Linezolida foi o primeiro fármaco da classe das oxazolidinonas a ser aprovado para o uso clínico. Esta nova oxazolidinona inibe a síntese protéica impedindo a formação do complexo de iniciação formado pelo mRNA, tRNA f-Met e a subunidade 50S do ribossomo bacteriano. Embora a resistência à linezolida possa ser mediada pelo produto do gene cfr ou por mutações nas proteínas ribossômicas L3, L4 e L22, o mecanismo de resistência mais comum envolve mutações no domínio V do gene rRNA 23S. Entre março de 2008 a dezembro de 2011, 38 cepas de estafilococos coagulase-negativos (SCNs) resistentes à linezolida (20 S. epidermidis, 14 S. haemolyticus, 3 S. hominis e 1 S. warneri) isoladas de hemoculturas e pontas de cateter de pacientes internados em dois hospitais terciários do Estado de São Paulo foram incluídas neste estudo para a determinação dos mecanismos de resistência e análise da estabilidade do fenótipo resistente. As cepas de SCNs apresentaram altos níveis de resistência à linezolida (CIMs de 16-128 µg/ml) e foram multi-resistentes, permanecendo sensíveis à vancomicina e teicoplanina. A mutação G2576T foi identificada no domínio V do gene rRNA 23S em todas as cepas de SCNs, exceto em uma cepa de S. haemolyticus. O gene cfr e mutações nas proteínas L4 e L22 não foram detectados. Em relação à proteína L3, todas as cepas de S. epidermidis do hospital A, incluindo a cepa controle sensível à linezolida, apresentaram a substituição Leu101Val, sugerindo que essa mutação seja um marcador clonal dessa população sem envolvimento com a resistência à linezolida. A única cepa proveniente do hospital B (S. epidermidis) foi selvagem para essa proteína ribossômica. Somente uma cepa de S. haemoyticus teve uma mutação no gene rplC, resultando na alteração Val154Leu. Em S. hominis, a mutação Phe147Ile foi identificada em uma cepa, enquanto a associação de Gly139Arg e Met156Thr foi observada nas outras duas cepas dessa espécie. A identificação dessas mutações na proteína L3 de cepas de S. haemoyticus e S. hominis resistentes à linezolida reforça o papel desses sítios na aquisição da resistência ao fármaco em Staphylococcus spp. No entanto, a presença de G2576T no rRNA 23S torna difícil determinar exatamente qual o envolvimento das mutações na L3 com os elevados níveis de resistência à linezolida apresentados por essas cepas. Na ausência da pressão seletiva do antimicrobiano, após 130 passagens, a resistência à linezolida mediada pela mutação G2576T permaneceu estável nas cepas de SCNs deste estudo, as quais, de acordo com os perfis de restrição do domínio V gerados por NheI, tinham tanto alelos rRNA 23S selvagens como mutados. O sequenciamento individual do domínio V das diferentes cópias do gene rRNA 23S mostrou G2576T em todas as cópias amplificadas por PCR: 4/4 e 5/5 em S. epidermidis e 3/3 em S. haemolyticus (CIMs de 16-32 µg/ml). A estabilidade da cópia rRNA 23S mutada foi observada mesmo em uma cepa S. epidermidis sensível à linezolida, a qual apresentou uma redução da CIM de 4 para 1 µg/ml mantendo seu único alelo mutado ao longo do processo de reversão ao fenótipo sensível. A similaridade genética foi determinada por PFGE e mostrou uma disseminação clonal das diferentes espécies de SCNs resistentes à linezolida. A análise das cepas de S. epidermidis por MLST mostrou a ocorrência do clone ST-2 (CC2) nos dois hospitais. O aumento da pressão seletiva devido a exposições cada vez mais frequentes à linezolida, provavelmente, favoreceu a seleção e a disseminação de clones endêmicos de SCNs com a mutação G2576T na instituição A desde 2008. De forma diferente, o uso mais restrito do fármaco na instituição B poderia explicar a ocorrência isolada de uma única cepa resistente desde 2005. / Linezolid was the first agent of the oxazolidinone class to be introduced clinically. This oxazolidinone inhibits protein biosynthesis by preventing the formation of the initiation complex that consists of the mRNA, the f-Met tRNA and the 50S subunit of the ribosome. Although linezolid resistance has been mediated by the cfr-encoded product or by ribosomal proteins (L3, L4 and L22), the most common mechanism of resistance involves mutations in the central loop of domain V of the 23S rRNA gene. From March 2008 to December 2011, 38 coagulase-negative staphylococci (CNS) strains (20 S. epidermidis, 14 S. haemolyticus, 3 S. hominis e 1 S. warneri) exhibiting resistance to linezolid were isolated from blood and catheter cultures from patients in two tertiary care hospitals in the State of São Paulo and were included in this study for the ascertainment of the resistance mechanisms to this antimicrobial agent and for the analysis of the stability of this resistance. The strains exhibited high-level resistance to linezolid (MICs 16-128 µg/ml) and all were multidrug resistant, remaining susceptible to vancomycin and teicoplanin. The G2576T mutation in domain V region of 23S rRNA was identified in all isolates, except in a linezolid-resistant S. haemolyticus strain. The cfr gene and mutations in ribosomal proteins L4 and L22 were not detected. Regarding L3 protein analysis, all S. epidermidis strains of hospital A, including the linezolid-susceptible control strain, showed the L3 Leu101Val mutation, suggesting that this alteration is probably not involved in linezolid resistance. The one strain from hospital B (S. epidermidis) was wild-type for this ribosomal protein. Only one S. haemolyticus strain had a mutation in the L3 protein, Val154Leu. Two S. hominis strains showed Gly139Arg/Met156Thr mutations whereas one strain had Phe147Ile in L3 protein. The identification of these mutations in L3 protein of the linezolid-resistant S. haemolyticus and S. hominis strains strengthens the role of these sites in the acquisition of linezolid resistance in Staphylococcus spp. However, the presence of G2576T in the 23S rRNA gene makes difficult to determine exactly the role of L3 mutations in conferring elevated linezolid MIC values showed by these clinical strains. In the absence of antibiotic pressure, after 130 passages, linezolid resistance was stable in the clinical strains of this study, which did not have all copies of the 23S rRNA gene mutated, according to the restriction of the domain V fragment with NheI enzyme. Sequencing of the individual copies of the 23S rRNA gene in the serially passaged strains showed G2576T in all amplified copies by PCR: 4/4 and 5/5 in S. epidermidis and 3/3 in S. haemolyticus strains (MIC of 16-32 µg/ml). The stability of the mutant rRNA copy was also observed in the linezolid-susceptible S. epidermidis strain (MIC of 4 µg/ml). After the passages in antibiotic-free medium, the linezolid MIC of this strain fell to 1 µg/ml and the G2576T mutation persisted in one 23S rRNA gene copy. The clonal relatedness of the strains was determined by PFGE and revealed a clonal dissemination of different CNS species. Regarding MLST analysis, all S. epidermidis strains belonged to the sequence type ST2 (CC2). Most likely, the increased selective pressure has contributed to the selection of endemic linezolid-resistant CNS clones showing the G2576T mutation that have been disseminated in the institution A since 2008. Differently, the restricted use of linezolid in the institution B could explain the occurrence of a single resistant strain since 2005.

