Anthony C.T. Liang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 73-76). / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Introduction to antibiotics --- p.2 / Chapter 1.2 --- Use of antibiotics in antimicrobial chemotherapy --- p.3 / Chapter 1.3 --- Drug resistance in bacteria --- p.4 / Chapter 1.4 --- Genetic of infections drug resistance --- p.6 / Chapter 1.5 --- Clinical importance of drug resistance --- p.8 / Chapter 1.6 --- Resistance studies on a clinically- isolated Shigella Species --- p.9 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Culture media for bacteria growth --- p.10 / Chapter 2.2 --- Large scale plasmid preparation by CsCl density gradient centrifugation --- p.12 / Chapter 2.3 --- Minipreps of plasmid DNA by the alkaline lysis method --- p.14 / Chapter 2.4 --- Elution of DNA using the Geneclean Kit --- p.16 / Chapter 2.5 --- Transformation of plasmid DNA into E.coli DH5α --- p.17 / Chapter 2.6 --- Antibiotic sensitivity test and screening of resistance colonies --- p.19 / Chapter 2.7 --- Agarose electrophoresis of DNA --- p.20 / Chapter 2.8 --- Restriction and ligation --- p.21 / Chapter 2.9 --- Protocol for studying substrate profiles of aminoglycoside-modifying enzyme (AME) --- p.23 / Chapter 2.10 --- DNA sequencing using the T7 sequencing Kit from Pharmacia --- p.26 / Chapter Chapter 3 --- Antibiotic resistance studies on multiple resistant Shigella spp / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Conjugation and transformation experiment --- p.35 / Chapter 3.3 --- Extraction of plasmid DNA from Shigella 2731and transconjugant 14R525(2731) --- p.37 / Chapter 3.4 --- Discussion --- p.39 / Chapter Chapter 4 --- Cloning and characterization of beta-lactamase gene of Shigella2731 / Chapter 4.1 --- Introduction --- p.40 / Chapter 4.2 --- Cloning of the beta-lactam gene in Shigella2731 --- p.42 / Chapter 4.3 --- "Resistance pattern of El, E2, and S1" --- p.43 / Chapter 4.4 --- "Plasmid DNA extraction of El, E2, and S1" --- p.44 / Chapter 4.5 --- Restriction mapping of the plasmid pSFlOO --- p.47 / Chapter 4.6 --- Discussion --- p.51 / Chapter Chapter 5 --- Cloning and characterization of the aminoglycoside resistance gene / Chapter 5.1 --- Introduction --- p.53 / Chapter 5.2 --- Cloning of the aminoglycoside resistance genes --- p.56 / Chapter 5.3 --- Substrate profile studies on aminoglycoside- modifying enzyme (AME) activity on transformant G and S --- p.57 / Chapter 5.4 --- Subcloning of plamsid DNA from transformant S --- p.60 / Chapter 5.5 --- "DNA sequencing of fragments A, B, and C" --- p.65 / Chapter 5.6 --- Discussion --- p.68 / Chapter Chapter 6 --- Conclusion --- p.79 / Chapter Chapter 7 --- Reference --- p.73

A study to assess the potentials of some commensal bacteria that belong to Staphylococcus, Acinetobacter and Stenotrophomonas genera as reservoirs of antibiotic resistance determinants in the environment of Nkonkobe Municipality of the Eastern Cape Province, South Africa, was carried out using standard microbiological and molecular techniques. A total of 120 Staphylococcus isolates which consisted of Staphylococcus haemolyticus (30%), Staphylococcus aureus (23.3%) from pig; Staphylococcus capitis (15%) from goat; Staphylococcus heamolyticus (5%) and Staphylococcus xylosus (15%) from cattle and other Staphylococci (11%) from dead chicken and pigs were isolated. About 23.3% of these isolates were coagulase positive and 76.7% were coagulase negative. This difference in prevalence along coagulase production divide was statistically significant (p < 0.05). Eighty-six Acinetobacter species (Acinetobacter baumannii/calcoaceticus and Acinetobacter haemolyticus) were also isolated from Alice and Fort Beaufort towns samples, while 125 Stenotrophomonas maltophilia isolates were from grass root rhizosphere (96%) and soil butternut root rhizosphere (4%). Between 75-100% of the Staphylococccus species were resistant to Penicillin G, tetracycline, sulphamethaxole and nalidixic acid; about 38 % were methicillin resistant, consisting of 12.6% methicillin resistant Staphylococcus aureus (MRSA) from pig and a total of 12% vancomycin resistant were observed. Also, 12% of the isolates were erythromycin resistant while 40.2 % were resistant to the third generation cephalosporin, ceftazidime. The antibiotic resistance genes vanA, VanB, eryA, eryB, eryC were not detected in all the phenotypically resistant Staphylococccus species, but mec A gene and mph genes were detected. In the Acinetobacter species, a wide range of 30-100% resistance to penicillin G, ceftriazone, nitrofurantoin, erythromycin, and augmentin was observed. Polymerase chain reaction (PCR) revealed the presence of Tet(B) and Tet(39) genes in these species, while Tet (A), Tet(M) and Tet(H) were absent. Also, 9.3% of the Acinetobacter species showed phenotypic production of extended spectrum beta lactamases (ESBLs) while 3.5% were positive for the presence of blaCTX-M-1 genes. The Stenotrophomonas maltophilia isolates showed varying resistance to meropenem (8.9%), cefuroxime (95.6 %), ampicillin-sulbactam (53.9%), ceftazidime (10.7%), cefepime (29.3 %), minocycline (2.2%), kanamycin (56.9%), ofloxacin (2.9%), levofloxacin (1.3%), moxifloxacin (2.8%), ciprofloxacin (24.3%), gatifloxacin (1.3%), polymyxin B (2.9 %), cotrimoxazole (26.1%), trimethoprim (98.6%), aztreonam(58%) and Polymyxin B (2.9 %). The isolates exhibited significant susceptibility to the fluoroquinolones (74.3-94.7 %), polymycin (97.1%) and meropenem (88.1%). Only sul3 genes were the only sulphonamide resistance gene detected among the trimethoprim-sulphamethoxazole resistant isolates. The observed multiple antibiotic resistance indeces (MARI) of >2 for Staphylococcus species, Acinetobacter species and Stenotrophomonas maltophilia suggest that they have arisen from high-risk sources where antibiotics are in constant arbitrary use resulting in high selective