views, using for it the ground control points obtained by field survey, where the minimum amount necessary of control points for DEM generation and other parameters as processing level, size of the windows, among others were tested. In the further step, the result of extraction was compared to Digital Elevation Models from ASTER (GDEM) and SRTM (Shuttle Radar Topographic Mission), this one generated from the Project TOPODATA from INPE (National Institute for Space Research). In the final step, the MDEs were evaluated with the checkpoints. Through statistical analysis the best MDEs were selected for orthorectification. The results showed that, for this specific case, all MDEs combination of Backward - Foward (generated from 6 control points) would be approved by submitting the lowest values of RMS (between 2.94 and 3 m) and, when using other MDEs from another combinations, obtained orthoimages also satisfies the Cartographic Accuracy Standard (PEC) for Class A 1:25,000 scale, as the orthoimages generated from the SRTM and GDEM. The chosen study area was a rural region of São Gabriel - RS, which has average altitude of 160 m, can be considered plane, which explains the good performance of the SRTM in conjunction with the MDEs generated from PRISM.

O objetivo deste trabalho foi a geração e avaliação de ortoimagens ALOS/PRISM, utilizandose para isso MDEs (Modelos Digitais de Elevação) obtidos através das diferentes combinações de visadas desse sensor de uma imagem com nível de processamento 1B1. Na primeira etapa foram gerados os MDEs a partir das combinações das três visadas, utilizando-se para isso pontos de controle obtidos através de levantamento de campo, onde foram testados a quantidade mínima necessária de pontos de controle para geração de MDE e outros parâmetros tais como nível de processamento, tamanho de janela, entre outros. Na etapa seguinte, comparou-se o resultado da extração com os Modelos Digitais de Elevação do ASTER (GDEM) e o SRTM (Shuttle Radar Topographic Mission), este gerado a partir do Projeto TOPODATA do INPE (Instituto Nacional de Pesquisas Espaciais). Na etapa final avaliaram-se os MDEs com os pontos de checagem. Através de análises estatísticas selecionaram-se os melhores MDEs para ortorretificação. Os resultados demonstraram que, para este caso específico, todos os MDES da combinação de visada Backward – Foward (gerados a partir de 6 pontos de controle) seriam aprovados por apresentarem os menores valores de RMS (entre 2,94 e 3m) e que, ao se utilizar outros MDEs oriundos de outras combinações, as ortoimagens obtidas também atendem ao Padrão de Exatidão Cartográfica (PEC) Classe A para escala 1:25.000, bem como as ortoimagens geradas com os Modelos de Elevação Globais SRTM e GDEM. A área de estudo escolhida foi uma região rural do município de São Gabriel – RS, que possui relevo com média altimétrica de 160m, podendo ser considerado plano, o que justifica o bom desempenho do SRTM em conjunto com os MDEs gerados a partir do PRISM. / The aim of this work was the generation and evaluation of orthoimages ALOS / PRISM, using the MDEs (Digital Elevation Models) obtained using different combinations of sensor views of an image with 1B1 processing level. In the first step the MDEs were created from combinations of three views, using for it the ground control points obtained by field survey, where the minimum amount necessary of control points for DEM generation and other parameters as processing level, size of the windows, among others were tested. In the further step, the result of extraction was compared to Digital Elevation Models from ASTER (GDEM) and SRTM (Shuttle Radar Topographic Mission), this one generated from the Project TOPODATA from INPE (National Institute for Space Research). In the final step, the MDEs were evaluated with the checkpoints. Through statistical analysis the best MDEs were selected for orthorectification. The results showed that, for this specific case, all MDEs combination of Backward - Foward (generated from 6 control points) would be approved by submitting the lowest values of RMS (between 2.94 and 3 m) and, when using other MDEs from another combinations, obtained orthoimages also satisfies the Cartographic Accuracy Standard (PEC) for Class A 1:25,000 scale, as the orthoimages generated from the SRTM and GDEM. The chosen study area was a rural region of São Gabriel - RS, which has average altitude of 160 m, can be considered plane, which explains the good performance of the SRTM in conjunction with the MDEs generated from PRISM.

Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities. The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined. The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins. The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed. In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.