Uma das causas mais freqüentes de insucesso terapêutico do câncer, está associada à Resistência a Múltiplas Drogas (MDR). A resistência a múltiplas drogas refere-se ao desenvolvimento de resistência simultânea a uma variedade de agentes citotóxicos que apresentam diferentes sítios de ação e estruturas químicas diversas. Os mecanismos MDR são representados por genes cujos produtos funcionam como bombas, reduzindo o acúmulo intracelular de drogas e a resposta ao tratamento. O linfoma é uma neoplasia comum na espécie, sendo muito responsiva à quimioterapia, embora a recidiva seja esperada e possa estar relacionada ao MDR. O presente estudo teve como objetivo avaliar a expressão dos genes MDR-1, MRP e LRP e de seus produtos, em cães com linfoma. Foram colhidas amostras de linfonodos periféricos de 15 cães com linfoma multicêntrico ao diagnóstico e na recidiva (durante a quimioterapia). A expressão dos genes e proteínas MDR foram determinados por RT-PCR e \"Dot Blotting\", respectivamente. As freqüências de expressão ao diagnóstico dos genes MDR, MRP e LRP foram 93,3% igualmente e as freqüências de seus produtos foram 85,8%; 71,5% e 85,8% respectivamente. Na recidiva, as freqüências de expressão dos genes MDR, MRP e LRP elevaram-se para 100% igualmente e as freqüências de suas proteínas foram 92,9% para P-gp e MRP e de 100% para LRP. Portanto, obteve-se uma alta freqüência na expressão do MDR-1/P-gp, MRP e LRP, em quase todos os cães, não só na recidiva, como também ao diagnóstico. É necessário verificar se estes mecanismos podem induzir o fenótipo MDR no linfoma canino multicêntrico. O estudo dos mecanismos MDR trará novas perspectivas para o tratamento de tumores como o linfoma canino. A modulação desses mecanismos por meio de drogas específicas ou por terapia gênica, pode contribuir para o sucesso do tratamento de neoplasias caninas ou humanas. / One of the most frequent causes of treatment failure in cancer is associated to Multiple Drug Resistance (MDR). MDR refers to simultaneous resistance to a variety of cytotoxic agents that have different targets and diversal chemical structures. MDR mechanisms are represented by genes wich encodes proteins that work like pumps, reducing intracellular drug acumulation and the response to treatment. Lymphoma is a common neoplasia in dogs wich is very responsive to chemotherapy, although relapses are expected, including those related to MDR. The aim of this study was to evaluate the possible expression of MDR-1, MRP and LRP genes and their proteins, in dogs with lymphoma. Lymph node samples at diagnosis and at relapse (during chemotherapy) from 15 dogs with multicentric lymphoma were obtained. The expression of MDR genes and proteins were determined by RT-PCR and Dot Blotting. Expression of MDR, MRP and LRP gene were 93,3% at diagnosis and their products were 85,8%; 71,5% and 85,8% respectively. Expression of MDR, MRP and LRP gene increased to 100% at relapse and their proteins were 92,9% to P-gp and MRP and 100% to LRP. High frequency of MDR-1/P-gp, MRP and LRP expressions were found in almost all dogs, not only at relapse but also at diagnosis. It\'s necessary to analyse if these mechanisms can induce the MDR fenotype in canine multicentric lymphoma. The study of MDR mechanisms will conduct the treatment of tumors like canine lymphoma to new perspectives. The modulation of these mechanisms by certain drugs or gene therapy, may contribute to the success in treatment of canine or human neoplasias.

Para avaliar a sensibilidade da colistina e polimixina B de isolados de P. aeruginosa multirresistentes foram utilizadas diferentes metodologias, como microdiluição, Etest e disco-difusão, cujos resultados evidenciaram que a microdiluição continua sendo o método de referência. Do total de 109 isolados, não houve resistência para colistina e apenas um isolado mostrou-se resistente para a polimixina B. Para determinar a linhagem clonal destes isolados foi empregada a técnica de eletroforese em campo pulsado, a qual evidenciou a existência de um padrão molecular predominante durante o período de estudo e a presença de 7 grupos de isolados de P. aeruginosa multirresistentes em 2003 / -

A resistência bacteriana emergiu como importante problema de saúde pública no mundo. Nesta pesquisa, a distribuição das espécies e a evolução da sensibilidade aos antibióticos entre isolados clínicos obtidos em um hospital terciário foram analisadas no período de 2007 a 2012. As bactérias isoladas foram identificadas por análises bioquímicas convencionais. Segundo as recomendações do Clinical and Laboratory Standards Institute (CLSI) compatíveis ao ano do processamento microbiológico, o perfil de sensibilidade foi determinado pelo método de disco difusão, entretanto para a sensibilidade a vancomicina utilizou-se a concentração inibitória mínima (CIM). Durante o período da pesquisa totalizou-se 4.464 resultados de culturas distribuídos em 2007 (865), 2008 (981), 2009 (485), 2010 (539), 2011 (704) e 2012 (890). Com relação aos cocos Gram-positivos e as enterobactérias, Staphylococcus aureus e Eschericia coli foram as bactérias mais frequentemente isoladas, respectivamente. Dos antibióticos da classe dos beta-lactâmicos, piperacilina + tazobactam e aztreonam mostraram os melhores resultados de atividade antibacteriana. Todas as cepas isoladas de enterobactérias foram sensíveis aos carbapenêmicos. As cepas de Pseudomonas aeruginosa foram mais sensíveis ao imipenem do que ao meropenen, no entanto a redução dos perfis de sensibilidade foi evidenciada para ambos os antibióticos: imipenem (69,6% para 41,7%) e meropenem (63,3% para 25,0%). Todas Burkloderoderia cepacea e Acinetobacter