pressure. The presence of tetracycline resistance genes in Acinetobacter species justifies the observed phenotypic resistance to oxytetracycline and intermediate resistance to minocycline. High phenotypic resistance and the presence of some resistance genes in Staphylococcus species is a possible threat to public health and suggests animals to be important reservoirs of antibiotic resistance determinants in the environment. Indiscriminate use of antibiotics induces this kind of antibiotic resistance and should be discouraged. Personal hygiene is encouraged as it reduces the load of Acinetobacter species contacted from the environment that may be difficult to control. Commensal Stenotrophomonas maltophilia are as important as their clinical counterparts due to their roles in opportunistic infection, antibiotic resistance and their associated genes, especially sul gene. Personal hygiene is hereby advocated especially when in contact with soil, plants and plants’ rhizospheric soil

Siu Pak-yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 158-174). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.vi / List of Figures and Tables --- p.xiii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Characteristics of Arsenic Compound --- p.1 / Chapter 1.1.1 --- Arsenic Compounds are Used as Poison --- p.1 / Chapter 1.1.2 --- Arsenic Compounds are Used as Medicine --- p.2 / Chapter 1.2 --- Arsenic Trioxide is a Traditional Chinese Medicine --- p.3 / Chapter 1.3 --- Properties of Arsenic Trioxide --- p.5 / Chapter 1.4 --- Use of Arsenic Trioxide in Cancer Treatment --- p.7 / Chapter 1.4.1 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.1 --- Characteristics of Acute Promyelocytic Leukemia --- p.7 / Chapter 1.4.1.2 --- Treatment of Acute Promyelocytic Leukemia with All-Trans Retinoic Acid --- p.10 / Chapter 1.4.1.3 --- Treatment of Acute Promyelocytic Leukemia with Arsenic Trioxide --- p.11 / Chapter 1.4.1.4 --- Action Mechanism of Arsenic Trioxide --- p.13 / Chapter 1.4.2 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Non-APL Leukemia --- p.15 / Chapter 1.4.3 --- Arsenic Trioxide as a Therapeutic Agent in the Treatment of Solid Tumors --- p.16 / Chapter 1.5 --- Human Hepatocellular Carcinoma --- p.16 / Chapter 1.5.1 --- The Incidence of Liver Cancer --- p.16 / Chapter 1.5.2 --- Classification of Liver Cancer --- p.17 / Chapter 1.6 --- Aim of the Project --- p.17 / Chapter 1.6.1 --- In Vitro Study of the Effect of Arsenic Trioxide on HepG2 Cells --- p.19 / Chapter 1.6.2 --- In Vivo Study of the Effect of Arsenic Trioxide by Tumor-Bearing Nude Mice Model --- p.20 / Chapter 1.6.3 --- "In Vitro Study of the Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.22 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Cell Lines and Culture Medium --- p.24 / Chapter 2.1.1.1 --- Cell Lines --- p.24 / Chapter 2.1.1.2 --- Culture Medium --- p.25 / Chapter 2.1.2 --- Chemicals --- p.26 / Chapter 2.1.3 --- Reagents and Buffers --- p.27 / Chapter 2.1.3.1 --- Phosphate Buffered Saline (PBS) --- p.27 / Chapter 2.1.3.2 --- "3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) Solution" --- p.27 / Chapter 2.1.3.3 --- Reagents for DNA Fragmentation Assay --- p.21 / Chapter 2.1.3.3.1 --- DNA Lysis Buffer --- p.27 / Chapter 2.1.3.3.2 --- Tris-EDTA (TE) Buffer --- p.27 / Chapter 2.1.3.3.3 --- Tris-Acetate (TAE) Buffer --- p.28 / Chapter 2.1.3.3.4 --- Proteinase K and Ribonuclease A (RNase A) --- p.28 / Chapter 2.1.3.3.5 --- 6X DNA Loading Dye --- p.28 / Chapter 2.1.3.3.6 --- One Hundred Base-Pair DNA Ladder --- p.28 / Chapter 2.1.3.4 --- Reagents for Western Blot Analysis --- p.29 / Chapter 2.1.3.4.1 --- SDS Lysis Buffer --- p.29 / Chapter 2.1.3.4.2 --- 4X Lower Gel Buffer --- p.29 / Chapter 2.1.3.4.3 --- 4X Upper Gel Buffer --- p.29 / Chapter 2.1.3.4.4 --- 10X SDS Running Buffer --- p.29 / Chapter 2.1.3.4.5 --- 2X SDS Sample Loading Dye --- p.30 / Chapter 2.1.3.4.6 --- Electroblotting Buffer --- p.30 / Chapter 2.1.3.4.7 --- Tris-Buffered Saline with 01% Tween-20 (TBS-T) --- p.30 / Chapter 2.1.3.4.8 --- Lysis Buffer for Detection of the Release of Cytochrome C --- p.31 / Chapter 2.1.3.5 --- Propidium Iodide (PI) --- p.31 / Chapter 2.1.3.6 --- "5，5 ´ة,6,6´ة-tetrachloro-1,1',3,3 '-tetraethylbenzimidazolyl carbocyanine Iodide (JC-1)" --- p.31 / Chapter 2.1.3.7 --- Reagents for In Vivo Study --- p.32 / Chapter 2.1.3.7.1 --- Saline --- p.32 / Chapter 2.1.3.7.2 --- Homogenizing Buffer --- p.32 / Chapter 2.1.3.7.3 --- 10% Buffered Formalin --- p.32 / Chapter 2.1.3.7.4 --- Acid Alcohol --- p.32 / Chapter 2.1.3.7.5 --- Scott's Tap Water --- p.32 / Chapter 2.1.3.7.6 --- 0.5% Aqueous Eosin --- p.33 / Chapter 2.2 --- Methods --- p.33 / Chapter 2.2.1 --- MTT Assay --- p.33 / Chapter 2.2.2 --- Trypan Blue Exclusion Assay --- p.34 / Chapter 2.2.3 --- Analysis of Cell-Cycle Phase Distribution by Flow Cytometry with PI Staining --- p.34 / Chapter 2.2.4 --- DNA Fragmentation Assay --- p.35 / Chapter 2.2.5 --- Quantification of Apoptosis by Flow Cytometry with Annexin V-PI Staining --- p.36 / Chapter 2.2.6 --- Assessment of the Change in Mitochondrial Membrane Potential (ΔΦm) --- p.37 / Chapter 2.2.7 --- Western Analysis --- p.38 / Chapter 2.2.8 --- Glucose Uptake Assay --- p.40 / Chapter 2.2.9 --- ATP Production Assay --- p.41 / Chapter 2.2.10 --- In Vivo Study --- p.44 / Chapter 2.2.10.1 --- Animal Model --- p.44 / Chapter 2.2.10.2 --- Cell Line --- p.44 / Chapter 2.2.10.3 --- Treatment with Arsenic Trioxide --- p.44 / Chapter 2.2.10.4 --- Assessment of the Anti-Cancer Activity of Arsenic Trioxide --- p.45 / Chapter 2.2.10.5 --- Tissue Sample Preparation --- p.45 / Chapter 2.2.10.5.1 --- Preparation of Plasma --- p.45 / Chapter 2.2.10.5.2 --- Preparation of Liver Tissue Homogenate --- p.46 / Chapter 2.2.10.5.3 --- Preparation of Cytosolic Fraction --- p.46 / Chapter 2.2.10.6 --- Measurement of the Plasma Enzyme Activity --- p.46 / Chapter 2.2.10.6.1 --- "Plasma Creatine Kinase (CK) Activity, Plasma Lactate Dehydrogenase (LDH) Activity, Plasma Alanine Transaminase (ALT) Activity and Plasma Asparate Transaminase (AST) Activity" --- p.46 / Chapter 2.2.10.7 --- Preparation of Tissue for Light Microscopic Study --- p.48 / Chapter 2.2.10.8 --- Measurement of the Basal Reduced Glutathione (GSH) Level of Liver Tissue --- p.51 / Chapter 2.2.10.9 --- "Measurement of the Activity of Antioxidant Enzyme, Glutathione S-Transferase (GST) of Liver Tissue" --- p.53 / Chapter 2.3 --- Statistical Analysis --- p.54 / Chapter Chapter 3 --- "In Vitro Study of Arsenic Trioxide on Acute Promyelocytic Leukemia Cell Line, NB-4" / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Principle of Flow Cytometry with Annexin V-PI Staining --- p.56 / Chapter 3.