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.

Appendix included in back. / Errata posted on back end cover. / Bibliography: leaves 236-268. / xxii, 268, [7] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A contribution to the knowledge of multidrug resistance and its role in acute leukaemia. / Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and molecular genetics, 1997

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

Thesis (Ph. D.)--Northeastern University, 2008. / Bouvé College of Health Sciences, School of Pharmacy. Includes bibliographical references (p. 181-188).

Thesis (Ph.D.)--Northeastern University, 2008. / Bouvé College of Health Sciences, School of Pharmacy. Includes bibliographical references (p. 181-188).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 158-177).

Orientador: Eduardo Mello de Capitani / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T04:22:55Z (GMT). No. of bitstreams: 1 Nunes_ElizabeteAbrantes_D.pdf: 12821896 bytes, checksum: 69c5877e5bd28eeddfa61b9e06a31e01 (MD5) Previous issue date: 2005 / Resumo: A tuberculose, a resistência aos medicamentos antituberculose e o HIV são hoje em dia três grandes endemias, com tendências similares e com prejuízos incalculáveis para a humanidade, em particular nos países de baixos recursos. Obiectivos do trabalho: Determinar o padrão de resistência aos medicamentos antituberculose, em pacientes HIV positivos, portadores de doença pulmonar por micobactéria tuberculosa e micobactéria não tuberculosa na região de Maputo e a prevalência de MOTT nesta população. População e métodos: estudo elaborado em dois hospitais da cidade de Maputo, Moçambique. Foram estudados 503 doentes com tuberculose pulmonar e HIV+. Obteve-se 282 amostras da expectoração e ou lavagem brônquica nas quais foi solicitado; baciloscopia, cultura de BK, cultura de micobactérias nào tuberculosas e teste de sensibilidade para os antibacilares. Foram também avaliadas as características clínicas, radiográficas, a contagem de CD4 e o perfil hematológico. Resultados: Em 229 (98,7%) dos isolados, o M tuberculosis foi a principal micobactéria identificada. As micobactérias não tuberculosas, surgiram em apenas 3 (1,3%) casos, identificadas na expectoração e com clínica compatível. Dos 282 doentes, 232 (82%) apresentaram sensibilidade aos medicamentos antituberculose (MAT) e 50 (17%) resistência a qualquer MAT. Quanto ao padrão de resistência 27 (13,6%) eram casos novos e 21 (26,6%) casos previamente tratados. Resistências mais observadas a qualquer MAT, foram de 13,6% nos CN e 26,6% nos PT. As resistências gerais aos diferentes MAT foram: 10 H- 14,9%, 20 S- 7,8%, 30 R - 6,4%. A resistência à R foi aumentada tanto nos CN como nos PT. A tuberculose multiresistente combinada, foi de 5,7%, sendo nos CN, 3% e PT, 11,4%. Factores de risco de resistência e de TB-MR, foram identificados: tratamento anterior de TB e CD4<200. Estes doentes apresentaram mediana de CD4 de 151 cels/mm3, mediana de Hgb de 7,8g1dl e de CTL 1140. Do ponto de vista radiológico o padrão atípico foi o mais frequente e cavidades foram observadas num grupo reduzido, predominando nos PT e nos casos que apresentaram poliresistência aos MAT.O sarcoma de kaposi pulmonar surgiu em 4,8% dos doentes e as infecções fiíngicas/bacterianas em 27,9% Conclusões: A tuberculose multiresistente (TB-:MR)apresentou níveis elevados pelo que se deve reduzir o risco de transmissão da TB com medidas de controle da transmissão nosocomial e na comunidade e ampliar a DOTS estratégia a um maior número de população. Face à resistência elevada à H, aconselhamos a introdução de um 30MAT na fase de manutenção no regime de tratamento dos CN, teste de sensibilidade aos MAT no início dos retratamentos. A profilaxia com H em HIV+ na prevenção de TB e tratamento de infecção latente deverá ser analisada com cuidado devido á elevada resistência à H. Uso de cotrimoxazol para redução das causas de morte associadas ao HIV!TB. Tratamento antiretroviral e aconselhamento para teste voluntário de HIV em todos os doentes TB / Abstract: Tuberculosis, multidrug-resistant tuberculosis and mv are, today, the three greatest endemics with similar trends and immeasurable impairment to humanity,particularin low resources countries. Obiectives: Determine the resistance pattern to anti-tuberculosis drugs (ATD) in mv positive patients with pulmonary disease by mycobacteria tuberculosis and mycobacteria non tuberculosis in the Maputo region and the prevalence of MOTT in this population. Population and methods: the study was conducted in 503 patients with pulmonary tuberculosis and mv positive in two hospitaIs of Maputo City, Mozambique. Two hundred and eighty two (282) sputum samples and/or bronchial wash were submitted for testing of baciloscopy, BK culture, MOTT and Drug Susceptibility Testing (DST) for ATD. Clinical and radiographic characteristics, CD4 counting and hematological profile were also evaluated. Results: M tuberculosis was the main organism identified in 229 (98,7%) of the isolated samples. Non-tuberculosis mycobacteria were identified in sputum of only 3 (1,3%) of the cases with compatible clinic. Drug sensitivity was observed in 232 (82%) patients and resistance in 50 (17%) of the 282 sampled cases. In relation to the drug resistance pattems, 27 (13,6%) were in New Cases (NC) and 21 (26,6%) in Previously Treated cases (PT). More observed resistance to ATD was recorded in 13,6% in NC and 26,6% in PT. General resistant to different ATD was: 1° H- 14,9%, 2° S- 7,8%, 3° R- 6,4%. The resistance to R increased both in the NC as in PT. Overall, the MDR-TB was 5,7%, being 3% in NC and 11,4% in PT. The risk factors identified for resistance and MDR-TB were: previous treatment to TB and CD4 <200. These patients presented a median CD4 of 151 cells/mm3, a median of Hgb of 7,8 g/dl and of CTL 1140. From a radiological point ofview, the atipic pattem was the most frequent and cavities were observed in a small group predominant1yin PT and in-the cases that presented poliresistance to ATD. The pulmonary Kaposi sarcoma was observed in 4,8% ofthe patients and the fungall bacterial infections in 27,9%. Conclusions: The high levels of MDR-TB recorded in this study suggests that the risk of TB transmission should be reduced through control measures of nosocomial transmission and in the community and amplify the DOTS strategy to a greater population number. As a result of the high resistance to H, it is advised to introduce one of the 3o ATDin the maintenance phase of the treatment regime of NC, and sensitivity tests to ATD in the beginning of retreatment. Prophylaxis with H in HIV positive for TB prevention and latent infection treatment should be analyzed carefully, due to the high resistance to H. The use of cotrimoxazol to reduce the death causes associated to lllV/TB. Anti-Retroviral Treatment (ATRV) is important and also counceling for HIV voluntary testing in all TB patients / Doutorado / Clinica Medica / Doutor em Clínica Médica

Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (&phi;SPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (&phi;SPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.

Philosophiae Doctor - PhD / Ovarian cancer is called the “Silent Killer” as it is often diagnosed in advanced stages of the disease or misdiagnosed which ends with a poor prognostic outcome for the patient. A high rate of disease relapse, a high incidence-to-mortality ratio as well as acquired multidrug resistance makes it necessary to find alternative diagnostic- and therapeutic tools for ovarian cancer. Nanotechnology describes molecular devices with at least one dimension in the sub- 1μm scale and has been suggested as a possible solution for overcoming challenges in cancer multidrug resistance as well as early diagnosis of the disease. One-pot synthesized gold nanoparticles were used to demonstrate in vitro drug delivery of doxorubicin in a manner which overcame the cytoprotective mechanisms of a multidrug resistant ovarian carcinoma cell line (A2780cis) by inducing apoptosis mediated by caspase-3 within 3h of treatment. The gold nanoparticles were further functionalized with nitrilotriacetic acid and displayed specific interaction with a 6xHis-tagged cancer targeting peptide, chlorotoxin. Proprietary indium based quantum dots were functionalized with the same surface chemistry used for gold nanoparticles and bioconjugated with chlorotoxin. Wide field fluorescence studies showed the peptide-quantum dot construct specifically targeted enhanced green fluorescent tagged matrix metalloproteinase-2 transfected A2780cis cells in a specific manner. The cytoprotective multidrug resistant mechanisms of the ovarian carcinoma was overcome successfully with a single dose of doxorubicin loaded gold nanoparticles and tumour specific targeting was demonstrated using quantum dots with a similar surface chemistry used for the gold nanoparticles.

>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.