baumanii demonstraram resistência ao meropenem, entretanto as cepas de Acinetobacter iuwoffi foram sensíveis aos carbapenêmicos. Aumentos semelhantes nos perfis de sensibilidade das cepas de Escherichia coli, Klebsiella pneumoniae e Klebsiella oxytoca (70% para 86,7%) foram observados para ciprofloxacina e levofloxacina. Da classe dos aminoglicosídeos, a amicacina mostrou melhor atividade antibacteriana do que a gentamicina. Nas amostras analisadas deste hospital não houve ocorrência de Enterococcus spp. resistente a vancomicina (VRE). Ainda, todas as cepas de cocos Gram-positivos foram sensíveis a vancomicina e teicoplamina. No geral os antibióticos apresentaram resultados preocupantes, uma vez que para as bactérias reconhecidas nos cenários das infecções hospitalares nenhuma foi sensível 100% a todas as classes de antibióticos. A situação da sensibilidade microbiana aos antibióticos é caótica tendo cada vez mais limitada a sua utilização na terapêutica / Bacterial resistance has emerged as an important public health problem in the world. In this study, the distribution of species and the evolution of antibiotic susceptibility among clinical isolates in a tertiary hospital were analyzed in the period from 2007 to 2012. Bacterial isolates were identified by conventional biochemical analyzes. According to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) supported a year of microbiological processing, the sensitivity was determined by the disk diffusion method, however for sensitivity to vancomycin was used the minimum inhibitory concentration (MIC). During the research period, 4,464 culture results were obtained and distributed in 2007 (865), 2008 (981), 2009 (485), 2010 (539), 2011 (704) and 2012 (890). With respect to Gram-positive cocci and Enterobacteriaceae, Staphylococcus aureus and Escherichia coli were the most frequently isolated bacteria, respectively. From beta-lactams class, piperacillin + tazobactam and aztreonam showed the best results of antibacterial activity. All isolated strains of Enterobacteriaceae were susceptible to carbapenems. Pseudomonas aeruginosa strains were more sensitive to imipenem than the meropenen, however reducing the sensitivity profile was observed for both antibiotics imipenem (69.6% to 41.7%) and meropenem (63.3% for 25.0%). All Burkloderoderia cepacia and Acinetobacter baumannii were resistant to meropenem, however Acinetobacter iuwoffi strains were susceptible to carbapenems. Similar increases in the susceptibility of Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca strains (70% to 86.7%) were observed for ciprofloxacin and levofloxacin. From aminoglycosides class, amikacin showed better antibacterial activity than gentamicin. In the samples analyzed in this hospital there was no occurrence of Enterococcus spp. resistant to vancomycin (VRE). Furthermore, all strains of Gram-positive cocci were susceptible to vancomycin and teicoplanin. Overall antibiotics showed worrying results, since none was recognized for bacteria in the nosocomial infection scenarios 100% sensitive to all classes of antibiotics. The situation of microbial sensitivity to antibiotics is becoming chaotic having limited their use in therapy

A tuberculose, causada por Mycobacterium tuberculosis, ainda é uma emergência de saúde pública global. O surgimento das cepas multirresistentes (MDR) e das cepas extensivamente resistentes (XDR) agravam a situação, diminuindo o número de fármacos disponíveis para o tratamento. Embora a isoniazida seja uma das primeiras moléculas introduzidas no tratamento da tuberculose, diferentes mecanismos de resistência têm sido propostos e o tema ainda não foi totalmente esclarecido. Neste trabalho foi realizada a caraterização estrutural e biofísica de 7 mutantes da proteína InhA identificadas a partir de isolados clínicos de M. tuberculosis resistentes à isoniazida. Os ensaios de calorimetria de titulação isotérmica (ITC) mostram diminuições nos valores da constante de dissociação (Kd) dos mutantes para os NADH em aproximadamente cinco vezes quando comparado com a proteína selvagem. As estruturas cristalográficas dos mutantes de InhA mostram novas moléculas de água que parecem estar envolvidas nas variações entrópicas e entálpicas observadas em dados calorimétricos. Estes resultados corroboram e sugerem que a diminuição na afinidade pelo NADH e a desestabilização do tetrâmero de InhA podem ser fenômenos associados a resistência à isoniazida. / Tuberculosis, caused by the infection of Mycobacterium tuberculosis, still remains as a global health emergency. The emergence of multidrug-resistant strains (MDR) and extensively drug-resistant strains (XDR) strains further aggravates the crisis, reducing the limited number of drugs available for the treatment of the disease. Even though isoniazid was one of the first drugs introduced in the antitubercular therapy, many resistance mechanisms were proposed and the subject is still not clear. In this work, a structural and biophysical characterization of seven mutant InhA proteins identified in clinical M. tuberculosis strains resistant to isoniazid were performed. Isothermal titration calorimetry (ITC) assays showed a decrease in the dissociation constant (Kd) values of the InhA mutants by up to almost five-fold when compared to the wild-type protein. Crystallographic structures of InhA mutants showed new water molecules that appear to be involved in the entropic and enthalpic variations described by the thermodynamic assays. These results corroborate and suggest that the decrease in affinity for NADH and the destabilization of the InhA tetramer may be the phenomena associated to isoniazid resistance.