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of NB-4 Cells --- p.59 / Chapter 3.4 --- Study of the Action Mechanism of Arsenic Trioxide upon Treatment of NB-4 Cells --- p.61 / Chapter 3.5 --- Summary --- p.63 / Chapter Chapter 4 --- "In Vitro Study of Arsenic Trioxide on Human Hepatocellular Carcinoma Cell Line, HepG2" / Chapter 4.1 --- Introduction --- p.64 / Chapter 4.2 --- The Effect of Arsenic Trioxide on Cell Proliferation of HepG2 Cells by MTT Assay --- p.66 / Chapter 4.3 --- The Effect of Arsenic Trioxide on HepG2 Cells at Clinically Achievable Concentration --- p.68 / Chapter 4.3.1 --- The Cytotoxicity of Arsenic Trioxide on HepG2 Cells by Trypan Blue Exclusion Assay --- p.68 / Chapter 4.3.2 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution --- p.71 / Chapter 4.3.3 --- The Underlying Mechanism of the Cytotoxic Effect of Arsenic Trioxide 一 Necrosis or Apoptosis? --- p.74 / Chapter 4.3.3.1 --- DNA Fragmentation Assay --- p.74 / Chapter 4.3.3.2 --- Flow Cytometry with Annexin V-PI Staining --- p.76 / Chapter 4.3.3.3 --- Brief Conclusion --- p.78 / Chapter 4.3.4 --- The Study of the Mechanism of Apoptotic Pathway --- p.78 / Chapter 4.3.4.1 --- Activation of Caspase-3 upon Arsenic Trioxide Treatment --- p.79 / Chapter 4.3.4.2 --- The Participation of Mitochondria in Arsenic Trioxide-Induced Apoptosis --- p.81 / Chapter 4.3.4.2.1 --- The Change in Mitochondrial Membrane Potential upon Arsenic Trioxide Treatment --- p.81 / Chapter 4.3.4.2.2 --- The Study of the Release of Cytochrome C from the Mitochondria to Cytosol upon Treatment with Arsenic Trioxide --- p.85 / Chapter 4.3.4.2.3 --- Brief Conclusion --- p.87 / Chapter 4.4 --- Arsenic Trioxide Mediated Its Effect via Other Action Mechanisms --- p.87 / Chapter 4.4.1 --- The Effect of Arsenic Trioxide on the Expression of Glucose Transporters 1 and2 --- p.88 / Chapter 4.4.2 --- The Effect of Arsenic Trioxide on Glucose Uptake --- p.91 / Chapter 4.4.3 --- The Effect of Arsenic Trioxide on ATP Production --- p.93 / Chapter 4.4.4 --- Brief Conclusion --- p.93 / Chapter 4.5 --- Summary --- p.95 / Chapter Chapter 5 --- In Vivo Study of Arsenic Trioxide on HepG2-Bearing Nude Mice / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Treatment with Arsenic Trioxide --- p.97 / Chapter 5.3 --- Assessment of the Anti-Tumor Effect of Arsenic Trioxide --- p.99 / Chapter 5.4 --- The Effect of Arsenic Trioxide toward Normal Tissues --- p.103 / Chapter 5.4.1 --- The Effect of Arsenic Trioxide on Liver --- p.104 / Chapter 5.4.1.1 --- Morphological Study --- p.104 / Chapter 5.4.1.2 --- Enzymatic Study --- p.107 / Chapter 5.4.1.3 --- Brief Conclusion --- p.107 / Chapter 5.4.2 --- The Effect of Arsenic Trioxide on Heart --- p.110 / Chapter 5.4.2.1 --- Morphological Study --- p.110 / Chapter 5.4.2.2 --- Enzymatic Study --- p.112 / Chapter 5.4.2.3 --- Brief Conclusion --- p.112 / Chapter 5.5 --- Involvement of the Glutathione Redox System --- p.115 / Chapter 5.5.1 --- Basal GSH Level --- p.115 / Chapter 5.5.2 --- The Activity of Glutathion S-Transferase --- p.117 / Chapter 5.5.3 --- Brief Conclusion --- p.117 / Chapter 5.6 --- Summary --- p.120 / Chapter Chapter 6 --- "In Vitro Study of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" / Chapter 6.1 --- Introduction --- p.121 / Chapter 6.2 --- The Effect of Doxorubicin on the Parental HepG2 Cells and R-HepG2 Cells by MTT Assay --- p.123 / Chapter 6.3 --- The Effect of Arsenic Trioxide on Cell Proliferation of R-HepG2 Cells by MTT Assay --- p.126 / Chapter 6.4 --- The Effect of Arsenic Trioxide on Cell-Cycle Phase Distribution of R-HepG2 Cells --- p.129 / Chapter 6.5 --- Trioxide on R-HepG2 Cells ´ؤ Necrosis or Apoptosis? --- p.131 / Chapter 6.5.1 --- DNA Fragmentation Assay --- p.131 / Chapter 6.5.2 --- Flow Cytometry with Annexin V-PI Staining --- p.133 / Chapter 6.5.3 --- Brief Conclusion --- p.133 / Chapter 6.6 --- Examination of the Probable Involvement of Arsenic Trioxide as a Substrate of P-Glycoprotein --- p.135 / Chapter 6.7 --- Summary --- p.137 / Chapter Chapter 7 --- Discussion / Chapter 7.1 --- The Significance of the Study of Arsenic Trioxide in the Treatment of Arsenic Trioxide --- p.138 / Chapter 7.2 --- Comparison of Preparation of Drug in Present Study with Others --- p.140 / Chapter 7.3 --- Effect of Arsenic Trioxide on Human Hepatocellular Carcinoma --- p.142 / Chapter 7.4 --- Mechanism Study of Arsenic Trioxide --- p.142 / Chapter 7.5 --- Dosage of Arsenic Trioxide Used in In Vivo Study --- p.152 / Chapter 7.6 --- Cytotoxicity of Arsenic Trioxide toward Normal Tissues --- p.153 / Chapter 7.7 --- "Effect of Arsenic Trioxide on Multidrug-Resistant Human Hepatocellular Carcinoma Cell Line, R-HepG2" --- p.154 / Chapter 7.8 --- Conclusions and Future Prospect --- p.156 / Chapter Chapter 8 --- References / Chapter 8.1 --- English References --- p.158 / Chapter 8.2 --- Chinese References --- p.174 / Chapter 8.3 --- Online References --- p.174

