Chemotherapy is used in the treatment of cancer. Unfortunately, drugs often fail due to multidrug resistance (MDR) caused by P-glycoprotein (P-gp1or ABCB1) and the multidrug resistance-associated protein (MRP1 or ABCC1). These proteins bind and transport drugs out of cancer cells, thereby conferring MDR. / The second chapter of this thesis addresses an unexplained phenomenon that accompanies P-gp1 expression, collaterally sensitive to verapamil. The collective results of this work demonstrated that treatment of cells that over-express P-gp1 with verapamil induces apoptosis. Furthermore, the findings show that the ATPase activity of P-gp1 was activated by verapamil. The degree of ATPase activation was proportional to the level of apoptosis and the increased demand for ATP resulted in the production of reactive oxygen species (ROS). Finally, the production of ROS led to cell death mediated by apoptosis in that experimental model system. / Chapters three and four are devoted to understanding the binding characteristics of MRP1 with two of its physiological substrates, glutathione (GSH) and leucotriene C4(LTC4). Photoreactive derivatives of these substrates were synthesised to address this objective, IAAGSH and IAALTC4. Photolabelling and transport studies showed that these derivatives have similar binding characteristics as the native compounds. In addition, photolabelling of MRP1 occurred with a high specificity with both compounds. IAAGSH and IAALTC 4 were also used to determine the locations of GSH and LTC4 binding sites. This was accomplished using MRP1-variants containing hemagglutinin (HA) epitopes at specific locations in the amino acid sequence. Through photoaffinity labelling, immunoprecipitation, and trypsin digestion, a map of binding sites for IAAGSH or IAALTC4 was obtained. Both LTC4 and GSH bound to transmembrane (TM) regions 10-11 and 16-17 which have been previously implicated in drug binding. Furthermore, novel binding sites for both substrates were discovered. IAALTC4 photolabelled a novel site within the first five TMs (TMD0) of MRP1, whereas IAAGSH labelled two cytoplasmic regions (L1 and L0). These may represent specific binding sites for LTC4 and GSH. / The work within this thesis explores some of the biochemical characteristics of Pgp1 and MRP1 that are not directly related to drug resistance and may lead to new strategies in cancer treatment.

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 200-242). Also available on the World Wide Web.

A epilepsia, doença descrita pela primeira vez em 2000 a.C., tem a crise convulsiva ou epiléptica como fenômeno paroxístico, e a International League Against Epilepsy define a crise epiléptica como \"manifestação excessiva e/ou hipersincrônica, normalmente autolimitada, da atividade dos neurônios no cérebro\". Há 40 anos surgiram os medicamentos antiepilépticos, mas a resistência múltipla a fármacos antiepilépticos (FAEs) é um problema significante que afeta pelo menos 30% dos pacientes portadores dessa doença devastadora. O mecanismo exato da fármaco-resistência desenvolvida em pacientes epilépticos ainda é desconhecido, porém uma possível causa seria a inadequada acumulação intraparenquimal do fármaco antiepiléptico relacionada a expressão aumentada da glicoproteína-P (PgP). Neste contexto, nosso objetivo foi investigar a correlação da expressão da PgP, codificada pelo gene ABCB1, no córtex temporal de 12 pacientes fármaco-resistentes frente aos FAEs fenobarbital, carbamazepina, fenitoína e lamotrigina; comparamos também a expressão da PgP nesse mesmo grupo de pacientes, selecionados no Centro de Cirurgia de Epilepsia (CIREP) frente a um grupo controle composto por indivíduos não epilépticos que evoluíram para óbito examinados no Serviço de Verificação de Óbito do interior (SVOi). Utilizamos a Cromatografia Líquida de Alta Eficiência para determinar as concentrações dos FAEs no plasma e no cérebro, sendo que a metodologia utilizada no tecido cerebral foi desenvolvida e validada especialmente para esse fim. Essas concentrações foram então determinadas e a razão entre as duas medidas foi comparada com a expressão de PgP no tecido cerebral. Analisando os resultados concluímos que não há correlação linear entre a razão dos fármacos estudados e a expressão de PgP no córtex temporal de pacientes com epilepsia refratária. / Epilepsy, desease described for the first time in 2000 B.C., presents the convulsion or epileptic seizure as its paroxysmal event, and the International League Against Epilepsy defines the epileptic seizure as \"excessive and/or hypersynchronous manifestation, usually self-limiting, from brain neurons activity\". Forty years ago the antiepileptic drugs (AEDs) emerged, but multiple resistance to AEDs is a significant problem which affects at least 30% of epileptic patients. The mechanism underlaying pharmacoresistance is still unknown, but a possible cause is the inadequate accumulation of AEDs in the brain tissue related to P-glycoprotein over expression. In this context, our aim was to investigate the correlation between PgP expression, codified by ABCB1 gene, in temporal cortex of 12 pharmacoresistant patients towards AEDs phenobarbital, carbamazepine, phenytoin and lamotrigine; we also compared PgP expression between the same group of patients, selected inside Epilepsy Surgery Center (CIREP), and a control group composed by non-epileptic deceased individuals examined at the Death Verification Service from our Medicine School (SVOi). We used High Performance Liquid Chromatography to determine AEDs concentrations in plasma and brain, and the methodology applied to brain quantification was specially developed for this purpose. These two concentrations were then determined and the ratio between them was compared with PgP expression in brain tissue. Analyzing the results we concluded that there is no linear correlation between the ratio of the AEDs studied and PgP expression in temporal cortex of patients with pharmacoresistant epilepsy.