Introdução: Infecções por Enterobactérias resistentes aos carbapenêmicos (ERC), em especial produtoras de Klebsiella pneumoniae carbapenamase tipo KPC hoje são endêmicas em diversas regiões do mundo, seu tratamento é ainda um grande desafio em particular de isolados resistentes à polimixina. Objetivos: Descrever as características clínicas, microbiológicas e moleculares das infecções por ERC. Método: Estudo de coorte prospectiva, realizado no Hospital Universitário de Londrina, Paraná, Brasil, entre março de 2011 a dezembro de 2012. Foram acompanhados pacientes >= 18 anos, que apresentaram infecção por ERC. Dados demográficos e clínicos como idade, sexo, diagnóstico à admissão e presença de co-morbidades de acordo com critérios de Charlson, internação em Unidade de Terapia intensiva e scores APACHE e SOFA desses pacientes, colonização prévia por ERC, cirurgia prévia à infecção, diálise, uso prévio de antimicrobianos e sítio de infecção foram coletados. Foram avaliados os antimicrobianos utilizados para tratamento das infecções por mais de 48 horas nos seguintes pontos: monoterapia ou terapia associada, tempo de início (menor e maior que 12 horas). A identificação do agente foi realizada por método automatizado (Vitek II - bioMerieuxR) e a concentração inibitória mínima dos antibióticos por técnica de microdiluição em caldo, pesquisa de gene blaKPC pela técnica de Polimerase Chain Reaction e sinergismo entre drogas utilizadas em tratamento combinado por meio do método Time Kill. A clonalidade, por Pulsed Field gel eletroforese e analisada por dendograma pelo Bionumerics. Foram realizadas análise bivariada e regressão logística multivariada com técnica de Forward Stepwise para detectar fatores de risco para resistência a polimixina e mortalidade. O nível de significância adotado foi de 5%, utilizando os programas Epi Info 7.0 e SPSS. Resultados: No período de estudo, 127 pacientes apresentaram infecções por ERC, idade média de 55,7 (± 18) anos e 88 (69.3%) do sexo masculino. Infecções de trato respiratório (52-42%) e trato urinário (51 - 40,2%) foram as mais freqüentes, 27 (21,3%) resistentes à polimixina, 113 (89%) das enterobactérias eram K. pneumoniae e 96 (75,6%) tinham gene blaKPC.. Cinquenta e cinco (43,3%) eram polimicrobianas, a maioria (28,3%) co-infecção por Acinetobacter baumannii. A taxa de mortalidade hospitalar foi 61,4%, sendo 34,6% relacionada à infecção e não houve diferença significativa entre os grupos sensíveis (34%) e resistentes à polimixina (37%), p=0.46. Os fatores de risco independentes para óbito foram choque (OR 27.40; IC95% 1.68-446.82; p= 0.02) e diálise (OR 13.26; IC95% 1.17-149.98; p= 0.03); para resistência à polimixina: uso prévio de carbapenem ( OR 2.95; IC95% 1.12-7.78; p= 0.02) e para óbito nessa população: diálise (OR 7,58; IC95% 1,30-43,92; p= 0.02). Terapia combinada, tempo de início de antibiótico sensível e sinergismo in vitro não tiveram impacto significativo na mortalidade. Conclusão: O uso prévio de carbapenêmico foi o único fator associado com a resistência à polimixina nesse estudo. Os fatores associados ao óbito entre os pacientes com infecções por enterobactérias resistentes à polimixina foram fatores de gravidade, como diálise e choque. Nenhuma opção terapêutica, em especial a associação de drogas e nem o tempo de início do tratamento, interferiu na mortalidade deste grupo de pacientes / Introduction: Infections due to Carbapenem resistant Enterobacteriaceae (CRE), particularly Klebsiella pneumoniae producing carbapenemase type KPC, have been endemic in several regions around the world. Their treatment remains a major challenge, particularly for isolates resistant to polymyxin. Objectives: To describe the clinical, microbiological and molecular characteristics of infections by CRE. Methods: Prospective cohort conducted at the University Hospital of Londrina, Paraná, Brazil, from March 2011 to December 2012. All hospitalized patients >= 18 years old who developed infection by CRE were followed until death or discharge. We collected and analyzed the following clinical data: age, sex, diagnosis at admission, presence of comorbidities according to the Charlson criteria, admission in Intensive Care Unit, APACHE and SOFA scores, previous colonization by CRE, previous surgery, dialysis, prior antibiotic use and infection site; furthermore, we also evaluated the time between the blood culture collect and the first antimicrobial dose administration (start time - smaller or longer than 12 hours) as well as whether the treatment was monotherapy or combine therapy for more than 48 hours. The microbiological identification was performed by automated method (Vitek II - bioMerieuxR) and the minimum inhibitory concentration of antibiotics by broth microdilution technique, research blaKPC gene by the technique of Polymerase Chain Reaction and synergism between the drugs used in the combination therapy by Time Kill method. The clonality was carried out by pulsed-field gel electrophoresis and analyzed by dendrogram by BioNumerics. Bivariate analyses and multivariate logistic regression with forward stepwise technique were performed to detect risk factors for resistance to polymyxin and mortality. The level of significance was 5%, using Epi Info 7.0 and SPSS programs. Results: During the study period, 127 patients developed infections by CRE, mean age 55.7 (± 18) years and 88 (69.3%) were male. Respiratory tract infections 52 (42%) and urinary tract 51 (40.2%) were the most frequent. Twenty seven (21.3%) agents were resistant to polymyxin; 113 (89%) were K. pneumoniae and 96 (75.6%) had blaKPC gene. Fifty-five (43.3%) were polymicrobial, the majority (28.3%) co-infection by Acinetobacter baumannii. The hospital mortality rate was 61.4% and 34.6% of the death were related to infection. There was no difference in mortality rate between sensitive (34%) versus resistant (37%)(p = 0.46) to polymyxin. The independent risk factors for death were shock (OR 27.40; 95% CI 1.68-446.82; p = 0.02) and dialysis (OR 13:26; 95% CI 1.17-149.98; p = 0.03); and for resistance to polymyxin were previous use of carbapenem (OR 2.95; 95% CI 1.12-7.78; p = 0.02). The risk factor for death in our study was dialysis (OR 7.58; 95% CI 1.30 to 43.92; p = 0.02). Combine therapy, start time and sensitive and antibiotic synergy in vitro had no significant impact on mortality. Conclusion: In our study, previous carbapenem use was the only factor associated with resistance to polymyxin. Furthermore, dialysis and shock were the only factors associated with death among patients with infections caused by CRE resistant to polymyxin. No therapeutic option, especially the combination of drugs and the start time decreased the higher mortality rates in this group of patients