Neoadjuvant chemotherapy has become standard practice in patients with high-risk early breast cancer as it improves rates of breast conservation surgery and enables prediction of recurrence and survival by using response to treatment as a surrogate. Previous studies have focused on generating molecular datasets to develop prediction models of response, though little is known on how tumours and their microenvironments are modulated by neoadjuvant chemotherapy. The thesis aims at molecularly characterising tumour changes during neoadjuvant chemotherapy in a cohort of 168 patients. Serial tumour samples at diagnosis, and, when available, midway through chemotherapy and on completion of treatment were profiled by shallow whole genome sequencing, deep exome sequencing and transcriptome sequencing, resulting in the generation of an unprecedented genomics dataset with tumours in situ while patients received chemotherapy. Molecular predictors of response to chemotherapy were inferred from the diagnostic biopsy. Several novel observations were made, including previously undescribed associations between copy number alterations, mutational genotypes, neoantigen load, HLA genotypes and intra-tumoural heterogeneity with chemosensitivity. Possible mechanisms of chemoresistance included LOH at the MHC Class I locus, decreased expression of MHC Class I and II genes and drug influx molecules, as well as increased expression of drug efflux pumps. A complex relationship between proliferation, tumour microenvironment composition (TME) and response to treatment was explored by deconvoluting bulk RNAseq data and performing digital pathology orthogonal validation. Clonal and microenvironment dynamic changes induced by/associated with chemotherapy were then modelled. Two types of genomic responses were identified, one in which the clonal composition was stable throughout treatment and another where clonal emergence and/or extinction was evident. Validation by multi-region deep sequencing confirmed the dynamics of the clonal landscape. Clonal emergence was shown to be associated with higher proliferation and decreased immune infiltrate, with an increase in genomic instability and homologous recombination deficiency during treatment. The immune TME composition and activity mirrored response to treatment, with cytolytic activity and innate and adaptive immune infiltrates linearly correlating with the degree of residual disease remaining after chemotherapy. Finally, the circulating tumour DNA (ctDNA) genomic landscape was explored by using shallow whole genome sequencing and targeted sequencing of plasma DNA. Tumour mutations detected on exome sequencing were also detected in ctDNA in plasma, supporting the use of liquid biopsies as a biomarker for monitoring response to therapy and detection of minimal residual disease.

Malaria persists today as a significant burden for a large part of the world. However, over the past few decades, a concerted effort by governments, non-governmental organizations, researchers, and community health workers worldwide has yielded progress in reducing the deadly impact of this disease. Today, some of these gains are threatened by the rise of antimalarial drug resistance, a recurring problem that has impeded global malaria reduction efforts before. Research on Plasmodium falciprum resistance to the numerous antimalarial compounds used today and in the past has made significant progress on determining which specific mutations modulate drug susceptibility and to what degree they do so. To gain a comprehensive understanding of drug resistance, we need to elucidate how and why it arises. Therefore, it is important to elucidate whether some malaria parasites acquire resistance-conferring mutations faster than others and why the native function of the genetic factors involved lend themselves to modulating drug resistance. For instance, resistance to multiple antimalarial therapies has repeatedly emerged in Southeast Asia. We investigated the long-held hypothesis that this was due to the ability of these parasites to mutate significantly faster than non-Southeast Asian strains. Elucidating whether this hypermutability phenotype accurately represents Southeast Asian parasite evolvability is important, as it can inform when resistance would be expected to next arise, particularly in the Greater Mekong Subregion in Southeast Asia. Here, we have adapted a fluctuation assay to Plasmodium falciparum and determined that some contemporaneous Cambodian parasites exhibit a mild mutator, but not a hypermutator, phenotype. We also show that this is likely driven by mutations in DNA repair genes carried predominantly by multidrug resistant Southeast Asian parasites. One of the most common genes in which drug resistance-conferring mutations occurs is the P. falciparum chloroquine resistance transporter (pfcrt). Mutations in pfcrt are associated with parasite susceptibility to many of the antimalarial compounds that have been used in a clinical setting to date. However, beyond its role in drug resistance, little is known about the native function of PfCRT. To facilitate the study of pfcrt, we have designed a zinc-finger nuclease (ZFN)-based gene engineering system that introduces a single double-strand break in intron 1 of pfcrt. Our ZFN strategy enables replacing nearly any endogenous pfcrt locus with a user-defined recombinant pfcrt allele. We show that our method of pfcrt allelic replacement is fast, efficient, and reliable. We used this system to generate a unique mutant parasite encoding a pfcrt-L272F mutation, which enlarges the parasite digestive vacuole, the lysosome-like organelle used to catabolize host-derived hemoglobin for amino acid salvage. Our results provide clear evidence that PfCRT is associated with the terminal steps of hemoglobin degradation, overall parasite fitness, and the balance of osmolytes across the digestive vacuole membrane. Bringing clarity to the native function of PfCRT can reveal how and why this single genetic factor has been and continues to be involved in the resistance to many different antimalarial compounds.