>Magister Scientiae - MSc / Purpose: One of the leading causes of death reported in women worldwide is breast cancer. Manytumours, including breast cancer, associated with poor prognosis, have received a renewed focus and increased perspective with regard to drug discovery and innovation towards developing rational combination regimens of first-line anticancer drugs with novel compounds that target diverse hallmarks of the cancer phenotype. Multidrug resistance (MDR), which has been found to significantly decrease the efficacy of anticancer drugs and causes tumor recurrence, has been a major challenge in clinical cancer treatment with chemotherapeutic drugs for decades. Several mechanisms of overcoming drug resistance have been postulated and the well known P-glycoprotein (P-gp) including other drug efflux transporters are considered to be critical in pumping anticancer drugs out of cells which in turn results in unsuccessful chemotherapy treatments. The endoplasmic reticulum (ER) is an interconnecting organelle which synthesizes proteins and its quality control processes ensures the proper protein folding, post-translational modifications and conformation of secretory and trans-membrane proteins. Previous studies demonstrated that geldanamycin (GA), a benzoquinone ansamycin antibiotic, the antibiotic, tunicamycin (TM) and the sesquiterpene lactone, thapsigargin (TG) have been found to cause ER stress and consequently, cellular arrest. GA is known to manifest anti-cancer activity through the inhibition of Hsp90-chaperone, TM interferes with N-glycosylation of newly synthesized proteins triggering the unfolded protein response, while TG inhibits intracellular Ca2+ ATPases resulting in increased cytosolic Ca2+. Cellular stress conditions, lead to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen which results in a unfolded protein response (UPR) to maintain cell survival in cancer cells. ERS has been previously reported to enhance MDR1 transcriptional induction and P-gp transport function in cancer cells, however, prolonged endoplasmic reticulum stress conditions and inadequate unfolded protein response force cells undergo apoptosis. In this study, we examined the effects of GA, TG and TM alone and in combination to determine the cellular response of the MCF-7 breast carcinoma cell line with regard to proliferation and P-gp-mediated drug efflux activity and apoptosis. Methods: Analyses of MCF-7 breast carcinoma cells exposed to Endoplasmic Reticulum Stress (ERS) inducers geldanamycin, thapsigargin and tunicamycin, alone and in combination, included growth curves alone and in the presence of 24 hour IC50 inhibitory concentrations of the 3 ERS inducers alone, dose-response curves (MTT cytotoxicity assays) of the ERS alone and in combination, analysis of P-glycoprotein-mediated efflux pump activity in the presence of the ERS inducers alone and in combination (Calcein-AM efflux assays), analysis of viability, cytotoxicity and early apoptosis via caspase-3/7 expression (Triplex assay) and morphological staining of apoptotic and/or necrotic cells in the presence of IC50 inhibitory concentrations of the ERS inducers alone with Annexin V-FITC. Results: This study investigated the effects of Endoplasmic Reticulum Stress (ERS) inducers on growth and proliferation of MCF-7 breast carcinoma cells in culture. The MCF-7 cell line was exposed to different concentrations of ERS inducers alone and in combination with each other. All responses occurred in a dose- and time- dependent manner. When combined at equimolar log dose concentrations, integrated effects yielded enhanced cytotoxic properties as IC50 values were drastically decreased in combination as opposed to single ERS inducer responses. Combined effect on P-glycoprotein-mediated drug efflux activity yielded minor but insignificant decreases in efflux pump activity at different time intervals as opposed to the increase in cellular efflux in the presence of the ERS inducers alone at different time intervals. Caspase-3/7 apoptotic protein expression was increased as log doses of ERS inducers alone were increased, leading to cell necrosis at higher cytotoxic concentrations. The determined IC50 growth inhibitory concentrations after 24 hours were confirmed by the Annexin V-FITC demonstrating early apoptotic, necrotic and viable cells in the presence of the ERS inducers alone. Conclusion: This study demonstrated a significant growth inhibition of MCF-7 breast carcinoma cells upon exposure to ERS inducers alone. Results suggested that when ERS inducers are used in combination, their efficacy is enhanced as 50 percent inhibitory concentrations were considerably lower in combination as opposed to when used alone. The present study is consistent with previous studies with geldanamycin, and was the 1st to investigate the effects of geldanamycin, thapsigargin and tunicamycin in combination and with reference to P-gp efflux activity. Results suggested that in combination, efflux activity may be reduced, and efficacy may be enhanced. To enhance efficacy would be a major breakthrough in cancer drug discovery and development-targeting specific populations of cancer cells and reducing ERS-induced toxicity to normal cells and vital organs.