by To Siu Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 84-92). / Abstract also in Chinese. / Declaration --- p.i / Abstract --- p.ii / Acknowledgement --- p.vi / Abbreviations --- p.vii / List of Figures --- p.viii / List of Tables --- p.xii / Content --- p.xiv / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- Osteosarcoma --- p.1 / Chapter 1.1.1. --- Incidence / Chapter 1.1.2. --- Age and Sex Distribution / Chapter 1.1.3. --- Clinical Features / Chapter 1.1.4. --- Treatment / Chapter 1.2. --- Adriamycin --- p.9 / Chapter 1.2.1. --- Drug Action / Chapter 1.2.2. --- Pharmacology / Chapter 1.3. --- Multidrug Resistance --- p.11 / Chapter 1.4. --- P-glycoprotein --- p.13 / Chapter 1.4.1. --- Nature / Chapter 1.4.2. --- Tissue Distribution / Chapter 1.4.3. --- Relation with MDR / Chapter 1.5 --- Multidrug Resistance Protein --- p.16 / Chapter 1.6. --- Reactive Oxygen Species --- p.17 / Chapter 1.6.1. --- Problems Arising from ROS / Chapter 1.6.2. --- Oxidative Stress and Diseases / Chapter 1.6.3. --- Defense System / Chapter 1.6.4. --- Antioxidative Enzymes / Chapter 1.6.5. --- Relation with MDR / Chapter 1.7. --- Topoisomerase II --- p.22 / Chapter 1.8. --- Methods to Detect MDR --- p.24 / Chapter 1.8.1. --- P-glycoprotein Immunohistochemistry / Chapter 1.8.2. --- Adriamycin Binding Assay / Chapter 1.9. --- Aims of Study --- p.25 / Chapter 2. --- MATERIALS AND METHODS --- p.27 / Chapter 2.1. --- Clinical Study --- p.27 / Chapter 2.1.1. --- Patients Recruitment / Chapter 2.1.2. --- Adriamycin Binding Assay / Chapter 2.1.3. --- P-glycoprotein Immunohistochemistry / Chapter 2.1.3.1. --- Sample and Control Preparation / Chapter 2.1.3.2. --- Immunohistochemical Procedure / Chapter 2.1.4. --- Tumour Necrosis Assessment / Chapter 2.2. --- Effect of Adriamycin on Osteosarcoma Cells --- p.32 / Chapter 2.2.1. --- Establishment of Adriamycin Adapted Osteosarcoma Cells / Chapter 2.2.1.1. --- Maintenance and Subculture of SaOS-2 Cell Line / Chapter 2.2.1.2. --- Storage of Cell Line / Chapter 2.2.1.3. --- Adriamycin Treatment / Chapter 2.2.2. --- KB-V1 Cell Culture / Chapter 2.2.3. --- Adriamycin Binding Assay / Chapter 2.2.4. --- P-glycoprotein Immunohistochemistry / Chapter 2.2.4.1. --- Sample and Control Preparation / Chapter 2.2.4.2. --- Immunohistochemical Procedures / Chapter 2.2.5. --- Thymidine Incorporation Assay / Chapter 2.2.5.1. --- Assay Procedures / Chapter 2.2.6. --- Catalase Assay / Chapter 2.2.6.1. --- Assay Procedures / Chapter 2.2.6.2. --- Unit Calculation / Chapter 2.2.7. --- Glutathione Peroxidase Assay / Chapter 2.2.7.1. --- Assay Procedures / Chapter 2.2.7.2. --- Unit Calculation / Chapter 2.2.8. --- Protein Determination / Chapter 2.3. --- Statistical Analysis --- p.45 / Chapter 3. --- RESULTS --- p.46 / Chapter 3.1. --- Clinical Study --- p.46 / Chapter 3.1.1. --- Patients Recruitment / Chapter 3.1.2. --- Correlation of Adriamycin Sensitivity to Tumour Necrosis / Chapter 3.1.3. --- Correlation of P-glycoprotein Expression to Tumour Necrosis / Chapter 3.1.4. --- Correlation of P-glycoprotein Expression to Adriamycin Sensitivity / Chapter 3.2. --- Effect of Adriamycin on Osteosarcoma Cells --- p.63 / Chapter 3.2.1. --- Adriamycin Sensitivity and P-glycoprotein Expression / Chapter 3.2.2. --- Thymidine Incorporation Rate / Chapter 3.2.3. --- Intracellular Concentration of Catalase / Chapter 3.2.4. --- Intracellular Concentration of Glutathione Peroxidase / Chapter 4. --- DISCUSSIONS --- p.71 / Chapter 4.1. --- Clinical Study --- p.71 / Chapter 4.1.1. --- Patients Recruitment / Chapter 4.1.2. --- Correlation between Adriamycin Sensitivity and Tumour Necrosis / Chapter 4.1.3. --- Correlation between P-glycoprotein Expression and Tumour Necrosis / Chapter 4.1.3.1. --- P-glycoprotein Is Induced During Chemotherapy / Chapter 4.1.3.2. --- P-glycoprotein Cannot Serve As a Prognostic Factor / Chapter 4.1.4. --- Correlation Between Adriamycin Sensitivity and P-glycoprotein Expression / Chapter 4.2. --- Effect of Adriamycin on Osteosarcoma Cells --- p.76 / Chapter 4.2.1. --- Adriamycin Sensitivity and P-glycoprotein Expression / Chapter 4.2.2. --- Proliferation Rate / Chapter 4.2.3. --- Antioxidative Enzymes Activities / Chapter 5. --- CONCLUSION --- p.82 / Chapter 6. --- FURTHER STUDY --- p.83 / Chapter 7. --- BIBLIOGRAPHY --- p.84 / Chapter 8. --- APPENDIX - SOLUTIONS PREPARATION --- p.93