O desenvolvimento de resistência a múltiplas drogas na terapêutica do câncer é um importante obstáculo para o tratamento efetivo. Os mecanismos de resistência a múltiplas drogas ocasionam a redução intracelular de agentes quimioterápicos e, por conseqüência, estão envolvidos no fracasso no tratamento do câncer. Os principais genes envolvidos neste fenômeno são: o gene MDR1(multiple drug resisctance), que codifica uma glicoproteína de alto peso molecular, a P-gp; o gene MRP1, que codifica uma glicoproteína de 190 Kda, denominada proteína associada à resistência a múltiplas drogas; e o gene da LRP (proteína relacionada à resistência de pulmão). Alguns estudos sugerem que o colesterol pode estar envolvido diretamente com o fenômeno de resistência a múltiplas drogas, e que os lipídeos podem influenciar várias e complexas funções no MDR, por afetarem o transporte de drogas através da membrana plasmática. Além disso, células tumorais tem maior necessidade de colesterol devido a uma taxa de multiplicação mais elevada que as células normais. Neste estudo analisou-se a expressão dos genes MDR1, MRP1 e LRP em células de sarcoma uterino resistentes à doxorrubicina, e a influência de lipoproteínas. Houve aumento da expressão dos genes MDR1, MRP1 e LRP nas células tratadas com a LDL, sendo mais expressivo o gene MDR1. A HDL diminuiu a expressão dos genes MRP1 e LRP. No entanto, o gene MDR1 teve sua expressão diminuída somente em concentrações maiores. As células cultivadas em meio sem soro fetal apresentaram um elevado aumento na expressão destes genes. Em conclusão, as lipoproteínas podem modular a expressão dos genes MDR1, MRP1 e LRP e, assim, atuar na resistência a múltiplas drogas. / The development of multidrug resistance in anticancer therapy is an obstacle in the efficiency of the treatment. The multidrug resistance mechanism causes reduction of intracellular chemotherapeutical drugs. Therefore, it leads to treatment failure. There are three main multidrug resistance genes: MDR1, which codifies the P-gp (a high weight glycoprotein); MRP1, which codifies a 190 Kda glycoprotein; and, the LRP (lung resistance related protein) gene. Several reports suggest that cholesterol may be directly involved with the multidrug resistance phenomenon and that lipids may affect many complex functions in this regard, as the activity of the drug transport across the plasmatic membrane. Moreover, tumor cells have great cholesterol necessity due to the high cell multiplication rate. Here we described the MDR, MRP, LRP gene expression of a doxorubicin-resistant uterine sarcoma cell line under the influence of lipoproteins. LDL increased the expression of all genes, mainly MDR1. Treatment with HDL led to reduction of MRP and LRP expression. However, the MDR gene expression decreased only by higher concentrations of HDL. Cells grown in serumdeprived medium led to an increased expression of all the studied genes. Therefore, lipoproteins may modulate the MDR, MRP, LRP gene expression and, consequently, the cell resistance to drugs.

Acute lymphoblastic leukemia (ALL) is an aggressive hematologic malignancy that results from the unregulated growth of B-cell and T-cell lymphoid progenitors. Despite the implementation of risk-stratification and improved multi-agent therapeutic regimens, 20% of pediatric and 50% of adult patients fail to achieve remission and end up relapsing. NT5C2 (5’ cytosolic nucleotidase II) is the most frequently mutated gene specifically found in relapsed ALL. NT5C2 mutations are present in 20% of relapsed T-ALLs and 3-10% of relapsed B-ALLs and present as heterozygous gain of function alleles exhibiting increased nucleotidase activity. As NT5C2 can dephosphorylate and inactivate the cytotoxic metabolites generated by 6-mercaptopurine, a chemotherapy used in the treatment of ALL, these NT5C2 activating mutations can contribute to thiopurine chemotherapy resistance (Tzoneva, Perez-Garcia et al. 2013). Here we perform an extensive structure-function study to understand how relapse-associated NT5C2 mutations result in increased nucleotidase activity and contribute to chemotherapy resistance in ALL. Crystallization of 15 NT5C2 WT and mutant structures as well as enzymatic, structural modeling, and genetic screens identified three regulatory mechanisms of NT5C2, which are disrupted by these gain of function alleles. Class I NT5C2 mutations lock the protein in an active configuration through stabilization of the helixA region, which allows for substrate processing and catalysis. Class II NT5C2 mutations disrupt an intramolecular switch off domain involving the arm region and the intermonomeric positively charged pocket. And a single C-terminus truncating mutant creates a third class of mutations, which show increased nucleotidase activity due to the loss of the C-terminus blockade against allosteric activation. These studies provide new insight into the regulatory controls that mediate NT5C2 activity providing a framework for the development of targeted inhibitors for the treatment of relapsed ALL. In addition to looking at relapse associated NT5C2 mutations on a structural level, we also explored how NT5C2 mutations shape the clonal architecture and evolutionary dynamics during tumor initiation and disease progression in ALL. To formally address these questions, we developed a murine NOTCH1-driven T-ALL with conditional knock-in of the Nt5c2R367Q mutation, the most recurrent mutation found in relapsed ALL, from the endogenous locus. Using this model, we confirmed that Nt5c2+/R367Q lymphoblasts show increased resistance to 6-MP in vitro and in vivo. We also found that Nt5c2+/R367Q mutant lymphoblasts exhibit impaired cell fitness and decreased leukemia initiating cell capacity. Metabolomic profiling and guanosine rescue experiments show that this decrease in cell fitness is due to excess clearance of purine metabolites out of the cell as a result of deregulated Nt5c2 nucleotidase activity. However, in the context of 6-MP therapy, Nt5c2+/R367Q mutant cells are positively selected for in mixed population studies in vitro and in vivo. These results identify a clear selective advantage for NT5C2 mutant cells in the context of 6-MP chemotherapy. In addition, NT5C2 mutant chemoresistant cells show collateral sensitivity to inhibition of inosine monophosphate dehydrogenase (IMPDH) with mizoribine, which further disrupts guanosine production pointing to a potentially selective therapy against NT5C2 mutant cells. We also show here the initial development of a small molecule NT5C2 inhibitor for the treatment of relapsed ALL. Using a malachite green based NT5C2 nucleotidase assay, we performed a small molecule high throughput assay and identified HTP_2 as a lead compound with low micromolar inhibitory activity against NT5C2 R367Q mutant recombinant protein. HTP_2 can reverse 6-MP resistance in Nt5c2+/R367Q mouse lymphoblasts and NT5C2 R29Q mutant expressing human cell lines. Interestingly, HTP_2 treatment also results in increased sensitivity to 6-MP therapy in NT5C2 wild-type cells, suggesting a role for wild-type NT5C2 activity in the clearance of 6-MP and supporting a potential therapeutic use for NT5C2 inhibitors in potentiating the effects of 6-MP based chemotherapy in NT5C2 wild-type cells as well. NT5C2 knockdown cells and Nt5c2 knockout mice show no apparent toxicities suggesting that systemic inhibition of NT5C2 could be fairly well tolerated. In all, this work presents a framework for the development of a high affinity NT5C2 inhibitor for the reversal of 6-MP resistance in relapsed ALL patients. These studies presented here address the role of NT5C2 mutant proteins as drivers of resistance and as therapeutic targets in relapsed ALL. Improved understanding of the molecular mechanisms responsible for increased NT5C2 nucleotidase activity and on the process of clonal evolution during disease progression provide important insight into the mechanism driving ALL resistance and relapse. The identification of IMPDH inhibition as a collateral vulnerability in NT5C2 mutant ALL cells and the development of a first-in-class NT5C2 inhibitor serve as framework for the development of new combination therapies aimed at curtailing the emergence of these thiopurine-resistant relapse driving clones in ALL.