In the past forty years advancements in fluorescence-based methods including imaging (e.g. confocal and multi-photon) and quantitative spectroscopies (e.g. Fluorescence Correlation Spectroscopy) have been applied to systems ranging from solutions to in vivo models: such methods possess the ability to monitor fluorescence intensity fluctuations and offer the potential to unravel biophysical and biochemical phenomena. A major disadvantage associated with these methods is their ever-increasing cost resulting in the development of image analysis tools that offer the potential to exploit hidden information contained in confocal images.The hypothesis pertaining to this thesis is that image analysis tools developed in recent years exemplified by Raster Image Correlation Spectroscopy (RICS), Spatial Intensity Distribution Analysis (SpIDA) and Fluorescence Intensity Gaussian Mixture Model Analysis (FIGMMA) will provide a new insight into current pharmaceutical problems. The application of these methods to the quantification of protein aggregation, monomer/dimer equilibria, p-glycoprotein efflux activity and transcytosis are presented in this thesis.Protein aggregation poses a major challenge to the biotechnology industry which currently lacks analytical capabilities to profile broad particle size ranges. An in-house RICS (ManICS) software was validated against Dynamic Light Scattering and Fluorescence Correlation Spectroscopy (FCS) to determine Bovine Serum Albumin (BSA) aggregate population distributions under accelerated stability conditions. Initial stages implicated in the growth of aggregates are vital to the mechanistic assessment of protein aggregation. Hence, real-time in situ examination of monomer loss and aggregation of BSA was performed at 50 °C to enable continuous assessment with imaging and subsequent SpIDA analysis. Results obtained from this study suggested reversible fluctuation between monomers and dimers for up to four hours.To correlate membrane receptor and transporter expression with activity and enable the comparison of expression in multiple cell lines, population densities of p-glycoprotein transporters and transferrin receptors were determined using SpIDA in samples subjected to immunofluorescence labelling.The Calcein retention assay is a routine approach to determining multidrug resistance associated with p-glcoprotein efflux and the traditional plate reader approach omits microscopic aspects of p-glycoprotein Calcein-AM uptake and efflux. Confocal microscopy and data obtained from image analyses supported the subcellular and intercellular assessment of Calcein accumulation in MDR1-transfected and control cell lines as a function of time and verapamil concentration. Finally, live cell imaging of transferrin vesicular transport and Cell TraceTM Calcein red-orange AM internalisation in combination with traditional Transwell® assays were assessed to compare their transcellular transport and intracellular concentrations in multiple cell lines. Images obtained enabled visualisation of internalisation and following analysis using SpIDA, RICS and FIGMMA the number of intracellular vesicles and dynamic parameters of Cell TraceTM Calcein red-orange diffusion and intracellular concentration were determined.In conclusion, image analysis tools were applied to providing new parametric insights into a number of pharmaceutically-relevant processes and in some instances this is the first example of such studies. Despite current phenomenal advances in image acquisition capabilities, there remains a broad scope for the validation of image analysis tools and their application to a multitude of areas of interest to pharmaceutical and biomolecular research.

The full text of this thesis/dissertation is not available online. Please <a href="mailto:upetd@up.ac.za">contact us</a> if you need access. Read the abstract in the section 00front of this document. / Dissertation (MSc (Medical Immunology))--University of Pretoria, 2006. / Immunology / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第20065号 / 農博第2194号 / 新制||農||1045(附属図書館) / 学位論文||H28||N5021(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)准教授 赤松 美紀, 教授 植田 和光, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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