by Chung Lung Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 149-157). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abbreviations --- p.ii / Abstracts --- p.v / List of figures --- p.ix / List of tables --- p.xii / Contents --- p.xiii / Contents / General Introduction --- p.1 / Chapter CHAPTER ONE --- CISPLATIN RESISTANCE MECHANISMS / Chapter 1.1 --- INTRODUCTION --- p.3 / Chapter 1.1.1 --- History of Cisplatin as An Anticancer Drug --- p.3 / Chapter 1.1.2 --- Active Mechanisms of Cisplatin --- p.8 / Chapter 1.1.3 --- Formation of DNA Adducts --- p.8 / Chapter 1.1.4 --- Cisplatin Resistance Mechanisms --- p.9 / Chapter 1.1.4.1 --- Intracellular Accumulation of Cisplatin --- p.11 / Chapter 1.1.4.2 --- Glutathione-S-transferase and Glutathion --- p.12 / Chapter 1.1.4.3 --- Metallothionein --- p.16 / Chapter 1.1.4.4 --- Cell Cycle Perturbation --- p.16 / Chapter 1.1.4.5 --- P-glycoprotein --- p.17 / Chapter 1.1.4.6 --- Multidrug Resistant Protein --- p.19 / Chapter 1.1.4.7 --- Topoisomerase II --- p.20 / Chapter 1.1.4.8 --- DNA Repair --- p.22 / Chapter 1.1.4.9 --- Induction of Programme Cell Death --- p.23 / Chapter 1.2 --- OBJECTIVES --- p.27 / Chapter 1.3 --- MATERIALS AND METHODS / Chapter 1.3.1 --- Materials --- p.28 / Chapter 1.3.2 --- Methods --- p.31 / Chapter 1.3.2.1 --- Cell Lines --- p.31 / Chapter 1.3.2.2 --- Drug Sensitivity Assay --- p.31 / Chapter 1.3.2.3 --- Platinum Uptake --- p.32 / Chapter 1.3.2.4 --- Cell Cycle Analysis --- p.32 / Chapter 1.3.2.5 --- Western Blot Analysis --- p.33 / Chapter 1.3.2.6 --- Glutathione Content Determination --- p.36 / Chapter 1.3.2.7 --- DNA Fragmentation --- p.36 / Chapter 1.3.2.8 --- JC-1 Staining --- p.37 / Chapter 1.3.2.9 --- HE and DCF Staining --- p.38 / Chapter 1.3.2.10 --- Quantitative RT-PCR --- p.38 / Chapter 1.4 --- RESULTS / Chapter 1.4.1 --- Cisplatin Sensitivity of A431 Cells by MTT Assay --- p.40 / Chapter 1.4.2 --- Cross-resistance to Anti-cancer Drugs --- p.40 / Chapter 1.4.3 --- Quantitation of Cisplatin Accumulation in A431 Cells by AAS --- p.44 / Chapter 1.4.4 --- Drug Detoxification Agent --- p.45 / Chapter 1.4.5 --- Detection of Cell Cycle Arrest by Flow Cytometer --- p.47 / Chapter 1.4.6 --- Expression of Drug Resistance Related Genes --- p.48 / Chapter 1.4.7 --- Detection of Apoptosis by DNA Fragmentation --- p.50 / Chapter 1.4.8 --- Role of Mitochondria and Reactive Oxygen Species by Flow Cytometer --- p.52 / Chapter 1.4.9 --- Detection of Apoptotic mRNA Level by Quantitative RT-PCR --- p.57 / Chapter 1.4.10 --- Detection of Apoptotic Protein Level by Western Blot Analysis --- p.57 / Chapter 1.5 --- DISCUSSIONS --- p.59 / Chapter CHAPTER TWO: --- THE INTERACTION BETWEEN DRUG RESISTANCE MECHANISMS AND GROWTH FACTOR SIGNALLING PATHWAY / Chapter 2.1 --- INTRODUCTION --- p.63 / Chapter 2.1.1 --- Structure of EGF and EGFR --- p.64 / Chapter 2.1.2 --- Growth Factor Signal Transduction Pathway --- p.69 / Chapter 2.1.3 --- Biological Effect of EGF --- p.69 / Chapter 2.1.3.1 --- Modification of Drug Sensitivity by EGF --- p.71 / Chapter 2.2 --- OBJECTIVES --- p.74 / Chapter 2.3 --- MATERIALS AND METHODS / Chapter 2.3.1 --- Materials --- p.75 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Cell Lines --- p.76 / Chapter 2.3.2.2 --- Drug Sensitivity Assay --- p.77 / Chapter 2.3.2.3 --- Northern Blot Analysis --- p.77 / Chapter 2.3.2.4 --- Southern Blot Analysis --- p.78 / Chapter 2.3.2.5 --- Others --- p.78 / Chapter 2.4 --- RESULTS / Chapter 2.4.1 --- Sensitivity to EGF --- p.79 / Chapter 2.4.2 --- EGFR Expression Levels --- p.80 / Chapter 2.4.3 --- EGF Induced Protein Phosphorylation Pattern --- p.84 / Chapter 2.4.4 --- Effect of EGF on A431 Cells --- p.86 / Chapter 2.4.5 --- Response of Cells to Agents Targeting on EGF Signalling Pathway --- p.91 / Chapter 2.4.6 --- Response of Cells to Other Growth Factors --- p.97 / Chapter 2.4.7 --- Sensitivity of Cells to Different Anti-cancer Drugs --- p.99 / Chapter 2.4.8 --- Drug Resistance Mechanisms --- p.103 / Chapter 2.4.9 --- 5-Fluorouracil Sensitivity in A431 Cells --- p.108 / Chapter 2.4.10 --- Cisplatin Sensitivity in A431 Cells --- p.113 / Chapter 2.5 --- DISCUSSIONS --- p.117 / Chapter CHAPTER THREE: --- IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENE IN A431 CELLS BY DIFFERENTIAL DISPLAY / Chapter 3.1 --- INTRODUCTION --- p.122 / Chapter 3.2 --- MATERIALS AND METHODS / Chapter 3.2.1 --- Materials --- p.128 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Identification of Differentially Expressed Genes by RT-PCR / Chapter 3.2.2.2 --- Cloning of a Differentially Expressed cDNAs --- p.129 / Chapter 3.2.2.3 --- Screening and Sequencing of cDNA Inserts --- p.130 / Chapter 3.2.2.4 --- Rapid Amplification of cDNA Ends (RACE) --- p.131 / Chapter 3.2.2.5 --- Amplifcation Reaction --- p.131 / Chapter 3.2.2.6 --- Cloning and Sequencing of the RACE Fragment --- p.132 / Chapter 3.3 --- RESULTS / Chapter 3.3.1 --- Identification of novel cDNA by mRNA differential display --- p.133 / Chapter 3.4 --- DISCUSSIONS --- p.145 / General Conclusion --- p.147 / References --- p.149

by Cheung Yuen-Nei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 194-208). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.vi / List of Figures --- p.xii / List of Tables --- p.xv / Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1 --- Introduction --- p.2 / Chapter 1.1 --- Characteristic of Saponins --- p.3 / Chapter 1.1.1 --- Occurrence of Saponins --- p.3 / Chapter 1.1.2 --- General Properties of Saponins --- p.3 / Chapter 1.1.2.1 --- Emulsifying Agents --- p.3 / Chapter 1.2.2.2 --- Forming Complex with Cholesterol --- p.4 / Chapter 1.1.2.3 --- Hemolytic Property --- p.4 / Chapter 1.1.3 --- Structure of Saponins --- p.5 / Chapter 1.1.3.1 --- Categories of Saponins --- p.5 / Chapter 1.1.3.1.1 --- Triterpene Saponins --- p.5 / Chapter 1.1.3.1.2 --- Steroid Saponins --- p.5 / Chapter 1.1.3.2 --- Monodesmosidic and Bidesmosidic Saponins --- p.7 / Chapter 1.1.4 --- Biological and Pharmacological Properties of Saponins --- p.9 / Chapter 1.1.4.1 --- Anti-microbial Activity --- p.9 / Chapter 1.1.4.1.1 --- Anti-fungal Activities --- p.9 / Chapter 1.1.4.1.2 --- Anti-bacterial Activities --- p.10 / Chapter 1.1.4.1.3 --- Anti-viral Activities --- p.10 / Chapter 1.1.4.2 --- Insecticidal Activity --- p.10 / Chapter 1.1.4.3 --- Molluscicidal Activity --- p.10 / Chapter 1.1.4.4 --- Hypocholesterolemic Activity --- p.11 / Chapter 1.1.4.5 --- Anti-ulcer Activity --- p.11 / Chapter 1.1.4.6 --- Contraceptive Activity --- p.12 / Chapter 1.1.4.7 --- Immunomodulatory Activities --- p.12 / Chapter 1.1.4.7.1 --- Direct Immunostimulation --- p.12 / Chapter 1.1.4.7.2 --- Acting as Immuno-adjuvants --- p.13 / Chapter 1.1.4.8 --- Anti-tumor Activity --- p.14 / Chapter 1.1.4.8.1 --- Anti-carcinogenesis --- p.15 / Chapter 1.1.4.8.2 --- Suppression of Tumor Growth --- p.16 / Chapter 1.1.5 --- Anti-tumor Activity of Steroid Saponins --- p.18 / Chapter 1.1.5.1 --- Diosgenin Steroid Saponin --- p.18 / Chapter 1.1.5.2 --- Hong Kong Compounds --- p.18 / Chapter 1.1.5.3 --- Hong Kong18 --- p.21 / Chapter 1.2 --- Human Hepatocellular Carcinoma (HCC) --- p.24 / Chapter 1.2.1 --- The Incidence of Liver Cancer --- p.24 / Chapter 1.2.2 --- Classification of Liver Cancer --- p.24 / Chapter 1.2.3 --- Human Hepatocellular Carcinoma Cell Lines --- p.25 / Chapter 1.2.3.1 --- Human Hepatocellular Carcinoma Cell Line HepG2 --- p.25 / Chapter 1.2.3.2 --- Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.27 / Chapter 1.2.3.2.1 --- Mechanisms of Multidrug Resistance --- p.28 / Chapter 1.2.3.2.2 --- Structure and Characteristics of P-glycoprotein --- p.29 / Chapter 1.2.3.2.3 --- Methods in Dealing with P-glycoprotein Over-expressed MDR Cells --- p.31 / Chapter 1.3 --- Objectives of the Project --- p.32 / Chapter 1.3.1 --- Study of the Anti-tumor Activities of Hong Kong 18 on Human Hepatocellular Carcinoma Cell Line HepG2 and Unravel the Underlying Mechanisms --- p.32 / Chapter 1.3.2 --- Study of the Anti-tumor Activities of Hong Kong 18on Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 and Unravel the Underlying Mechanisms --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Cell Culture --- p.34 / Chapter 2.1.1.1 --- Cell Lines --- p.34 / Chapter 2.1.1.2 --- Culture Media --- p.35 / Chapter 2.1.2 --- Reagents and Buffers --- p.36 / Chapter 2.1.2.1 --- Phosphate Buffered Saline (PBS) --- p.36 / Chapter 2.1.2.2 --- Reagents and Buffers for DNA Fragmentation --- p.36 / Chapter 2.1.2.3 --- Reagents and Buffers for Western Analysis --- p.37 / Chapter 2.1.2.4 --- Reagents and Buffer for Caspases Activities --- p.39 / Chapter 2.1.2.5 --- Fluorescent Dyes used for Flow Cytometry --- p.39 / Chapter 2.1.3 --- Chemicals --- p.39 / Chapter 2.2 --- Methods --- p.46 / Chapter 2.2.1 --- MTT Assay --- p.46 / Chapter 2.2.2 --- Determination of Cell Viability --- p.46 / Chapter 2.2.3 --- Purification of Macrophages from balb/c Mice --- p.47 / Chapter 2.2.4 --- Hemolysis Assay --- p.47 / Chapter 2.2.5 --- In vivo Studies of the Toxicity of HK18 --- p.48 / Chapter 2.2.6 --- DNA Fragmentation Assay --- p.50 / Chapter 2.2.7 --- Detection of Apoptotic and Necrotic / Late Apoptotic Cells Death by Flow Cytometry with Annexin V-FITC / PI --- p.51 / Chapter 2.2.8 --- Detection of Mitochondrial Membrane Potential by JC-1 Fluorescent Dye --- p.52 / Chapter 2.2.9 --- Detection of Intracellular Ca Level by Flow Cytometry with Fluo-3 Fluorescent Dye --- p.52 / Chapter 2.2.10 --- Detection of Intracellular Hydrogen Peroxide Level by Flow Cytometry with DCF Fluorescence Dye --- p.53 / Chapter 2.2.11 --- Simultaneous Detection of Mitochondrial Membrane Potential and Intracellular Ca2+ or Mitochondrial Membrane Potential and Intracellular Hydrogen Peroxide --- p.54 / Chapter 2.2.12 --- Western Analysis --- p.55 / Chapter 2.2.12.1 --- Total Protein Extraction --- p.55 / Chapter 2.2.12.2 --- Extraction of Cytosolic Proteins --- p.59 / Chapter 2.2.13 --- Determination of Caspases Enzymatic Activity --- p.63 / Chapter 2.2.14 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.67 / Chapter 2.2.14.1 --- RNA Extraction by TRIzol Reagent --- p.67 / Chapter 2.2.14.2 --- Reverse Transcription --- p.68 / Chapter 2.2.14.3 --- Polymerase Chain Reaction --- p.68 / Chapter 2.3 --- Statistic Analysis --- p.71 / Chapter Chapter 3 --- Cytotoxicity of HK18 --- p.72 / Chapter 3.1 --- Cytotoxicity of HK18 on HepG2 Cells --- p.73 / Chapter 3.1.1 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by MTT Assay --- p.73 / Chapter 3.1.2 --- Study of the Cytotoxic Activity of HK18 on HepG2 Cells by Tryphan Blue Exclusion Assay --- p.76 / Chapter 3.2 --- Cytotoxicity of HK18 on R-HepG2 Cells --- p.78 / Chapter 3.2.1 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by MTT Assay --- p.78 / Chapter 3.2.2 --- Study of the Cytotoxic Activity of HK18 on R-HepG2 Cells by Tryphan Blue Exclusion Assay --- p.81 / Chapter 3.3 --- Cytotoxicity of HK18 on Macrophages --- p.83 / Chapter 3.4 --- Hemolytic Activity of HK18 --- p.85 / Chapter 3.5 --- In vivo Study of the Toxicity of HK18 --- p.87 / Chapter Chapter 4 --- Mechanistic Study of HK18 on HepG2 Cells --- p.90 / Chapter 4.1 --- Hallmarks of Apoptosis Induced by HK18 on HepG2 Cells --- p.91 / Chapter 4.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on HepG2 Cells --- p.91 / Chapter 4.1.2 --- Induction of DNA Fragmentation by HK18 of HepG2 Cells --- p.97 / Chapter 4.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in HepG2 Cells --- p.99 / Chapter 4.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of HepG2 Cells --- p.99 / Chapter 4.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in HepG2 Cells --- p.101 / Chapter 4.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on HepG2 Cells --- p.105 / Chapter 4.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated HepG2 Cells --- p.108 / Chapter 4.