Cheung Tsz Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 187-203). / Abstracts in English and Chinese. / Acknowledgements --- p.is / Abbreviations --- p.II / Abstracts --- p.V / List of Figures --- p.IX / List of Tables --- p.XIII / Contents / Chapter Chapter 1. --- General Introduction / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- Growth Factor --- p.2 / Chapter 1.3 --- Growth Factor and Growth Factor Receptor --- p.4 / Chapter Chapter 2. --- Alteration of EGF Responses and EGFR Signaling in EGF-conditioned A431 cells / Chapter 2.1 --- Background Information / Chapter 2.1.1 --- Epidermal Growth Factor (EGF) --- p.6 / Chapter 2.1.2 --- Epidermal Growth Factor Receptor (EGFR) --- p.10 / Chapter 2.1.2.1 --- The Structure of EGFR --- p.10 / Chapter 2.1.2.2 --- The EGFR Family --- p.11 / Chapter 2.1.2.3 --- EGFR Activation --- p.13 / Chapter 2.1.3 --- The Intracellular Signal Transduction Pathways in EGFR Signaling --- p.18 / Chapter 2.1.3.1 --- The Ras/Raf/MAPK Pathway (MAPK pathway) --- p.19 / Chapter 2.1.3.2 --- The Jak/Stat Pathway --- p.23 / Chapter 2.1.3.3 --- The PI3K/Akt Pathway --- p.28 / Chapter 2.1.4 --- EGFR and Cancer --- p.31 / Chapter 2.1.5 --- EGFR-targeted Cancer Therapy --- p.35 / Chapter 2.1.5.1 --- Monoclonal Antibody (MAb) --- p.36 / Chapter 2.1.5.2 --- Immunotoxin Conjugates --- p.37 / Chapter 2.1.5.3 --- Bispecific Antibody --- p.37 / Chapter 2.1.5.4 --- Small-molecule EGFR Tyrosine Kinase Inhibitor (EGFR-TKI) --- p.38 / Chapter 2.1.5.5 --- Antisense Oligonucleotide --- p.39 / Chapter 2.2 --- Objectives --- p.41 / Chapter 2.3 --- Materials and Methods / Chapter 2.3.1 --- Materials --- p.42 / Chapter 2.3.2 --- Methods / Chapter 2.3.2.1 --- Cell Lines --- p.44 / Chapter 2.3.2.1.1 --- Establishment of Epidermal Growth Factor (EGF)-conditioned A431 Cells (EGF-conditioned Cells) ´ؤ AC Cells --- p.44 / Chapter 2.3.2.2 --- Growth Curve between A431 Parent Cells and EGF-conditioned Cells --- p.45 / Chapter 2.3.2.3 --- Epidermal Growth Factor (EGF) Sensitivity Assay --- p.45 / Chapter 2.3.2.4 --- Western Blot Analysis --- p.47 / Chapter 2.3.2.4.1 --- Protein Samples Preparation --- p.47 / Chapter 2.3.2.4.2 --- Protein Assay (by BCA Protein Assay Reagent) --- p.48 / Chapter 2.3.2.4.3 --- Protein Electrophoresis --- p.49 / Chapter 2.3.2.4.4 --- Electroblot (Protein Transfer) --- p.50 / Chapter 2.3.2.4.5 --- Antibody Probing (Immunoblotting) --- p.51 / Chapter 2.4 --- Results / Chapter 2.4.1 --- Growth Curve --- p.53 / Chapter 2.4.2 --- EGF Responses of A431 Parent Cells and EGF-conditioned Cells by MTT Assay --- p.55 / Chapter 2.4.3 --- The EGFR Expression Levels in A431 Parent Cells and EGF-conditioned Cells by Western Blot Analysis --- p.57 / Chapter 2.4.4 --- EGF-induced Protein Tyrosine Phosphorylation Pattern in A431 Parent Cells and EGF-conditioned Cells by Western Blot Analysis --- p.59 / Chapter 2.4.5 --- The Expression Profiles of EGFR Signaling Molecules in A431 Parent Cells and EGF-conditioned Cells by Western Blot Analysis --- p.61 / Chapter 2.4.5.1 --- The Ras/Raf/MAPK Pathway --- p.62 / Chapter 2.4.5.2 --- The Jak/Stat Pathway --- p.63 / Chapter 2.4.5.3 --- The PI3K/Akt Pathway --- p.64 / Chapter 2.4.6 --- The Cellular Responses to the Modifiers that Targeting the EGFR Signaling --- p.68 / Chapter 2.4.6.1 --- The Sensitivity of A431 Parent Cells and EGF-conditioned Cells to Various Signaling Modifiers --- p.69 / Chapter 2.4.6.2 --- The Influence of EGFR Signaling Modifiers on EGF --- p.76 / Chapter 2.5 --- Discussion --- p.85 / Chapter Chapter 3. --- The Inter-relationship between the Differential Anti-cancer Drugs Sensitivity and Alteration of EGFR Signaling in EGF-conditioned A431 Cells / Chapter 3.1 --- Background Information / Chapter 3.1.1 --- Drug Resistance and its Mechanisms in Tumor Cells --- p.90 / Chapter 3.1.2 --- Anti-cancer Drugs ´ؤ Introduction / Chapter 3.1.2.1 --- Camptothecin (CPT) --- p.93 / Chapter 3.1.2.2 --- Methotrexate (MTX) --- p.95 / Chapter 3.1.2.3 --- 5-fluorouracil (5-Fu) --- p.98 / Chapter 3.1.2.4 --- Vincristine (VCR) and Taxol --- p.104 / Chapter 3.1.2.5 --- Cisplatin (cis-DDP) --- p.108 / Chapter 3.2 --- Objectives --- p.110 / Chapter 3.3. --- Materials and Methods / Chapter 3.3.1 --- Materials --- p.112 / Chapter 3.3.2 --- Methods / Chapter 3.3.2.1 --- Cell Lines --- p.115 / Chapter 3.3.2.2 --- Determination of Drug Sensitivity by MTT Assay --- p.115 / Chapter 3.3.2.2.1 --- Determination the Influence of EGFR Signaling Modifiers on the Differential Anticancer Drugs Sensitivity by MTT Assay --- p.115 / Chapter 3.3.2.3 --- Semi-quantitative RT-PCR --- p.116 / Chapter 3.3.2.3.1 --- Preparation of RNA Samples --- p.116 / Chapter 3.3.2.3.2 --- RT-PCR --- p.117 / Chapter 3.3.2.4 --- DNA Fragmentation Assay --- p.118 / Chapter 3.3.2.5 --- Western Blot Analysis --- p.120 / Chapter 3.3.2.6 --- Northern Blot Analysis --- p.120 / Chapter 3.4 --- Results / Chapter 3.4.1 --- The Responses to Various Anti-cancer Drugs / Agents in A431 Parent Cells and EGF-conditioned Cells --- p.122 / Chapter 3.4.2 --- The Expressions of Classical Cellular Drug Resistant Factors in EGF-conditioning-associated Differential Anti-cancer Drugs Sensitivity --- p.126 / Chapter 3.4.2.1 --- Camptothecin Sensitivity --- p.126 / Chapter 3.4.2.2 --- Methotrexate Sensitivity --- p.130 / Chapter 3.4.2.3 --- 5-fluorouracil Sensitivity --- p.135 / Chapter 3.4.2.4 --- Vincristine and Taxol Sensitivity --- p.141 / Chapter 3.4.3 --- EGFR Signaling Modifiers and Differential Anti-cancer Drugs Sensitivity by MTT Assay --- p.143 / Chapter 3.4.3.1 --- Methotrexate --- p.143 / Chapter 3.4.3.2 --- Vincristine --- p.147 / Chapter 3.4.3.3 --- Taxol --- p.149 / Chapter 3.5 --- Discussion --- p.153 / Chapter Chapter 4. --- Identification of Differentially Expressed Genes in A431 Parent Cells and EGF-conditioned Cells by Differential Display (DD) / Chapter 4.1 --- Introduction 一 Differential Display (DD) --- p.156 / Chapter 4.2 --- Objectives --- p.160 / Chapter 4.3 --- Materials and Methods / Chapter 4.3.1 --- Materials --- p.161 / Chapter 4.3.2 --- Methods / Chapter 4.3.2.1 --- Cell Lines --- p.163 / Chapter 4.3.2.2 --- RT-PCR-based mRNA Differential Display --- p.163 / Chapter 4.3.2.2.1 --- Preparation of RNA Samples --- p.163 / Chapter 4.3.2.2.2 --- Identification of Differentially Expressed Genes by RT-PCR --- p.164 / Chapter 4.3.2.2.3 --- Reamplification of cDNA Probes --- p.164 / Chapter 4.3.2.2.4 --- Subcloning of the Differentially Expressed cDNAs --- p.165 / Chapter 4.3.2.2.4.1 --- Preparation of the Ultra-competent E.coli Cells for Transformation --- p.165 / Chapter 4.3.2.2.4.2 --- Preparation of Cloning Vector --- p.166 / Chapter 4.3.2.2.4.3 --- Transformation --- p.166 / Chapter 4.3.2.2.5 --- Verification of cDNA Differentially Expression by Colony-PCR and Northern Blot Analysis --- p.167 / Chapter 4.3.2.2.5.1 --- Colony-PCR --- p.167 / Chapter 4.3.2.2.5.2 --- Preparation of Cloned Plasmid cDNA and Bacterial Glycerol Stocks --- p.167 / Chapter 4.3.2.2.5.3 --- Preparation of cDNA Probes for Northern Blot Analysis --- p.168 / Chapter 4.3.2.2.5.4 --- Northern Blot Analysis --- p.168 / Chapter 4.3.2.2.6 --- Sequencing of the Desired Cloned cDNA Inserts --- p.170 / Chapter 4.3 --- Results --- p.171 / Chapter 4.4 --- Discussion --- p.180 / Chapter Chapter 5. --- General Conclusion and Future Perspectives / Chapter 5.1 --- General Conclusion --- p.182 / Chapter 5.2 --- Future Perspectives --- p.185 / References --- p.187

Drug resistance is a serious problem in health care in general, and in malaria treatment in particular, rendering many of our previously considered ‘wonder drugs’ useless. Recently, large sums of money have been allocated for the continuous development of new drugs to replace the failing ones. We seem to be one step behind the evolution of antimalarial resistance; is it possible to get one step ahead? Are interventions which slow down the evolution and spread of drug-resistant malaria parasites achievable? In this thesis, I address these issues with experimental data, using the well-established rodent malaria model Plasmodium chabaudi to understand the selective advantages and disadvantages drug-resistant parasites endure within a vertebrate host and the selective pressures various drug treatment regimes exert on these parasites. Competitive interactions between drug-resistant and drug-sensitive parasites were observed within the host, with resistant parasites having a competitive disadvantage in the absence of drug treatment. The frequency of resistant parasites at the start of the infection was an important determinant of the strength of selection: the lower their frequency, the stronger the competitive suppression in non-treated hosts and the greater their competitive release following drug treatment. Genetically similar genotypes, one resistant and one sensitive, showed similar dynamics following drug treatment. Multiplicity of infection did not have an effect on the within-host dynamics: a larger number of co-infecting susceptible genotypes did not lead to greater competitive suppression or release of resistant parasites. Lastly, various drug treatment regimes were compared. Conventional drug treatment resulted in the greatest selective advantage for drug-resistant parasites, while less aggressive treatments were equally as effective, or even better, at improving host health and reducing overall infectiousness. These studies demonstrate that altering the within-host ecology of drug-resistant parasites by administering drugs and hence removing the drug-sensitive competitors has a large influence on the transmission potential of drug-resistant parasites. Furthermore, this thesis provides proof of principle that other drug treatment regimes different from those currently in use could better control drug-resistant parasites, without compromising other treatment goals. In the case of malaria, less drugs may mean extending the useful lifespan of that drug.