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated HepG2 Cells --- p.114 / Chapter 4.2.1.5 --- HK18 Induced Cytochrome c and AIF Released from Mitochondria of HepG2 Cells --- p.120 / Chapter 4.3 --- Downstream Biochemical Changes Induced by HK18 on HepG2 Cells --- p.123 / Chapter 4.3.1 --- Activation of Caspase 3 of HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.123 / Chapter 4.3.2 --- Induction of Caspases Activities of HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.125 / Chapter 4.4 --- Down-regulation of Anti-apoptotic Bcl-2 Family Members of HepG2 Cells by HK18 --- p.129 / Chapter Chapter 5 --- Mechanistic Study of HK18 on R-HepG2 Cells --- p.133 / Chapter 5.1 --- Hallmarks of Apoptosis Induced by HK18 on R-HepG2 Cells --- p.134 / Chapter 5.1.1 --- Induction of Phosphatidylserine Externalization by HK18 on R-HepG2 Cells --- p.134 / Chapter 5.1.2 --- Induction of DNA Fragmentation by HK18 of R-HepG2 Cells --- p.137 / Chapter 5.2 --- Study of the Underlying Mechanisms of HK18 Induced Apoptosis in R-HepG2 Cells --- p.139 / Chapter 5.2.1 --- The Role of Mitochondria in HK18 Induced Apoptosis of R-HepG2 Cells --- p.139 / Chapter 5.2.1.1 --- HK18 Induced Mitochondrial Membrane Depolarization in R-HepG2 Cells --- p.139 / Chapter 5.2.1.2 --- Addition of Bongkrekic Acid Reduced HK18 Cytotoxicity on R-HepG2 Cells --- p.142 / Chapter 5.2.1.3 --- Elevation of Intracellular Hydrogen Peroxide Level in HK18 Treated R-HepG2 Cells --- p.144 / Chapter 5.2.1.4 --- Elevation of Intracellular Ca2+ Level in HK18 Treated R-HepG2 Cells --- p.146 / Chapter 5.3 --- Downstream Biochemical Changes Induced by HK18 on R-HepG2 Cells --- p.148 / Chapter 5.3.1 --- Activation of Caspase 3 of R-HepG2 Cells by HK18 as Demonstrated by Western Blot --- p.148 / Chapter 5.3.2 --- Induction of Caspases Activation of R-HepG2 Cells by HK18 as Demonstrated by Enzymatic Activity Assays --- p.150 / Chapter 5.4 --- Down-regulation of the Anti-apoptotic Bcl-2 Protein of R-HepG2 Cells by HK18 --- p.154 / Chapter 5.5 --- HK18 was Not a Substrate of P-glycoprotein Contents --- p.156 / Chapter Chapter 6 --- Discussion --- p.158 / Chapter 6.1 --- Cytotoxicity of HK18 --- p.159 / Chapter 6.1.1 --- HK18 was Cytotoxic to the Human Hepatocellular Carcinoma Cell Line HepG2 and Multidrug Resistant Human Hepatocellular Carcinoma Cell Line R-HepG2 --- p.159 / Chapter 6.1.2 --- Study of the Toxicity of HK18 --- p.160 / Chapter 6.2 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on HepG2 Cells --- p.161 / Chapter 6.2.1 --- Apoptotic Cell Death Induction of HK18 on HepG2 Cells --- p.161 / Chapter 6.2.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.162 / Chapter 6.3 --- Mechanistic Studies of the Cytotoxic Effects of HK18 on R-HepG2 Cells --- p.181 / Chapter 6.3.1 --- Apoptotic Cell Death Induction of HK18 on R-HepG2 Cells --- p.181 / Chapter 6.3.2 --- Studies of the Underlying Mechanisms of HK18 Induced Apoptosis of HepG2 Cells --- p.181 / Chapter Chapter 7 --- Future Perspectives --- p.190 / Chapter Chapter 8 --- References --- p.193

Orientador: Fernando Rogério Pavan / Banca: Katiany Rizzieri Caleffi Ferraciolli / Banca: Tais Maria Bauab / Resumo: Tuberculose (TB) é a doença infecciosa que mais mata pessoas no mundo e é causada principalmente pelo bacilo Mycobacterium tuberculosis. Em 2016, 10,4 milhões de pessoas desenvolveram a doença e 1,8 milhão morreu em sua decorrência. Atualmente o principal agravante deste cenário é a resistência do bacilo aos antimicrobianos disponíveis para o tratamento. Entre os principais mecanismos responsáveis pela resistência aos antimicrobianos, as mutações em genes que codificam dos alvos dos fármacos se destacam. Fluoroquinolonas e aminoglicosídeos são duas classes de antimicrobianos de 2ª linha utilizados no tratamento de TB e atuam na proteína DNA girase e no ribossomo bacteriano impedindo o processo de transcrição e síntese proteica respectivamente. Mutações nos genes gyrA e rrs que codificam estes alvos podem ser responsáveis por tal resistência. Para que medidas de saúde pública possam ser tomadas para otimizar o tratamento, é preciso conhecer a que os isolados clínicos são resistentes e qual o mecanismo envolvido neste processo. Para isso, uma biblioteca com 100 isolados clínicos coletados entre 2007 e 2009 no hospital de referência Clemente Ferreira da cidade de São Paulo foi avaliada em relação a resistência a fluoroquinolonas e aminoglicosídeos. A primeira etapa foi a determinação da concentração inibitória mínima (CIM) de três antibióticos da classe das fluoroquinolonas (ofloxacino, moxifloxacino e gatifloxacino) e três da classe dos aminoglicosídeos (amicacina, canamicina e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tuberculosis (TB) is a most deadly infectious disease of people in the world and is mainly caused by the Mycobacterium tuberculosis bacillus. In 2016, 10.4 million people developed disease and 1.8 million died. Currently, the main aggravating factor of this scenario is the resistance of the bacillus to the antimicrobials available for treatment. Among the main mechanisms for resistance to antimicrobials, the mutations in genes that code the targets of the drugs stand out. Fluoroquinolones and aminoglycosides are two classes of antimicrobials of 2nd line for TB treatment and they act on protein DNA gyrase and on the bacterial ribosome impeding the process of transcription and protein synthesis respectively. Mutations in the gyrA and rrs genes encoding these targets may be explain the resistance. Public health guidelines are taken to optimize treatment and for this it is necessary to know what clinical isolates resistant and what mechanism are is involved in the process. For this, a library with 100 clinical isolates collected between 2007 and 2009 at a Clemente Ferreira reference hospital in the city of São Paulo was evaluated for resistance to fluoroquinolones and aminoglycosides. A first stage was the determination of the minimum inhibitory concentration (MIC) of three fluoroquinolone antibiotics (ofloxacin, moxifloxacin and gatifloxacin) and three of the class of aminoglycosides (amikacin, kanamycin and streptomycin) against 100 clinical isolates using a microdilution assay in 96-well plates. According to the MIC, clinical isolates resistant to at least one antimicrobial were selected for the study of the possible mechanism of resistance. Mutations in the gyrA and rrs genes were scree ned using the GenoType MTBDRsl kit because it may explain the resistance... (Complete abstract click electronic access below) / Mestre