Chai, Stella. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 108-126). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.v / PATENT AND PUBLICAION --- p.vii / CONFERENCE ABSTRACTS AND PRESENTATIONS --- p.viii / AWARDS --- p.ix / ABBREVIATIONS --- p.x / LIST OF TABLES --- p.xii / LIST OF FIGURES --- p.xiv / TABLE OF CONTENT --- p.xviii / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Multidrug resistance (MDR) --- p.1 / Chapter 1.1.1 --- Cancer --- p.1 / Chapter 1.1.2 --- Mechanisms of MDR in cancer --- p.1 / Chapter 1.1.2.1 --- Drug entry --- p.3 / Chapter 1.1.2.2 --- Drug metabolism --- p.3 / Chapter 1.1.2.3 --- Drug sequestration --- p.4 / Chapter 1.1.2.4 --- Mechanisms activated after nuclear entry --- p.5 / Chapter 1.1.2.5 --- Evasion of drug-induced apoptosis --- p.5 / Chapter 1.1.3 --- Approaches in treating MDR --- p.5 / Chapter 1.1.3.1 --- Overcoming MDR by inhibiting transporters --- p.6 / Chapter 1.1.3.2 --- Overcoming MDR by altering signaling pathway --- p.6 / Chapter 1.1.4 --- ATP Binding Cassette (ABC) Transporters --- p.7 / Chapter 1.1.4.1 --- P-glycoprotein (P-gp) --- p.7 / Chapter 1.1.4.2 --- Multidrug resistance-associated protein 1 (MRP 1) --- p.9 / Chapter 1.1.4.3 --- Breast cancer resistant protein (BCRP) --- p.10 / Chapter 1.1.4.4 --- ABC drug transporters and drug absorption --- p.11 / Chapter 1.2 --- The Use of Traditional Chinese Medicine (TCM) in circumventing P-gp-mediated MDR --- p.12 / Chapter 1.2.1 --- Active ingredients in TCM - Alkaloid --- p.12 / Chapter 1.2.2 --- Active ingredients in TCM - Saponin --- p.14 / Chapter 1.2.3 --- Active ingredients in TCM - Flavonoid --- p.15 / Chapter 1.2.4 --- Active ingredients in TCM - Others --- p.17 / Chapter 1.3 --- Polyoxypregnane compounds (POPs) --- p.17 / Chapter 1.3.1 --- Characterization --- p.17 / Chapter 1.3.2 --- POPs isolated from M. tenacissima --- p.18 / Chapter 1.4 --- Objectives of Current Study --- p.22 / Chapter CHAPTER 2. --- EFFECTS OF POLYOXYPREGNANE COMPOUNDS ON VIABILITY AND PROLIFERATION OF HUMAN RESISTANT CANCER CELLS --- p.24 / Chapter 2.1 --- Materials and Methods --- p.25 / Chapter 2.1.1 --- "Chemicals, Materials and Reagents" --- p.25 / Chapter 2.1.2 --- Methods --- p.26 / Chapter 2.1.2.1 --- Cell Lines and Cell Culture --- p.26 / Chapter 2.1.2.2 --- Preparation of POPs --- p.27 / Chapter 2.1.2.3 --- Sulforhodamine B assay --- p.27 / Chapter 2.1.2.4 --- Statistical analysis --- p.29 / Chapter 2.2 --- Results --- p.29 / Chapter 2.2.1 --- Effects of POPs on the viability of parental SW620 and P-gp-overexpressing resistant SW620/Ad300 cells --- p.29 / Chapter 2.2.2 --- Effects of POPs on the viability of parental MCF-7 and MRP1-overexpressing resistant MCF-7/VP cells --- p.33 / Chapter 2.2.3 --- Effects of POPs on the viability of parental MCF-7 and ABCG2-overexpressing resistant MCF-7/FLV1000 cells --- p.37 / Chapter 2.3 --- Discussion --- p.41 / Chapter 2.3.1 --- Structure activity relationship (SAR) --- p.43 / Chapter 2.3.2 --- Nine compounds relating to P-gp-mediated MDR --- p.46 / Chapter CHAPTER 3. --- MECHANISM OF NINE SELECTED POPS IN MODULATING P-GP-MEDIATED MDR --- p.49 / Chapter 3.1 --- Materials and Methods --- p.49 / Chapter 3.1.1 --- "Chemicals, Materials and Reagents" --- p.49 / Chapter 3.1.2 --- Methods --- p.53 / Chapter 3.1.2.1 --- Cell Lines and Cell Culture --- p.53 / Chapter 3.1.2.2 --- Extraction of nine POPs from M. tenacissima --- p.54 / Chapter 3.1.2.3 --- Sulforhodamine B (SRB) assay --- p.55 / Chapter 3.1.2.4 --- Flow cytometry assay --- p.55 / Chapter 3.1.2.5 --- P-gp ATPase assay --- p.56 / Chapter 3.1.2.6 --- Immuno-blot/Western blot analysis --- p.58 / Chapter 3.1.2.7 --- Reverse transcription and quantitative real-time PCR --- p.59 / Chapter 3.1.2.8 --- Statistical analysis --- p.60 / Chapter 3.2 --- Results --- p.60 / Chapter 3.2.1 --- Effects of nine selected POPs on the viability of sensitive human breast cancer MCF-7 cells --- p.60 / Chapter 3.2.2 --- Effects of nine selected POPs on the viability of MDR 1 -transfected HEK1 MDR1 cell line and its control vector transfected cell line HEK293 pcDNA3 --- p.61 / Chapter 3.2.3 --- Effects of nine selected POPs in inhibiting efflux of P-gp substrate --- p.64 / Chapter 3.2.4 --- Effects of nine selected POPs in modulating P-gp ATPase activity --- p.68 / Chapter 3.2.5 --- Effects of nine selected POPs in regulating P-gp protein expression --- p.69 / Chapter 3.2.6 --- MDR1 mRNA expression in various cell lines --- p.72 / Chapter 3.3 --- Discussion --- p.72 / Chapter 3.3.1 --- Effective POPs are targeting specifically P-gp overexpression --- p.73 / Chapter 3.3.2 --- Mechanistic understanding the circumvention of MDR by the effective POPs --- p.74 / Chapter 3.3.2.1 --- Relative potency for the reversal of P-gp-mediated MDR --- p.75 / Chapter 3.3.2.2 --- Inhibition of P-gp-mediated drug efflux across cell membrane by the effective POPs --- p.75 / Chapter 3.3.2.3 --- Stimulation of ATPase by the effective POPs --- p.76 / Chapter 3.3.2.4 --- No effect of POPs on the alteration of P-gp expression --- p.77 / Chapter 3.3.2.5 --- An overall summary of the mechanism of MDR reversal by the effective POPs --- p.78 / Chapter 3.3.3 --- Implication in drug disposition and drug-drug interactions --- p.79 / Chapter 3.3.4 --- Additional information for the structure activity relationship (SAR) --- p.80 / Chapter CHAPTER 4. --- EFFECTS OF CRUDE EXTRACT AND THREE MAJOR POLYOXYPREGNANES (POPS) OF MARS DEN I A TENACISSIMA --- p.81 / Chapter 4.1 --- Materials and Methods --- p.82 / Chapter 4.1.1 --- "Chemicals, Materials and Reagents" --- p.82 / Chapter 4.1.2 --- Methods --- p.82 / Chapter 4.1.2.1 --- "Preparation of M. tenacissima extract, artificial mixture and three fractions" --- p.82 / Chapter 4.1.2.2 --- Sulforhodamine B assay --- p.85 / Chapter 4.1.2.3 --- "Biotransformation study of POP68, POP69 and POP70" --- p.85 / Chapter 4.1.2.4 --- HPLC-MS analysis --- p.86 / Chapter 4.1.2.5 --- Animal care and housing conditions --- p.87 / Chapter 4.1.2.6 --- Toxicity studies of fraction 2 in mice --- p.88 / Chapter 4.1.2.7 --- Statistical analysis --- p.89 / Chapter 4.2 --- Results --- p.89 / Chapter 4.2.1 --- "Effects of crude extract, artificial mixture on the viability of sensitive human breast cancer MCF-7 cells" --- p.89 / Chapter 4.2.2 --- "Effects of crude extract, artificial mixture on the viability of sensitive SW620 and P-gp-overexpressing resistant SW620/Ad300 cells" --- p.90 / Chapter 4.2.3 --- "Metabolites of POP68, POP69 and POP70 after incubation with human intestinal microbiota" --- p.91 / Chapter 4.2.4 --- Toxicity of fraction 2 in mice --- p.94 / Chapter 4.3 --- Discussion --- p.98 / Chapter CHAPTER 5. --- FINAL DISCUSSION AND CONCLUSIONS --- p.105 / REFERENCES --- p.108